B A Boronic

Acetonitrile AMP Antimicrobial peptide APBA 4-aminophenylboronic acid ARG, R Arginine ATP Adenosine triphosphate

CDCl3

Chloroform

ACKNOWLEDGEMENT

Preparing my PhD thesis has been a wonderful experience. During this period, I have had the opportunity to meet a lot of remarkable people. I have received support and encouragement from a great number of individuals that made my stay in France and Germany an unforgettable experience.

First, I would like to express my sincere gratitude to my PhD supervisors.

Prof. Sabine Szunerits, for her support, guidance and scientific discussions throughout my study period and also for the suggestions during the writing process of this thesis and the patience while correcting it. Prof. Nils Metzler-Nolte, also, for his guidance, scientific ideas and discussions for the past three years. I am especially grateful for your confidence and the freedom you gave me to do this work. I would never have come this far without your support, encouragement and possibility you both created initiating this co-tutelle project.

My sincere thanks go to Dr. Rabah Boukherroub for his suggestions and sharpening my scientific thinking. To Dr. Alexandre Barras for his valuable advice during my research work and guidance while exploring new areas that I was no familiar with and to Dr. Emerson Giovanelli for his useful suggestions and corrections throughout the process of writing this thesis.

A special thank goes to Dr. Jean Dubuisson for his generosity and letting me experience the research of human coronavirus and helping me to develop my background in this field. I would also like to express my special thanks to Dr. Karin Seron, who had helped me with the viral assays experiments. I want to thank all the NanoBioInterfaces group (NBI, France) and Inorganic chemistry I group (AC1, Germany) for providing a stimulating research environment and for their friendship, support and company during the past three years.

My heartfelt thanks go to Ioana Silvia Hosu and Milica Budimir for always being there during the good and bad days, listening to my problems and providing possible solutions. Without you, life in Lille would not have been the same. I will always cherish all those humour, stupid jokes and meaningful discussions. To Vedi without whom we would be just the fantastic trio instead of magnificent four. Anna, Vlad and Mathias thank you for being a great company (Slavic ACKNOWLEDGEMENT power), especially during the karaoke evenings. Many thanks to Quentin without whom my coffee breaks would not be the same.

I would like to thank Sugina for always remembering me when buying a coffee. Without you, my mornings would be miserable, especially when the day was starting earlier than usual. Also, many thanks to all the girls who were sharing the lab space with me for maintaining the ambience and peaceful working environment in the lab. Especially to Nicole for always entertaining me throughout the day. I would also like to express my thanks to Carsten and Matt who had helped me a lot with HPLC, often sacrificing their free time.

I would like to thank all the lab members from both groups, which I did not mention for stimulating research environment. I truly appreciate the nature of sharing the ideas among lab mates.

I extend my sincere thanks to all members of the Department of Chemistry at Ruhr University Bochum, all members of the NBI team and all those who contributed directly or indirectly to the dissertation. Especially to Dr. Laura Chambre and Prof. Amitav Sanyal for providing the cryogels.

Special thanks go to Université de Lille and Ruhr Universität Bochum for funding my PhD fellowship.

I would like to thank all the people I met on my way during the past three years who believed in me and make my PhD experience even more valuable as well as all my friends who supported my decision of studying abroad even if that meant being miles away from them.

I have no words to explain my gratitude to Antoine for his encouragement, continuous support and help he had offered me for the past 3 years.

RESUME/ABSTRACT/ZUSAMMENFASSUNG

This work investigates the potential antibacterial and/or antiviral activity of carbonbased nanostructures such as diamond nanoparticles and carbon quantum dots (CQDs) as well as reduced graphene oxide (rGO) in combination with cryogels. CQDs formed by hydrothermal synthesis from 4-aminophenylboronic acid as the carbon precursor showed to be efficient in the inhibition of the viral attachment of human coronavirus HCoV-229E-Luc to cells with an EC50 of 5.2±0.7 µg mL -1 . Mechanistic studies suggest that the CQDs are acting at the early stage of virus infection as well at the viral replication step. In parallel, we took advantage of the multivalent character of CQDs as well as nanodiamonds and modified them with short synthetic antimicrobial peptides (AMPs). Tests of these nanostructures against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli pathogens showed increased antibacterial activity when compared to AMPs alone. In the case of rGO combined with cryogels loaded with AMPs, bacterial eradication was achieved efficiently and on-demand using near-infrared light as external trigger to release AMPs.

II ZUSAMMENFASSUNG

Die zunehmende Antibiotikaresistenz und die begrenzte Entwicklung neuer Medikamente erfordern die Suche nach alternativen Strategien zur Ausrottung bakterieller Infektionen. Ähnliche Probleme stellen sich bei der Entwicklung antiviraler Therapeutika, da stetig neue Viren auftauchen und diese durch genetische Mutationen der Therapie entgehen können.

In dieser Arbeit wurde die potenzielle antibakterielle und/oder antivirale Aktivität von Nanostrukturen auf Kohlenstoffbasis wie Diamantnanopartikeln und Kohlenstoff-Quantenpunkten (CQDs) sowie reduziertem Graphenoxid (rGO) in Kombination mit Kryogelen untersucht. CQDs, die durch hydrothermale Synthese aus 4-Aminophenylboronsäure als Kohlenstoffvorläufer gebildet wurden, erwiesen sich als effizient bei der Hemmung der viralen Anheftung des humanen Coronavirus HCoV-229E-Luc an Zellen mit einem EC50 von 5,2±0,7μg mL -1 . Mechanistische Studien deuten darauf hin, dass die CQDs sowohl im frühen Stadium der Virusinfektion als auch im viralen Replikationsschritt wirken. Parallel dazu nutzten wir den multivalenten Charakter der CQDs sowie der Nanodiamanten und modifizierten diese mit kurzen synthetischen antimikrobiellen Peptiden (AMPs). Analysen dieser Nanostrukturen gegen gram-positive Staphylococcus aureus und gram-negative Escherichia coli-Erreger zeigten eine erhöhte antibakterielle Aktivität im Vergleich zu AMPs allein. Im Falle von rGO in Kombination mit AMPs beladenen Kryogelen wurde die bakterielle Eradikation effizient erreicht. Bei Bedarf wurde Nahes Infrarot Licht als externer Auslöser für die Freisetzung von AMPs verwendet.

OBJECTIVES

III

OBJECTIVES

Both, the increasing antibiotic resistance and the limited development of new antibacterial drugs, make imperative the search for alternative strategies to eradicate bacterial infections. Similar problems are faced in the development of antiviral therapeutics, due to the continued emergence of new viruses and their ability to escape therapy by genetic mutations.

Nanoparticles have attracted tremendous interest for the treatment of bacterial and viral infections mainly due to either their intrinsic antibacterial properties (e.g. silver nanoparticles)

or their multivalent nature. Indeed, multivalent interactions are omnipresent in biology and confer on biological systems dramatically enhanced affinities towards determined receptors.

Such multivalent binding interactions have lately been considered for the development of new therapeutic strategies against bacterial and viral infections. While a high synthetic effort is often needed for the development of such therapeutics, the integration of several molecules (otherwise named ligands) onto nanostructures turned out to be a viable alternative. Particles modified with numerous ligands have the additional advantage of giving rise to high local concentrations of the corresponding bound molecules.

In this thesis, different surface modification strategies have been applied and developed with the goal of obtaining nanostructures with antibacterial and antiviral properties. Carbonbased nano scaffolds, notably diamond nanoparticles (often also referred to as nanodiamonds), carbon quantum dots (CQDs) and 2D carbon nanomaterials such a reduced graphene oxide (rGO), were chosen herein as they possess unique biological and physicochemical properties.

Each nanostructure exhibits inimitable features and can be exploited in diverse biological applications, including antiviral and antibacterial applications.

The thesis is divided into two parts: the first part is devoted to the screening of a large number of peptide nanostructures and nanostructures against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli; the second part is an in-depth study of the possibility of functionalised CQDs to work as inhibitors for the interaction of the human coronavirus HCoV-229E with the cell membrane. After a general overview of the use of nanoparticles for biological applications, a brief description of the surface modification strategies of carbon nanomaterials towards biomedical applications is provided in Chapter 1. Introduction Nanotechnology, introduced almost half a century ago, is an active research area with both novel science and useful applications that has gradually established itself in the past two decades. Much of nanoscience and nanotechnology is concerned with producing new or enhanced materials in the form of nanoparticles (NPs) and nanostructured materials and represents an active area of research and a techno-economic sector with full expansion in many application domains. These nanostructures have gained prominence in technological advancements due to their tunable physicochemical characteristics (Figures 1.1) such as melting point, wettability, electrical and thermal conductivity, catalytic activity, light absorption and scattering resulting in enhanced performance over their bulk counterparts. The implementation and rapid development of chemical functionalization strategies on nanomaterials have, in addition, greatly increased the variety of available nanomaterials and their application. Through the manipulation of size, surface characteristics and the material used, the nanoparticles can be developed into smart systems, encasing therapeutic and imaging agents as well as bearing stealth property. Nanometre-sized materials are thus gaining massive attention for various application areas ranging from their use in energy devices and as catalytic scaffolds to their interest for biomedical applications(Figure 1.2) [2][3][4]. This is due to their unique physical and chemical properties which are barely found in bulk materials. In the biomedical field, the large surface-to-volume ratio of NPs is of value when engineering drug delivery systems, diagnostic platforms and nanotechnology-based imaging strategies. The high surface-to-volume ratio allows the integration of different molecules allowing the target delivery of drugs [6,7] and is responsible for the multivalent character of these nanostructures. A series of experiments and theoretical studies have also shown that the multivalent nature of nanoparticles allows distinguishing surfaces (cells) on the basis of their receptor concentration, rather than just on the basis of the presence of a suitable receptor [8,9]. The use of multivalent particles coated with a single type of ligand is very effective, provided that a cognate receptor has been identified that is sufficiently overexpressed in targeted cells.

There are various other reasons why using nanoparticles for therapeutic and diagnostic agents, as well as the advancement of drug delivery, is important and much needed. One of them is that that hydrophobic character of many drugs limits their oral or injectable administration, as they are not manufactured as the optimal formulation for each product. Products containing proteins or nucleic acids require a more innovative type of carrier system to enhance their efficacy and protect them from unwanted degradation. It is notable that the efficiency of most drug delivery systems is directly related to particle size (excluding intravenous and solution). Due to their small size and large surface area, drug nanoparticles show increase solubility and thus enhanced ability to cross the blood-brain barrier (BBB), enter the pulmonary system and be absorbed through the tight junctions of endothelial cells of the skin [10][11][12][13][14]. Additionally, the use of multifunctional theranostic agents with targeted delivery can solve problems associated with an increase in the drug doses and the resulting toxicity [15][16][17].

While a high surface-to-volume ratio is a fundamental characteristic of NPs for drug delivery, the ability of some NPs (e.g. gold nanorods, rGO) to convert light energy into heat also allows the remote manipulation of their environment. Such a capacity can cause intentional damages to the targeted tissues and/or favour the interaction with determined bioactive compounds. For example, thermosensitive drug delivery vehicles can be activated by photothermal therapy (PTT), allowing the on-demand drug release at a target site [18][19][20].

Understanding how the body handles the exogenous particulate matter is warranted.

Once, they enter systemic circulation, particle-protein interaction is the first phenomenon taking place before distribution into various organs. Absorption from the blood capillaries allows the lymphatic system to further distribute and eliminate the particles. If something is recognised as foreign, macrophages will engulf and clear it from the body. This tends to be the struggle with nanoparticle-based nanomedicines; however, clearance can be influenced by the size and surface characteristics of particles.

Currently, several therapeutic agents and technologies based on nanotechnology have been approved for clinical use (Table 1.1) or are under clinical trials, which underlines the already high interest in this field. NPs typically used for biomedical applications fall into three categories (Figure 1.3):

i) inorganic nanoparticles (gold, silica, iron oxide, metal quantum dots); ii) organic nanoparticles (polymeric liposomes, micelles, dendrimers); iii) carbon nanoparticles (fullerene, nanodiamonds, carbon nanotubes, carbon dots, graphene). In this thesis work, we have exploited different strategies for the surface modification of carbon nanoparticles. Our goal was to obtain nanostructures with antibacterial and antiviral properties. Therefore, carbon-based nanoparticles, notably carbon dots, nanodiamonds, and 2Dmaterials such a graphene oxide were chosen. Before discussing our results on the application of these nanomaterials, a general outline about a different class of nanomaterials focusing on carbon based materials is given below.

Inorganic nanoparticles

Inorganic particles comprise metal, metal oxides and semiconductors. One of the main reasons for their use in biomedicine is their intrinsic physical properties. As a few examples, we can cite iron oxide magnetic NPs, gold NPs exhibiting surface plasmon resonance, luminescent semiconductor quantum dots, and upconverting luminescent lanthanide-doped NPs [23][24][25][26][27][28]. All of these properties can be fine-tuned during the synthesis by engineering the size, shape, composition and structure of the inorganic core.

Basic requirements for biomedical applications of such nanostructures include water solubility and colloidal stability under physiological conditions while keeping their physicochemical properties steady. Another critical factor is their biocompatibility [29].

Biocompatible surface modifications enable water solubility and endow NPs with stability and biocompatibility. Surface modifications can modulate the pharmacokinetics and medical functions of NPs aiming at different tissue or organ lesions [30].

Synthetic strategies to obtain biocompatible NPs include in situ "one-pot" synthesis of hydrophilic particles [31,32] or post surface modification of hydrophobic NPs. Most common coating materials are polymers, zwitterionic molecules and biomolecules [33][34][35]. For improving solubility and biocompatibility among polymers, the most commonly used is PEG and PEG-copolymers [36]. PEG-based modifications can be roughly categorized into i) encapsulation strategies [36] ii) ligand exchange by coordinating with the anchoring groups strategies [37]. Apart from PEG molecules, zwitterions are another class of materials often used for coating of inorganic particles to increase their biocompatibility. The advantage compared to the PEG molecule is little apparent increase of hydrodynamic size under physiological conditions [38,39]. Zwitterions and other biomolecules such as proteins, nucleic acids, peptides can be coated onto the particle surface upon covalent or non-covalent interactions (Figure 1.4). The covalent coupling can be achieved by creating the bond directly or through crosslinker coupled with biomolecules with functional groups like -COOH, -NH2, -SH, maleimide [41,42]. The non-covalent attachment includes electrostatic interactions [43]which depend on the affinity of two differently charged species, biotin-avidin system [44] allowing incorporation of biomolecules as well as non-biomolecules such as DNA, antibodies, fluorescent dyes, hydrophobic interactions and base-pairing interactions [45,46].

Inorganic nanoparticles found their application in many different biomedical fields.

They have been extensively studied for their potential use as antimicrobial agents due to their direct and indirect antimicrobial properties (Table 1.2). Their mode of action varies between particles, but they exhibit bactericidal properties mostly through the generation of reactive oxygen species (ROS) although some are effective due to their physical structure and metal ion release (Table 1.

2).

One of the major types of NPs with antimicrobial properties that have been utilized for decades is metals/metal oxides NPs [47,48]. This group of NPs include silver (Ag), iron oxide (Fe3O4), titanium oxide (TiO2), copper oxide (CuO), zinc oxide (ZnO), and gold NPs.

Silver nanoparticles possess unique physicochemical properties that have a significant impact on their biological properties. It has been shown that silver nanoparticles can effectively prevent the growth of bacteria on the surface of agar plates but are becoming less effective in a liquid medium [49]. The difference is associated with the aggregation of colloidal silver in the presence of high salts concentration and other media components. These results suggest that the antimicrobial efficacy depends on the surface oxidation state of NPs and particles dispersion [49]. Next to that, size and shape play a significant role in their ability to interact with the bacterial surface [50,51]. Small and spherical NPs show higher affinity towards the surface of a cell membrane [52,[START_REF] Yamanaka | Bactericidal Actions of a Silver Ion Solution on Escherichia coli, Studied by Energy-Filtering Transmission Electron Microscopy and Proteomic Analysis[END_REF]. Although silver NPs are known to researchers for decades and have been extensively studied. Their bactericidal mechanism of action is not yet fully understood. In AgNPs there might be three sources of bactericidal activity: Ag, Ag + , AgNPs. All three of them can interlacing, making it difficult to determine what effect comes from each of them [52,[START_REF] He | Mechanisms of the pH dependent generation of hydroxyl radicals and oxygen induced by Ag nanoparticles[END_REF].

Impregnation of medical devices and wound dressing significantly reduced the infections rate in hospital settings [START_REF] Politano | Use of silver in the prevention and treatment of infections: silver review[END_REF], but the toxicity of the particles on mammalian cells is still in dispute [START_REF] Carlson | Unique Cellular Interaction of Silver Nanoparticles: Size-Dependent Generation of Reactive Oxygen Species[END_REF][START_REF] Xia | Comparison of the Abilities of Ambient and Manufactured Nanoparticles To Induce Cellular Toxicity According to an Oxidative Stress Paradigm[END_REF][START_REF] Lekamge | The Toxicity of Silver Nanoparticles (AgNPs) to Three Freshwater Invertebrates With Different Life Strategies: Hydra vulgaris, Daphnia carinata, and Paratya australiensis[END_REF].

Titanium oxide, similarly to silver NPs, has been studied for its antibacterial and antiviral activities [START_REF] Akhtar | Antibacterial and antiviral potential of colloidal Titanium dioxide (TiO2) nanoparticles suitable for biological applications[END_REF][START_REF] Baek | Effects of coating materials on antibacterial properties of industrial and sunscreen-derived titanium-dioxide nanoparticles on Escherichia coli[END_REF]. Their optical, dielectric, and photo-catalytic properties resulting from size quantization makes them promising structures which can be utilized as a self-cleaning and self-disinfecting material for surface coating in many application, e.g. self-cleaning or selfsterilizing surfaces for places such as medical centres. Their bactericidal properties are associated mostly with the formation of ROS [START_REF] Huang | Bactericidal mode of titanium dioxide photocatalysis[END_REF].

Zinc oxide NPs show broad-spectrum bactericidal activity. The particles show to successfully inhibit the growth of methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), and methicillin-resistant S. epidermidis (MRSE) strains and proved to be effective bactericidal agents that were not affected by the drug-resistant mechanisms of MRSA and MRSE [START_REF] Ansari | Characterization of clinical strains of MSSA, MRSA and MRSE isolated from skin and soft tissue infections and the antibacterial activity of ZnO nanoparticles[END_REF][START_REF] Kadiyala | Unexpected insights into antibacterial activity of zinc oxide nanoparticles against methicillin resistant Staphylococcus aureus (MRSA)[END_REF]. These inexpensive NPs proved to be effective in size dependency against a wide range of bacteria species. They possess the highest photocatalytic efficiency among all inorganic photocatalytic materials and are more biocompatible than TiO2 [START_REF] Fan | Zinc Oxide Nanostructures: Synthesis and Properties[END_REF]. Their white colour and ability to absorb UV light enhances its conductivity which significantly activates the interaction with bacteria. It has been proven that zinc oxide NPs have the ability to prevent the biofilm formation [START_REF] Mahamuni-Badiger | Biofilm formation to inhibition: Role of zinc oxidebased nanoparticles[END_REF] making them suitable coating material designated for medical and other application like, e.g. potential antibacterial agent in food preservation and packaging [START_REF] Espitia | Chapter 34 -Zinc Oxide Nanoparticles for Food Packaging Applications[END_REF].

Iron oxide in its bulk form is considered inert and has lack of any antimicrobial properties. However, when synthesized as nanosized particles, iron oxide NPs demonstrate antiadherent properties and significantly reduce both Gram-negative and Gram-positive bacterial colonization [START_REF] Gabrielyan | Antibacterial effects of iron oxide (Fe3O4) nanoparticles: distinguishing concentration-dependent effects with different bacterial cells growth and membrane-associated mechanisms[END_REF]. For biomedical applications, iron and iron oxide magnetic NPs with nanocrystalline magnetite (Fe3O4) cores is preferable because of its biocompatibility profile, biodegradability and facile synthesis. Roughly iron oxide can be divided into two classes, superparamagnetic iron oxide nanoparticles (SPIONs) and ultra-small superparamagnetic iron oxide nanoparticles (USPIONs), each with different mean hydrodynamic diameters, as well as different relaxometry properties. It has been shown that FeO-NPs can improve the stability and solubility of the encapsulated drug [START_REF] Cheng | A facile method to prepare superparamagnetic iron oxide and hydrophobic drug-encapsulated biodegradable polyurethane nanoparticles[END_REF], and no toxicity effect has been observed after cell uptake. Additionally, nano-therapy with smartly coated MPs possess a broad scope in diagnostic and therapeutic applications [START_REF] Ebrahimi | Novel drug delivery system based on doxorubicin-encapsulated magnetic nanoparticles modified with PLGA-PEG1000 copolymer[END_REF][START_REF] Ji | A Dual pH/Magnetic Responsive Nanocarrier Based on PEGylated Fe₃O₄ Nanoparticles for Doxorubicin Delivery[END_REF][START_REF] Attia | One-step synthesis of iron oxide polypyrrole nanoparticles encapsulating ketoprofen as model of hydrophobic drug[END_REF][START_REF] Ivashchenko | Synthesis and characterization of magnetite/silver/antibiotic nanocomposites for targeted antimicrobial therapy[END_REF].

Gold NPs possess effective antimicrobial properties due to its large surface area, high chemical stability, optical properties, oxidation resistance and biocompatibility [START_REF] Giljohann | Gold Nanoparticles for Biology and Medicine[END_REF][START_REF] Dykman | Gold nanoparticles in biomedical applications: recent advances and perspectives[END_REF][START_REF] Kim | Multifunctional Photonic Nanomaterials for Diagnostic, Therapeutic, and Theranostic Applications[END_REF]. The key characteristics that distinguish AuNPs from other nanostructures are their unique optical properties resulting from a physical phenomenon known as localised surface plasmon resonance (LSPR). LSPR is known to be highly sensitive to the geometric parameter (e.g. size, shape, symmetry) and material composition. In the case of spherical gold NPs, LSPR occurs in the visible region and can be shifted to the NIR region. As a result, the optical properties of gold nanostructures can be tuned and adjust to suit specific biomedical application [START_REF] Wiley | Synthesis of Silver Nanostructures with Controlled Shapes and Properties[END_REF]. Besides their surface can be easily modified by a wide variety of ligands via thiol or amine functions which allows the incorporation of biomolecules that can tailor their properties as well as introduce new functionalities to NPs [START_REF] Singh | Dual functionality nanobioconjugates targeting intracellular bacteria in cancer cells with enhanced antimicrobial activity[END_REF][START_REF] Rattanata | Gallic acid conjugated with gold nanoparticles: antibacterial activity and mechanism of action on foodborne pathogens[END_REF]. AuNPs, when decorated with antibiotics, show bactericidal properties to many multidrug-drug-resistant pathogens [START_REF] Silvero | Selective Photoinduced Antibacterial Activity of Amoxicillin-Coated Gold Nanoparticles: From One-Step Synthesis to in Vivo Cytocompatibility[END_REF]. Additionally, their activity can be enhanced by binding to nonantibiotic molecules and together with light energy can induce ROS production and mutation extensively use in therapy against cancer cells [START_REF] Farooq | Gold Nanoparticles-enabled Efficient Dual Delivery of Anticancer Therapeutics to HeLa Cells[END_REF][START_REF] Singh | Gold Nanoparticles in Diagnostics and Therapeutics for Human Cancer[END_REF].

Another group of inorganic particles which thanks to their properties found an application in the diverse biomedical field are quantum dots (QDs). They possess superior photo-stability, bright photoluminescence, narrow emission and broad absorption spectra. They are nanometer-scale semiconductor crystals composed of groups II to VI or III to V elements.

Their ability to absorb white light and then remit specific colour depends on the bandgap of the material. QDs usually consist of a semiconductor core, overcoated by a shell to improve optical properties and a cap enabling improved solubility in aqueous buffers. Their use for the biological application has cast some doubts due to their potential toxicity. There have been several studies reported that size, charge, coating ligands, oxidative, photolytic and mechanical stability each could contribute to the toxicity of cadmium containing QDs [START_REF] Zhang | Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements[END_REF]. In addition, the potential leakage of heavy metal ions from the core of the particles caused by photolysis and oxidation has a significant impact on the toxicity of QDs [START_REF] Zhang | Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements[END_REF][START_REF] Fang | Toxicity evaluation of CdTe quantum dots with different size on Escherichia coli[END_REF]. The additional layer on the surface of QDs (usually ZnS or CdS) not only protect the core from photo-oxidation minimizing cytotoxicity but also enhancing photostability [START_REF] Vasudevan | Core-shell quantum dots: Properties and applications[END_REF]. However, even with advances in synthesis, obtaining medically useful QDs is still problematic due to differences in optical properties from batch to batch. Moreover, additional functionalization is required for incorporation of biologically active molecules [START_REF] Medintz | Potential clinical applications of quantum dots[END_REF][START_REF] Matea | Quantum dots in imaging, drug delivery and sensor applications[END_REF].

Organic nanoparticles

Another class of nanomaterials used for biomedical purposes includes organic NPs.

They consist of a wide range of different materials like liposomes, polymers, lipids, capsules, gels [START_REF] Doshi | Designer Biomaterials for Nanomedicine[END_REF][START_REF] Mattu | 2 -Smart Polymeric Nanoparticles[END_REF]. These kinds of NPs are mostly made from organic matter, and their synthesis and design rely on non-covalent interactions that convert them into desired structures via selfassembly. The most important feature of these nanostructures is their ability to encapsulate other materials, such as hydrophobic drugs. The material used for their fabrication is by nature biodegradable, which makes them an attractive system for drug delivery.

Methods used for the preparation of organic nanoparticles can be divided into three categories [START_REF]Emulsion: Theory and practice[END_REF]100]: i) procedure based emulsification ii) nano-precipitation iii) drying process.

The first method is based on two steps process. The first step involves the preparation of nanodroplets of specific size wherein organic compound are formerly solubilized. In the second step, nanoparticles are formed by a different mechanism such as precipitation, gelation or polymerization. The second method is based on the nanoprecipitation where the organic compound is precipitating out of the solution through solvent displacement, self-assembly mechanism induced by ionic gelation or by the formation of polyelectrolyte complexes.

One of the most extensively used organic nanoparticles for drug delivery is liposomes.

Liposomes are spherical in shape particles that contain one or more lipid or phospholipids bilayer with the spacing in between the bilayers. Their structure contains lipophilic as well as hydrophobic and amphiphilic molecules which makes them suitable for incorporation of many In biomedical application, the optical properties of CQDs are of great importance. Next to the absorption and fluorescence properties, size-dependent photoluminescence is an exciting feature of the particles [154,155]. This phenomenon can be explained by the core-state emission induced by perfect carbon crystal with fewer defects, surface-state emissiondetermined by the hybridisation of the carbon backbone and/or molecular fluorescence-induced by fluorescence impurities. The quantum confinement effect occurs when the particles decrease in size, and their radius approaches the exciton Bohr radius. The quantisation of the energy levels occurs according to Pauli's exclusion principle (Figure 1.5A), which relates them more closely to the atoms than in the bulk material. A decrease in size results in an increase in the energy difference between the top of the valence band and the bottom of the conduction band so that more energy is needed to excite the dot. Consequently, more energy is released when it returns to its ground state (Figure 1.5B). In terms of application, it means that changing the size of the dots (controlling the size during the synthesis), any emission wavelength can be obtained from the same material (in a determined wavelength range depending on the material)

[156], which is a very useful property especially for bioimaging [157]. However, not only the size but also the surface functionalization can tailor the optical properties of carbon based quantum dots: modifying the nanoparticles with either electrondonating or electron-accepting molecules will cause the photoluminescence wavelength to shift.

Another important feature of CQDs for biomedical application is their cell cytotoxicity

[160] which is, in contrast to metallic nanoparticles for example, significantly lower due to their hydrophilic character and to the "green" reagents involved in their process of fabrication which The choice of the precursor or passivation agent used for the synthesis already defines which functional groups will (potentially) be exhibited at their surface. Carboxylic acid, hydroxyl or amino groups give us a possibility to covalently link biologically active material.

Next to covalent functionalization, non-covalent modification based on π-π interaction as well as van "click" reaction is its high selectivity and shows great potential in the fields of biomedicine.

Apart from covalent modifications, non-covalent functionalization is another approach.

Compared to covalent bonding, π-π interactions, complexation or non-covalent interactions have a lesser impact on the surface of NPs and also provide new targeting molecules.

Nanodiamonds

In contrast to CQDs, the carbon atoms in diamond all possess sp 3 hybridisation. The average size of these structures is 4-5 nm, and their quality and surface chemistry can be influenced by the considered synthetic approaches. The most popular synthesis conditions or processes include the detonation (D-NDs), the high pressure/temperature (HPHT-NDs) and the chemical vapour deposition (CVD-NDs). All NDs share the same core structure, which confers them properties similar to bulk diamond which are: excellent stability in harsh conditions, high refractive index and excellent thermal conductivity. They also provide versatile surface chemistry which allows to tailor them as a function of the desired application. In addition, they can undergo processes which can modify their sp 3 Even after the intense purification process, sp 2 carbon impurities might be still present in the cleaned D-NDs, causing a significant tendency to agglomeration [175]. Therefore, prior to utilisation, they need to undergo deagglomeration and fractionation. This step is usually achieved by high energy ball milling [177] followed by a fractionation which separates the different agglomerates according to their size.

To make nanodiamonds applicable, they very often need to undergo additional surface functionalization. These modifications will tune their future interaction with the surrounding media. The presence of hydroxyls, carboxylic acids, epoxides, as well as lactones, has been identified at the surface of NDs [178]. Various kinds of NDs have been commercialised up to date. However, we must not forget that the composition might differ as a function of the producer.

The main strategies used to modify of NDs' surface can be divided into covalent and non-covalent approaches (Figure 1 This connection creates another possibility for grafting molecules by applying the copper(I) catalysed azide-alkyne cycloaddition. The esterification reaction is commonly used for grafting biopolymers with the aim of improving colloidal stability and blood circulation [187]. Another approach consists in the reaction with siloxanes which exhibits several advantages such as the use of mild reaction conditions and an extended versatility. The product after the reaction is covered with a uniform shell, the thickness of which depends on the amount of precursor. This reaction is often used to enhance the surface area, hence to increase the loading capacity or to introduce amine functions which later will be used in a condensation reaction with a carboxylicgroup-bearing biomolecule [186]. The derivatisation of the hydroxyl group has been found to be an excellent method for modification and has been intensively explored. Barras NDs can be used as co-adjuvants, stimulating the pro-inflammatory or antiinflammatory signalling pathway [193,194]. They found their application not only as a platform for the delivery of antibodies but also as enhancers of host immune response. Recently it has been reported that NDs have been used in an influenza vaccine in conjugation with trimeric H-7 antigen (viral protein). This nanoconjugate resulted in a virus-like particle vaccine suspension which showed increased efficiency in vitro [194]. In addition, NDs can show an intrinsic bactericidal activity [195,196]. Ong and co-workers demonstrated that these bactericidal properties depend on the bacteria type, concentration, size and structure [197,198].

The toxicity of nanodiamonds is doubtful [199]. Indeed, NDs undergo various cleaning processes, and each of them depends on the considered producer. This induces multiple variants as far as the surface modifications are concerned. In vivo and in vitro studies have been carried

out in order to test their features such as cell vitality, in vivo physiological and mechanistic behaviour [200]. It has been reported that even at high doses, NDs conjugates did not increase systemic immune response nor increase or modify the serum indicators of liver toxicity [201].

Nanodiamonds, due to their excellent properties, biocompatible nature and their easy-to-modify surface, are excellent candidates as nanocarriers for biomolecules. In addition, nitrogen-vacancy defects centre in NDs makes them fluorescent. Therefore, they found their application as a non-photobleaching fluorescent probe for bioimaging experiments [202].

Graphene, Graphene oxide and reduced graphene oxide

Graphene and its derivatives such as graphene oxide (GO) and reduced graphene oxide (rGO) (Figure 1.10) have also attracted attention for biomedical applications [203,204].

Graphene is a two-dimensional material, consisting of carbon atoms with the sp 2 hybridisation.

Ever since its discovery in 2004, a vast number of studies has been performed followed up its The possibility of non-covalent functionalization via van der Waals forces and π-π interactions with organic molecules or polymers is an easy manner to modify these nanosheets.

The first one applied to molecules with high hydrophobic character while the other to the molecules with short or highly extended π system. In addition to that, the presence of oxygen groups on the surfaces and edges of GO allow ionic interactions and hydrogen bonding.

Electrostatic interactions employ two opposite charged materials commonly used as a convenient tool in the preparation of composites via self-assembly as such GO/rGO composites with biocompatible materials have been reported. For example, successful anchoring of lactoferrin, lactoferrin protected gold nanoclusters, chitosan has been reported and as prepared or nanosized drug carriers present many distinct advantages over conventional antibiotics, including improved pharmacokinetics (higher solubility, controlled release, site-targeted delivery) and accumulation while reducing the side effects proper to antibiotic therapy.

Additionally, nanocarriers can improve the stability of antibacterial agents in various physiological environments, and it is believed that it can help to overcome the antimicrobial drug resistance [4,5].

Antibacterial NPs are made up of metals, metal oxides, carbon-based materials, surfactant-based emulsions or naturally occurring compounds with antibacterial activity [6].

Their unique physicochemical properties and high surface-to-volume ratio make them To maximise the therapeutic effect, the therapeutic index and adverse reactions need to be optimised [12]. Additionally, the acquired resistance of infectious pathogens is another challenge in the discovery of new drugs. The exceptional properties of NPs such as their small and controllable size, or their structural/functional versatility, make them very promising platforms to overcome those limitations [12,13]. The use of NPs as delivery platforms show huge advantages over traditional antibiotics. Incorporating poorly water-soluble drugs onto the NPs may increase their solubility, extend their half-life, as well as their constant and stimuliresponsive release, which would ultimately lead to lower the corresponding administration frequency and dose [12][13][14]. Targeted delivery, together with the synergistic effect due to the co-delivery of multiple antimicrobials achieved by NP carriers, can also minimise the side effects resulting from the taken antibiotics [12,13].

For the past decade, several studies have significantly contributed to the development of novel nanoparticle-based platforms with different compositions and biological properties.

Amongst them, carbon-based materials like nanodiamonds (NDs) [15,16] and carbon dots (CQDs) [17,18] became a viable alternative as nanocarriers.

NDs have attracted increasing attention due to their large specific surface area, good biocompatibility [19,20], non-toxicity [21,22], high drug loading capacity [23], and inexpensive large-scale synthesis. Some of the advantages of NDs over other carbon vehicles such as carbon nanotubes are their optical transparency, their complete inertness and the scope of their functionalization possibilities, which make them highly adaptive as a function of the considered application [23][24][25].

CQDs, as another group of carbon materials, have attracted tremendous attention due to their chemical stability, very good dispersibility in aqueous media, biocompatibility, low toxicity, high cell membrane permeability, fluorescent properties [26,27], and facile surface functionalization [28,29]. Their low cytotoxicity, as well as their good photostability, are key advantages over other quantum dots. Furthermore, the presence of different functional groups on the CQD surface, together with fluorescent properties, makes them excellent drug delivery tools [18,29,30]. Surprisingly, there are only a few studies about the antibacterial use of CQDs as nanocarriers for antibiotic delivery [29,[31][32][33].

The work described in this chapter concerns the synthesis and functionalization of CQDs and NDs with short, cationic and synthetic antimicrobial peptides (synAMP), to investigate their antibacterial activity and to compare them to that of the free peptide. Peptides were modified in two distinct ways, which allowed to incorporate them by two different methods:

a) an amide bond formation via a condensation reaction; b) a copper(I)-catalysed azide-alkyne cycloaddition (CuAAC).

Antibacterial peptides are mostly cationic and target bacterial cell membrane causing the disintegration of the lipid bilayer structure [34,35]. Most of them are also amphipathic, i.e.

they contain both a hydrophobic and a hydrophilic part [36]. Such a peculiar structure helps them to bind to lipid components and phospholipid groups. Some other peptides can kill bacteria without interacting with the membrane. Instead, they do inhibit some of the important pathways inside the cells [37]. It was also reported that some antimicrobial peptides (AMPs)

were able to kill resistant strains of bacteria [38].

There are several limitations to the clinical application of naturally occurring AMP including, e.g. their toxicity, their sensitivity to proteases, as well as their isolation and purification, associated with high costs. This underlines the search for smaller synthetic AMPs that have no natural counterparts and low minimal inhibitory concentration (MIC). One of the advantages of using synAMP over naturally occurring peptides is the possibility to introduce chemical modifications that are not accessible through natural biosynthetic pathways, increased yield during the synthesis and reduced production cost.

In this work, we chose to synthesise a hexapeptide based on arginine (Arg, R) and tryptophan (Trp, W) with the sequence H-RWRWRW-NH2 (Figure 2.3), where arginine brings the charged moiety and tryptophan serves as a lipophilic bulk [39]. We also synthesised some derivatives by altering the described sequence. It is worth to notice that amongst synAMPs, the one chosen for these studies is one of the smallest candidates that prove to show antibacterial activity [40,41]. The mechanism of action of this peptide has already been investigated by Wenzel et al. [42]. Shortly, the hexapeptide incorporates into the bacterial cytoplasmic membrane, which contains negatively charged phospholipids. When attached to the cell membrane, it causes the detachment of cytochrome C (a protein involved in the respiratory chain) and MurG (an enzyme involved in the synthesis of the cell wall). The indifference of cytochrome C affects the synthesis of adenosine triphosphate (ATP) and consequently, the cellular energy, whereas the indifference of MurG affects the cell wall integrity.

The choice of this peptide was driven by its already recognised activity against some methicillin-resistant S. aureus (MRSA) and vancomycin-intermediate-resistant S. aureus (VISA) [43]. As it is also known to cause bacteria death through the disruption of a broad range of cellular processes, such a peptide can be envisioned as a key structure to address the issue of growing antimicrobial resistance.

Together with the multivalent character of nanoparticles which potentially can significantly increase the affinity to appropriate target compared to the monovalent reference, the conjugates might be a promising alternative to nowadays antibiotics by improving their antibacterial activity and stability against protease degradation. The antibacterial activity of peptides and carbon-peptides conjugates was investigated against two strains of pathogenic bacteria: E.coli and S.aureus.

Synthesis of short synthetic antimicrobial peptides (synAMPs) and their antibacterial activity

All the peptides were prepared by standard Fmoc/tBu solid-phase peptide synthesis (SPPS). To increase the overall positive charge on the peptides and their organometallic conjugates, they were synthesised as peptide amides. All the structures were purified by reverse-phase high-pressure liquid chromatography (RP-HPLC), analytical HPLC established their purity and the composition of the final product was confirmed by electron ionisation mass spectrometry (ESI-MS) (Table 2.1). Additionally, their antibacterial activity was assessed by determining their MIC values by the microdilution method (Table 2.

1).

We decided to use the H-RWRWRW-NH2 as a model structure, that amongst short cationic peptides showed the highest antibacterial activity [40,41]. Another factor that determined our choice was the ease of preparation and sequence modification without losing the antibacterial activity. In general, a set of 14 differently modified peptides was prepared.

The modifications were made in order to improve the antibacterial activity of the peptides (introduction of organometallic moiety on the N terminal end (2a-3b; 6a-7b) [44][45][46] and introduce the necessary linkage for the CuAAC reactions between the nanoparticles (introduction of propargyl group on the N terminal end (4a-4b), the addition of extra amino acid with propargyl group on the C terminal end (5a-7b). Small cationic synAMPs owe their antibacterial activity mainly to charge and lipophilic bulk. These two factors were taken into consideration while designing the sequences to avoid eukaryotic toxicity. All peptides were prepared in high purity and satisfying yield. The activity against pathogenic microbes was as expected, higher for Gram-positive bacteria than Gram-negative.

In the case of peptides (1a-1b; 4a-5b) which have a balanced content of charged and bulky lipophilic groups, there is no difference in the activity irrespective of the order of the amino acids. Peptides with the structure McC(O)-WRWRW-NH2 showed higher activity than McC(O)-RWRWR-NH2 where the one with ruthenocene showed an increase in antibacterial activity over the one that contained ferrocene.

Synthesis and surface modifications of nanoparticles prior to the formation of bioconjugates

Two different kinds of carbon particles were used as a scaffold to incorporate previously prepared peptides (1a-7b). Commercially available nanodiamonds (NDs) and carbon dots (CQDs). Both particles have hydroxyl, carboxy or amino groups on their surface, which enable to create amid bond between the peptides and create azido groups, necessary for the "click" reaction.

In this work, we synthesised amine rich carbon dots (CQDs-1), which surface was modified in two ways (Figure 2 CQDs-1 were prepared by a hydrothermal method previously reported by Zhu et al. [47] with small modifications. The reaction was conducted by carbonisation of the organic precursor with a sealed Teflon lined autoclave chamber at elevated temperature under reduced pressure for 5 h. The obtained product was left to cool down to be further purified by dialysis for 24 h, resulting in a dark brown solution with the yield of the reaction-around 60%.

The morphology and structure of CQDs-1 were examined.

Wavelength / nm

CQDs-1

The UV/Vis of CQDs-1 (Figure 2.6C) reveals an absorption maximum at ≈242 nm attributed to π-π* transition of C=C and a band at 344 nm attributed to n-π* transition of C=O and C=N bonds. [48,49] Furthermore, the QY of as-prepared carbon dots was as high as 61%

(0,33) (using quinine sulphate as a reference; QY, 0.54 in 0.12 M H 2 SO 4 ). Additionally, upon increasing the excitation wavelength, the emission gradually shifts to the red region with an increase in fluorescence intensity (Figure 2.6D). The phenomenon of excitation-dependent emission is typical for these nanostructures.

To The FTIR spectrum of as-prepared dots exhibits a distinct band at 3432 cm-1 attributed to stretching vibration of -OH. Another absorption band corresponding to stretching vibrations are the one of NH (3262cm -1 ), C=CH (3018cm -1 ), CH (2923cm -1 ), C=O (1771cm -1 and 1645cm -1 ), C-C (1297cm -1 ). The existence of the amine functional groups is confirmed by the presence of a band at around 1567cm -1 and 1396cm Modifications that have been made in order to enable the incorporation of peptide amides on the surface of as-prepared carbon dots were verified by FTIR and XPS. First one includes the conversion of amine groups presented on the surface into carboxyl capped carbon dots [50,51]. Briefly, the amino groups reacted with ClCH2COONa under strongly alkaline conditions. The suspension was further neutralised, resulting in CQDs-COOH (CQDs-2). As observed in the FTIR spectra, the sharp peak at around 1650 cm-1 was enhanced, which indicates that plenty of carboxyl groups were introduced after the treatment of CQDs-1. The increase in the oxygen content for CQDs-2 compared to CQDs-1 is 26.87% and 14.94% respectively, strongly indicating the successful introduction of additional carboxyl groups.

Additionally, we can observe an increase in negative zeta potential and hydrodynamic diameter (Table 2.2). Moreover, the quantum yield decreased significantly which can be attributed to the increase of electron withdrawing carboxyl groups (QY= 7% (0.038) using quinine sulphate as a reference; QY, 0.54 in 0.12 M H2SO4).

In case of CQDs-3, the successful formation of azide groups on the surface of nanoparticles was confirmed by the appearance of the band at 2110cm (Figure 2.9B). In addition, we can observe the increase in the atomic percentage of the N1s component as well as the increase in the potential on the surface from -9.9 for CQDs-1 to +7

for CQDs-3 (Table 2.2). Furthermore, we decided to quantify the free amino group on the surface of CQDs before and after the modification with the technique of so-called Keiser test.

The Keiser test makes the use of the formation of Ruhemann's Blue by the reaction of ninhydrin with the primary amino groups. The successful implementation of this colorimetric assay on the nanoparticles has been already reported [52]. We can see than the modification made on

CQDs-1 caused a decrease in the amount of free amino groups in both CQDs-2 and CQDs-3.

It may indicate the success of converting the surface from amino into carboxyl groups and successful coupling of azido moieties. It is also giving us more information about possible loading efficiency. The formation of the click conjugates for both carbon structure was performed in the same way using copper sulphate pentahydrate and ascorbic acid as catalysts for the reaction. In both cases, the process was accomplished in two steps. The first step consists of the introduction of the azide molecule on the surface of the nanoparticles, and the second step was achieved by using a previously prepared "clickable" peptides. The successful modification can be easily followed by the disappearance of the band at 2100 cm -1 for CQDs (Figure 2.12A) and 2123cm -1 for ND (Figure 2.12B). Additionally, for CQDs containing metallocene moieties, the characteristic band at 3071cm -1 was observed. The band at 1200 cm -1 and 1130 cm -1 are attributed to stretching rocking mode of -NH. For ND, the band assigned to stretching vibrations of -NH appears at 1540cm -1 .

(A) (B)

Figure 2.12: (A) FTIR spectra of CQDs-peptides (CQDs-3.4a, CQDs-3.5a, CQDs-3.6a, CQDs-3.7a); (B) FTIR spectra of ND-peptides (ND-3.4a, ND-3.5a, ND-3.6a, ND-3.7a) bioconjugates.

Following the changes in the zeta potential of the particles (Table 2.4), we can assume that the cationic peptide is incorporated on the surface, and its charge is not altered by additional interactions. For CQDs, the potential changes form 7±3 up to 26±1 and for all the particles stay in the same range. ND showed a higher charge on the surface, which is influenced by higher loading of the cationic peptide on the surface. As expected, the hydrodynamic diameter increases for all prepared nanoconjugates indicating the successful incorporation of peptides.

In the case of CQDs click particles, another factor indicating the success of the reaction is the decrease in the quantum yield. The changes are quite significant, decreasing from 33% for CQDs-3 to get as low as 9.8% after the modification. It is worth to notice that the peptides "b" are the reverse sequence of peptides "a" showing the same FTIR spectra.

Antibacterial activity of carbon-based conjugates

The antibacterial activity of all the compounds was assessed by determining their MIC values by microdilution assay. MIC value is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. In this work, one Gram-negative (E.coli DSM 30038) and one Gram-positive (S.aureus ATCC 43300), the bacterial strain was used.

The first group of compounds where the peptides were covalently linked showed no antibacterial activity against studied bacterial strains (conjugates: 1a-3b) (Table 2.5). Peptides used in this study owe their antibacterial properties to charge and lipophilic bulk. The unprotected guanidine groups on the arginine side chain could react with the nanoparticles during the coupling, resulting in the loss of the positive charge and increase lipophilicity. This indicates the importance of preserving these groups during the reaction.

Table 2.5: Antibacterial properties of carbon nanomaterials (CQDs-1; CQDs-2; ND-1) and covalently modified carbon-peptides conjugates (CQDs-2.1a-CQDs-2.3b; ND-1.1a-ND-1.3b).

n.a.: not active

To overcome this problem and protect the positive charge of the peptides, click chemistry was developed to link AMPs to the nanostructures. Increased antibacterial activity of the conjugates (Table 2.6) revealed the multivalent character of carbon nanostructures.

For the conjugates prepared by the "click" reaction, the activity is in general higher for Gram-positive bacteria. However, for some compounds, the activity for Gram-negative E.coli has been significantly increased. For carbon dots conjugates (CQDs-3.4a; CQDs-3.4b; CQDs-3.5a; CQDs-3.5b) we can observe 5 fold increase of activity and 4-fold increase in case of nanodiamonds (ND-3.4a; ND-3.4b; ND-3.5a; NDs-3.5b) with the MIC values ranging between 6.02µg mL -1 -6.59µg mL -1 (CQDs) and 7.65µg mL -1 -8.22µg mL -1 (NDs) respectively. The additional amino acid and the position of the propargyl group does not seem to alter the MIC values. This contrast to the presence of the metallocene group (conjugates: 6a-7b) where the activity in case of ND was slightly decreased, and carbon dots showed no activity in the studied range.

Considering Gram-positive bacteria S.aureus (MRSA) 5-fold increase for carbon dots and a 4-fold increase in the activity is also observed (conjugates: 6a-7b). In the case of peptides containing ferrocene or ruthenocene, the activity increases 3-fold for CQDs being approx.

12µg mL -1 with an exception for compound CQDs-3.7a where the MIC value (6.34µg mL -1 ) is close to the value of the free peptide (8µg mL -1 ). Nanodiamond conjugates showed a 2-fold increase in case of ND-3.6b and ND-3.7b, no significant difference for ND-3.6a and decreased

CQDs-2.2a

FcCO-WRWRW-NH2 n.a. n.a.

CQDs-2.2b

FcCO-RWRWR-NH2 n.a. n.a.

CQDs-2.3a

RcCO-WRWRW-NH2 n.a. n.a.

CQDs-2.3b

RcCO-RWRWR-NH2 n.a. n.a. ND-1 -n.a. n.a.

ND-1.1a

H-RWRWRW-NH2 n.a. n.a.

ND-1.1b

H-WRWRWR-NH2 n.a. n.a.

ND-1.2a

FcCO-WRWRW-NH2 n.a. n.a.

ND-1.2b

FcCO-RWRWR-NH 2 n.a. n.a.

ND-1.3a

RcCO-WRWRW-NH2 n.a. n.a.

ND-1.3b

RcCO-RWRWR-NH2 n.a. n.a.

activity for the compound ND-3.7a was observed. Lower activity compared to CQDs can be attributed to the higher loading of ND, resulting in a higher amount of ferrocene/ ruthenocene presented on the surface. Therefore, increasing the lipophilicity of the compounds.

Additionally, CuSO 4 that was used as a catalyst during the click reaction (compounds 4a-7b) was tested, showing no inhibition of growth in case of both bacterial strains.

Conclusions

A set of 28 different carbon-peptide nanostructure conjugates were successfully prepared, and their antibacterial activity was assessed by determining the MIC values against Gram-negative E.coli DSM 30083 and S.aureus ATCC 43300. The first group of compounds obtained by covalent linking showed no antibacterial activity against studied strains (conjugates: 1a-3b) while the second group where click chemistry was applied (conjugates: 4a-7b) showed a significant decrease in the minimal inhibitory concentrations for Gram-negative and Gram-positive bacteria. Amongst them, conjugates 4a-5b showed the best MIC values for the tested strains being 5-fold lower for peptide modified CQDs and 4-fold higher for peptide modified ND when compared to the peptide in solution.

The lack of activity in some particle-conjugates in the tested range is associated with the molecules loaded on the surface rather than particles themselves. The data collected in the (Table 2.3) and (Table 2.5) indicate that charge is a significant factor in the antibacterial activity. Even though the zeta potentials were decreased towards less negative charges indicating successful incorporation of peptides, it did not increase as much as expected, like for the "clicked" compounds suggesting that some if not all of the positive charge in the backbone of the loaded peptides was blocked.

In addition, it was shown that the position of the propargyl group, as well as the reverse order of amino acids in the sequence, does not influence the activity of tested compounds. This contrast the results obtained for the conjugates where the organometallic moiety was present.

The presence of ferrocene or ruthenocene results in loss of activity for the Gram-negative bacteria and only 2-3 fold higher for Gram-positive S.aureus with an exception for the conjugate 7a where the MIC value did not improve. This might be explained by the increased lipophilicity on the surface of nanoparticles due to the presence of ferrocene or ruthenocene.

Which indicated that the existence of organometallic moiety in case of as prepared conjugates is not favourable and better MIC values are obtained for the "simple" peptides.

Introduction

Advances in controlled drug release systems and devices have addressed many of the challenges encountered in traditional drug delivery [1][2][3][4][5][6][7]. Controlled drug release can be triggered either by internal or external stimuli [8][9][10][11]. These systems may provide a long-term solution to the main drawbacks of drug resistance development which is a widespread problem encountered in many treatments, especially in bacterial infections therapy due to increasing antibacterial resistance and severity of side effects [12,13].

Stimuli-responsive systems are considered to be effective for "on-demand" drug delivery under the influence of a particular stimulus, which can be endogenous or exogenous in nature [14]. Endogenous systems are systems that result in drug release upon changes in pH, temperature change, redox state etc. [8][9][10][11]. The limited control over drug release is the main obstacle in designing and in the efficient use of such systems. Exogenous stimuli like light, magnetic field, electric field and ultrasound have been successfully utilised helping to overcome these limitations [15]. Among them, light is considered the most attractive stimulus due to ease and spatiotemporal control [16][17][18][19]. Near-infrared (NIR) light has good tissue penetration due to limited attenuation. It is also considered safe for cells and tissues [17,20]. Therefore systems that are NIR light-responsive can be utilised as a potential drug carrier for drug delivery in clinical application [4]. In such systems, the release of a drug is achieved upon exposure to NIR light where photothermally active material convert absorbed light radiation into heat.

Depending on the material, the local application of heat will result in either physical or chemical change in the system, subsequently resulting in enhanced drug release [21]. Several thermoresponsive nanomaterials have been successfully applied in on-demand release systems such as gold nanorods, metallic nanoparticles, organic chromophores and carbon nanomaterials notable graphene-based materials [1,17,22]. Graphene oxide (GO) and its derivatives, notably reduced graphene oxide (rGO) have been used to fabricate drug delivery platforms either as discrete nanocarriers or have been incorporated into composite nanomaterial [23].

Modifications with nontoxic polymers enhance its biocompatibility and dispersibility.

Embedding GO into hydrogels would have in addition several advantages such as high loading capacity, protection of an active molecule together with on-demand release possibilities [21,24,25]. While hydrogels have been extensively explored, in recent years, the focus has shifted towards utilisation of the macroporous member of hydrogel family-cryogel [26]. The advantage of using cryogel over traditional hydrogel is its exceptional combination of highly porous characteristic with sufficient osmotic stability, better drug release due to higher porosity and swellability which allows for better transport of medium into their interiors [27].

We envisioned that graphene oxide (rGO) loaded cryogel patch, where the release of antibacterial agent can be triggered photothermally, might be an effective on-demand delivery platform with antibacterial properties for the ablation of skin related pathogens. The increased temperature during NIR irradiation is used in this approach to promote the release of the loaded drug either through enhanced diffusion out of the gel or by the disruption of the structure (Figure 3.1). For that purpose, we utilise a photothermal active cryogels consisting of rGO embedded in a gel composed of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic furfuryl groups. The furan groups served a dual-mode providing hydrophobic pockets and covalent linkage sites for maleimide-modified antimicrobial peptide. We took advantage of thermally controlled equilibrium of the Diels-Alder reaction, which at elevated temperature induce the reverse reaction and the reformation of maleimide and furan moieties whereas lower temperature favours the adduct formation.

Fabrication and photothermal properties of furan-modified cryogels with and without embedded rGO

A series of maleimide reactive cryogels with and without embedded rGO (Figure 3.2B)

were synthesised using varying amounts of poly(ethylene glycol) methyl ethermethacrylate (PEGMEMA) and furfuryl methacrylate (FuMA) monomers and an aqueous dispersion of rGO.

The fabrication of cryogels was carried out at -13°C under UV irradiation (365nm). 2,2dimethoxy-2-phenylacetophenone (DMPA) was used as a photoinitiator and poly(ethylene glycol) dimethacrylate (PEGDMA) as a crosslinker (Figure 3.2B). As can be seen from Table 3.1), increasing the FuMA/PEDMEM ratio resulted in a decrease of pore size diameter; The swelling capacity of the cryogels was investigated by recording the water uptake as a function of time (Figure 3.3). In general, all cryogels showed a decrease in swelling capacity with increased hydrophobic content (FuMA). Addition of rGO in the structure of as-prepared cryogels decreases the overall water uptake.

Loading of cryogels with antimicrobial agents

Loading of antimicrobials was accomplished in two ways : a) by simple absorption of the drug inside the gels via gel immersion into an aqueous solution of the antimicrobial agents; b) by Diels-Alder reaction between the furan containing cryogel and a maleimide containing antimicrobial (Figure 3.5A)

Loading of the antibacterial drug via absorption and subsequent release

The loading capacity of cryogel CG-rGO-40 for ampicillin was investigated first. The amount of antibiotic-loaded onto the gel was calculated from the solution before and after the loading using a calibration curve on HPLC. From Figure 3.5B, we can see that the concentration of antibiotic used in the initial solution is proportional to the amount loaded inside the cryogel. When 800 µg mL -1 of the antibiotic was used for loading, 114 µg mL -1 (14%) of ampicillin was found to be loaded into the CG-rGO-40 gel.

To trigger the release of ampicillin (Figure 3.5C), a laser at a power density of 1 W cm-2 was used and resulted in a total release of 56 µg mL -1 . In comparison, the passive release of extensively washed cryogels was low. The minimal inhibitory concentration for Gram-negative bacteria E.coli K12 was determined to be 10 µg ml -1 . The amount of released antimicrobial was higher than the minimum concentration inhibiting the growth of the bacteria (Figure 3.5C). In addition, Figure 3.5D shows that already after 5minutes of irradiation, the amount of released antibiotic is sufficient for complete bacterial ablation, while passively released amount is below the MIC value for the tested bacteria, which shows the importance of combining laser irradiation with rGO embedded cryogels in "on-demand" antibiotic release. ) and passively at 24 o C; (D) Comparison of the action of free ampicillin (2 µg mL -1 , negative control), ampicillin (10 µg mL -1 ; positive control) and released ampicillin after 10 min upon activation and passively when incubated with E. coli (110 8 CFU mL -1 ).

Loading of the antibacterial agent via Diels-Alder reaction and subsequent release

Another way of incorporating biologically active molecule into the cryogels is the possibility of furfuryl interaction with maleimide modified antimicrobial. In this section, we evaluated loading efficiency using HPLC calibration curve or fluorescent microscopy of the solution before and after loading. Fluorescent-maleimide dye was used as a model to validate the loading mechanism. Cryogel CG-rGO-40 was immersed in a solution of the dye for 24h, resulted in fluorescent cryogel with the loading efficiency of 40 ± 2% (Figure 3.6B). To confirm that the dye is covalently bonded to the furan moiety, the same amount of the dye was The amount of peptide integrated into CG-rGO-40 was calculated to be 198 µg mL -1 (33%) (Figure 3.7B) after 20h incubation (HPLC). Unfortunately, the release from the cryogel was <1% even after irradiation for 30 min at 70°C. These findings are comparable to those of using N-(5-fluoresceinyl)maleimide, where only 6% of the loaded dye was released (0.6% at 37 o C without radiation) (Figure 3.6C). One possible explanation could be the strong π-π stacking interaction between the peptide and the rGO inside the gel and/or electrostatic interaction between positively charged peptide and negatively charged reduce graphene oxide.

To validate this theory, an rGO-free gel (CG-40), was loaded with the peptide/dye, and photothermal release was achieved by using an rGO-covered Kapton patch on top of the cryogel (Figure 3.7A). Such a construct does not influence significantly the max temperature of the (194±6 µg mL -1 ) when heated to 70 °C for 240 min, while only 2.4 µg mL -1 (1.4 %) was released at 37 °Cas for the dye, using such a construct allows us to release 28% while heating and only 1,4% at 37 °C.

The activity of the released antibacterial peptide was tested on Gram-positive bacteria S. aureus ATCC43300. The MIC value for the maleimide-modified peptide was determined by microdilution method, and the obtained value was the same as for the peptide itself (16µg mL -1 ). While the amount of the released AMP at 37°C was not efficient to inhibit the growth of tested bacteria strain, the amount of the peptide-maleimide released upon heating of the cryogel up to 70°C already after 10min of irradiation was enough to reach the MIC value and ultimately decrease bacteria cell viability (Figure 3

.7E).

Before applying this approach for real application (e.g. would healing platform), the cell toxicity of the cryogel was determined using a HeLa cell line and the CCK-8 viability assay.

As shown in the (Figure 3.8), none of the prepared cryogels shows any cytotoxicity.

As expected for a PEG-based matrix [30].

Conclusions

Cryogels were fabricated using photo-polymerisation leading to embedded matrix comprised of hydrophilic poly(ethylene glycol) and hydrophobic furfuryl groups. Furfuryl groups were incorporated as control over the hydrophobicity as well as a handle for Diels-Alder conjugation. Ampicillin was loaded inside the matrix via adsorption, while a maleimidecontaining anti-microbial peptide was conjugated via the Diels-Alder reaction. While the system performed as expected for the release of conventional antibiotic like ampicillin, the study reveals that integration of rGO inside the cryogels hindered the release of AMPs due to the strong - interactions between the anti-microbial peptide and the rGO. To overcome this problem, a bilayer device structure was employed. The furan containing the rGO-depleted hydrogel was interfaced with NIR responsive Kapton patch modified with rGO. In case of cryogel loaded via absorption of commercially available ampicillin-total eradication of E.coli bacteria is achieved after 10 min. of NIR irradiation. Cryogels loaded via Diels-Alder reaction with AMP inhibited the growth of bacteria by 75% after 10 min. Increasing the irradiation time resulted in the higher release of a peptide, and after 30min, no bacteria could be detected. In order to apply this approach in the clinical application, the cytotoxicity of the cryogels was tested. Obtained results revealed no toxicity against HeLa cells. In addition to cytotoxicity, the antibacterial activity of as-prepared cryogels was tested showing no activity before antimicrobial loading.

3.5.

CHAPTER 4

Functional carbon quantum dots as a medical countermeasure to human coronavirus (HCoV)

Much of the content below served as a basis for a publication [1]. The author conceived and performed all syntheses and performed or coordinated all experiments. Antiviral assays were performed in collaboration with Dr.Karin Seron in the group of Dr. Jean Dubuisson (Institut Pasteur de Lille, U1019-UMR8204-CIIL-Center for Infection and Immunity of Lille).

Introduction

The eradication of viral infections is an ongoing challenge in the medical field, not only due to the problem of spreading but also owning to virus ability to escape therapy by genetic mutations. The lack of targeted antiviral therapeutics, as well as the constant emergence of new viruses, make the search for antiviral agents a challenging and extremely needed research task [2]. As part of a global strategy to prevent epidemics, some severe emerging pathogens with great epidemic potential have been identified by the World Health Organization (WHO) [3],

including, next to Ebola virus disease, the highly pathogenic human coronavirus (HCoV) infections. While circulating HCoVs (HCoV-229E, HCoV-OC43, HCoV-NL63 and HKU1) cause relatively mild common cold-like respiratory tract infections, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle-East respiratory syndrome coronavirus (MERS-CoV) lead to pneumonia, which requires hospitalisation and intensive care [4]. A total of 2266 laboratory-confirmed cases of MERS-CoV, including 804 associated deaths, have been declared to WHO until now, with a high case-fatality rate (35%) [5]. As the virus is circulating in animals and humans, it may undergo further adaptation and cause a pandemic. Therefore, therapeutic options are urgently needed.

The current treatments for MERS-CoV are extrapolated from SARS-CoV and H1N1

influenza outbreaks [6][7][8]. These include different combinations of small molecules with broad antiviral activity (e.g. ribavirin, corticosteroids, interferons (IFN)), and monoclonal or polyclonal antibody therapies [8,9]. The membrane-anchored glycoprotein S was found lately to be essential for the interaction between MERS-CoV and the host cell [9,10], and the development of MERS-CoV entry/fusion inhibitors targeting the S1 subunit is nowadays considered as a viable antiviral strategy.

CHAPTER 4 Functional carbon quantum dots as a medial countermeasure to human coronavirus (HCoV)

Recently, nanoscale materials have emerged as promising and efficient platforms to modulate the viral infection cycle [11]. Given that the attachment of viruses to host cells is favoured by multivalent interactions, the multivalent character of nanostructures and their high surface-to-volume ratio, both allowing the attachment of several ligands, make them well adapted to interfere with the viral attachment and to block the viral entry into cells.

We investigate here the potential of functional carbon quantum dots (CQDs) as inhibitors of host cell infections by HCoV-229E coronavirus as the model virus (Figure 4.1).

CQDs with an average diameter below 10 nm and excellent water dispersibility are highly attractive for nanomedical applications due to a lack of visible signs of toxicity in animals [12].

They can be synthesised quickly via several different, inexpensive and simple methods, and their excellent optical properties offer in vivo tracking possibilities. It was recently demonstrated that CQDs are suitable scaffolds to interfere with the entry of viruses into cells [13][14][15]. As an example, boronic acid-modified CQDs were able to inhibit HIV-1 entry by suppressing syncytium formation [14]. Additionally, CQDs functionalised with boronic acid and amine moieties have been demonstrated to interfere with the entry of herpes simplex virus type 1 [13]. Han and co-workers also reported the potential of CQDs as viral inhibitors by activation of type I interferon responses [15].

The present study reveals that boronic acid functions can be responsible for the anti-HCoV activity. CQDs derived from citric acid and ethylenediamine, further conjugated by "click" chemistry with boronic acid functions (-B(OH)2), display an effective 50% inhibition concentration EC50=52±8 µg mL -1 . Likewise, CQDs derived from 4-aminophenylboronic acid and phenylboronic acid without any further modification exhibit antiviral behaviour with an effective EC50 lowered down to 5.2±0.7 µg mL -1 . The underlying mechanism of action of these CQDs proved to be their interaction with the HCoV-229E S protein. Surprisingly, an equally large inhibition activity was observed at the viral replication step.

First-generation of CQDs inhibitors of host cell infections by

HCoV-229E coronavirus: Boronic acid-modified CQDs

4.2.2.

Cytotoxicity assay

The cell toxicity of CQDs-1, CQDs-4 and CQDs-5 were established on Huh-7 cell lines after 8 h (time points corresponding to HCoV-229E infections) and 24 h incubation. The CQDs toxicity was evaluated using cell viability assessment by the resazurin assay, based on the conversion of a non-fluorescent dye to a fluorescent molecule by mitochondrial and cytoplasmic enzymes. All CQDs are non-toxic to Huh-7 cells even at the highest concentration (100 µg mL -1 ) investigated when incubated for 8 and 24 h (Figure 4.4A). Neither the presence of boronic acid nor triazole units had a negative effect on cell toxicity.

The uptake mechanism proved to be the same for all of the nanostructures. Taking the example of CQDs-4 (which later proves to have antiviral activity), Huh-7 cells were fixed after

Wavelength / nm

The endocytosis of CQDs-4 was in addition quantitatively evaluated using flow cytometry by treating Huh-7 cells with 100 µg mL -1 of CQDs-4 for 1 h at 4 °C and for 1 h, 3 h

Antiviral assay of first-generation CQDs

The antiviral activity of CQDs-1, CQDs-4 and CQDs-5 was evaluated on Huh-7 cells monolayers, infected with HCoV-229E-Luc (Figure 4.5A). Addition of CQDs-1 1 h after the infection and further incubation for 6 h at 37 °C shows no inhibition of the infection. This contrasts to CQDs-4, where a concentration-dependent virus inactivation is observed with an estimated EC50=52±8 µg mL -1 (Figure 4.5B). Addition of mannose to CQDs-4 results in a complete loss of antiviral activity of the latter at low particle concentrations, with some antiviral activity above 50 µg mL -1 CQDs. These data reveal two important findings. First, it highlights that boronic acid functions, the mode of action of which is the selective and reversible formation of tetravalent boronate complexes with cis-diolsand thus glycan units [23] are interacting with HCoV-229E. CQDs-4 are in this context speculated to be pseudolectins, targeting the S protein of HCoV-229E via a lectin-carbohydrate binding mechanism, similar to that of the oligomannose-specific lectin Griffithsin [24]. Thus, the addition of mannose blocking the antiviral activity is in favour of this mechanistic assumption. The remaining little antiviral activity of mannose-saturated CQDs-4 might be due to the presence of the triazole function at the particles' surface. Indeed, control particles CQDS-5, bearing no boronic acid function but a triazole ring, display some antiviral activity, even though largely decreased when compared to CQDs-4.

(A) (B)

Second-generation of CQDs inhibitors of host cell infections by

HCoV-229E coronavirus

Formation and characterisation of CQDs-6, -7, -8

To validate if boronic acid functions can be formed directly on CQDs, hydrothermal carbonisation of phenylboronic acid and 4-aminophenylboronic acid were performed, resulting in CQDs-6 and CQDs-7, respectively (Figure 4.6A). As a control, hydrothermal carbonisation of aniline and poly(ethylene glycol) PEG600, both lacking boronic acid functions, was conducted. Unfortunately, several attempts to prepare CQDs from aniline as a starting material failed. The boron content is lower than that reported by Shen and Xi [26] but comparable to that reported by Wang et al. [27]. This indicates that some of the phenylboronic acid groups were carbonised under our experimental conditions. The low B content might also indicate doping rather than the presence of boronic acid functions. CQDs-7

particles depict binding energy bands at 284.

: zeta potential; 2 The hydrodynamic size was recorded at 37 °C; 3 PDI: Polydispersity index; 4 XPS was used to determine the atomic percentage of the elements respectively.

The FTIR spectra (Figure 4.9A) of CQDs-6, -7, -8 exhibit a distinct band at 3465 cm -1 attributed to the stretching vibration of -OH groups and bands around 2874 and 2924 cm -1 , due to CH2 stretching. The sharp band at 1618 cm -1 is assigned to graphitic C=C, and the C-H deformation mode is observed at 1460 cm -1 . The C=O band at ~1780-1650cm -1 is also visible in all cases. In the case of CQDs-7, the band at 1090 cm -1 might be due to C-B stretching modes.

This band is less defined in the case of CQDs-6, which might underline doping rather than the presence of boronic acid functions. The FTIR spectrum of CQDs-8 displays the C-O-C bands of the PEG units at 1043 cm -1 .

CQDs

Wavelength / nm

CQDs-8

CQDs-7

CQDs-6

h incubation. This might be due to the presence of NH2 groups on these particles. The uptake mechanism of these particles was comparable and is exemplified using CQDs-7 in Performing 11 B NMR analysis of CQDs-6 and CQDs-7 (Figure 4.12) and comparing the resulting spectra to those of the respective starting materials, 4-aminophenylboronic acid(4APBA) and phenylboronic acid (PBA) (Figure 4.12), reveals large differences in the chemical composition. 4-aminophenylboronic acid and phenylboronic acid both exhibit a strong signal at around 29 ppm, in accordance with literature data for -B(OH)2 functions [28,29]. The small-signal at about 20 ppm arises most likely from residual B(OR)3 often used in boronic acid synthesis [30]. The

Mechanism of action

We further investigated the mechanism of action of CQDs-4 and CQDs-7 on viral infection by performing a time-of-addition assay. CQDs (at 10 µg mL -1 ) were added at different time points during infection, as represented in Figure 4.13A. As expected, a strong inhibition of the infection was observed when CQDs were added 1 h after inoculation. Moreover, the inhibition activity of CQDs was stronger when the nanoparticles were added during the entry step, i.e. 30 min before and after inoculation and during inoculation. These results are in agreement with our hypothesis of the interaction of CQDs with HCoV-229E S protein, or the interaction of CQDs with entry factors. Surprisingly, a strong inhibitory activity of CQDs was also observed when they were added 5.5 h after the entry step, that is at the replication step.

The inhibition is not significantly different for the entry step compared to the replication step.

It suggests that, in addition to its major effect on HCoV-229E entry, CQDs can also affect the genomic replication of this virus. It could potentially be explained by an interaction between the CQDs and a cell surface protein leading to signal transduction affecting viral replication, or by interaction with cytosolic proteins as CQDs are internalised.

To determine if CQDs are interacting directly with viral particles, HCoV-229E-Luc was incubated with CQDs at 10 µg mL -1 for 30 min at 37 °C before inoculation. The inoculum was diluted ten times, leading to a final concentration of CQDs of 1 µg mL -1 , and an infection assay was performed. In parallel, Huh-7 cells were inoculated with HCoV-229E-Luc in the presence of CQDs at 1 or 10 µg mL -1 . The inoculum titers were kept constant in different conditions.

The results showed that the pre-incubation of the virus with CQDs at high concentration does not impair HCoV-229E infection, meaning that CQDs are not interacting with the virus before

Conclusions

The viral infection cycle produces important biological and structural changes in the host cell, resulting in cell damage. The possibility to interfere with viral attachment to cells as well as with viral replication to reduce viral infection and spreading is an appropriate antiviral approach. We presented here the antiviral performance of seven different CQDs with their main features summarised in Table 4.3. Three of these CQDs (CQDs-4, -6, -7) proved to interfere significantly with HCoV-229E-Luc infection in a concentration-dependent manner. While

CQDs-6 showed the most modest antiviral activity, the estimated EC50 value decreased considerably from CQDs-4 (boronic acid-modified quantum dots, derived from ethylenediamine/citric acid as carbon precursors; EC50 = 52±8 µg mL -1 ) to CQDs-7 (5.2±0.7 µg mL -1 ). The presence of boronic acid functions proved to be essential for CQDs-4 to exhibit antiviral activity, whereas CQDs-6 and CQDs-7 did not carry any substantial amount of boronic acid functions, as revealed by 11 B NMR and confirmed by mannose addition experiments. These findings reveal the complex nature of the identification of viral inhibitors for human coronaviruses such as HCoV-229E-Luc. Mechanistic studies suggest that the particles are acting at the early stage of the virus infection, especially through the inhibition of the virus entry which could itself be due to the inhibition of protein S-receptor interaction with the host cell membrane. In addition, all of the three different particles interfere with the viral replication step, something less common. These results are extremely encouraging to replace currently used antiviral agents such as ribavirin, and IFN is known to have major side effects such as confusion, short-term memory loss, deficits in executive functions, as well as extrapyramidal effects. Further experimental confirmations are required to know if this approach can be extrapolated to other coronaviruses (notably to the clinically relevant MERS-CoV) and to validate the potential of these nanostructures as alternative anti-MERS therapeutics or anti-MERS approaches able to confront this severe and life-threatening disease.

Additionally, the precise in vivo behaviour of such particles needs to be understood in the future, so that the different possible inhibition mechanisms can be elucidated.

CHAPTER 5

Conclusions and perspectives

Unprecedented antibacterial resistances, as well as new and more aggressive viruses, are continuously emerging, with a global impact on public health and society at large. This has made the search for antibacterial and antiviral drugs or therapeutics a very challenging research task. In the case of viral infections, the general absence of availability of specific antiviral therapeutics for a variety of viruses represents a considerable threat to the body's immune system as they hide inside cells, making it difficult for antibodies to reach them. In contrast to bacterial infections, which are mostly treated using antibiotics, immunisation against viral infections is not always possible. While vaccines against serious viral infections such as measles, mumps, hepatitis A, and hepatitis B do exist, some viruses are capable of mutating from one person to the next, making vaccination a difficult task as the viruses have already changed their format by the time the corresponding vaccines become available. We considered both antiviral and antibacterial aspects of this thesis. Special attention was given to carbon nanomaterials, considered as promising candidates for various biological applications because of their biocompatible nature and rich surface chemistry. Among the possible candidates, diamond particles (NDs), were considered as they represent an important class of highly stable nanomaterials limiting their decomposition or transformation to materials with potential toxicity and decreased activity. Furthermore, carbon quantum dots (CQDs), formed by green synthetic methods without the need of a metal catalyst were investigated as they show good biocompatibility, small hydrodynamic size (5-20 nm in diameter) and excellent hydrophilic character. These nanostructures were modified with short cationic synthetic antimicrobial peptides (synAMP) that are rich in arginine (Arg) and tryptophan (Trp). The peptides were coupled either through a condensation reaction or via a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) using an azido group originating from a previous chemical modification at the nanoparticle surface. Among the 28 investigated structures, conjugates 4a-5b showed the best MIC values for the tested bacteria strains (Gramnegative E.coli DSM 30083 and S.aureus ATCC 43300) with a 5-fold lower MIC value for peptide modified CQDs and 4-fold lower for peptide modified NDs, when compared to the free peptides in solution, reaching MIC values of ca. 3 µg mL -1 and 4 µg mL -1 respectively.

The collected data indicated that charge is a significant factor in the antibacterial activity as well as the presence of metallocenoyl group.

Additionally, rGO-embedded furan-modified cryogels conjugated with maleimidemodified ampicillin and synAMP were formed, and the possibility of on-demand release of the antibacterial agent to ablate bacteria was investigated. The cryogels were fabricated using photo-polymerisation reaction using hydrophilic poly(ethylene glycol) and hydrophobic furfuryl elements, where maleimide-containing anti-microbial peptide could be linked via the Diels-Alder reaction. While the system performed as expected for the release of ampicillin, the study reveals that integration of rGO inside the cryogels hindered the release of AMPs due to the strong - interactions between the anti-microbial peptide and the rGO.

We also considered CQDs as antiviral agents. We have developed a first and second generation of functional antiviral CQDs prepared by one-pot synthesis or by subsequent surface post-modification. The structure showed a concentration-dependent virus inactivation with an estimated EC50 of 52±8 µg mL -1 for the first generation, and 5.2±0.7 µg mL -1 for the second.

Our mechanistic studies revealed that the particles interact with the virus already at the early stage of the viral infection, inhibiting the virus entry. This behaviour could be due to the inhibition of the protein S-receptor interaction with the host cell membrane. In addition, the existence of another interaction occurring during the replication step was discovered. Asprepared CQDs could thus be a potential substitute for currently used antiviral agents. However, further experimental confirmations are required to validate their potential as alternative therapeutics as well as the possibility of this approach can to be extrapolated to other coronaviruses, notably the clinically relevant MERS-CoV.

Overall, the scope of application of the different carbon-based nanostructures is certainly broad. The third generation of functional antiviral CQDs is under development, using MERS-CoV specific peptides as surface anchors. This will allow specific targeting of the inhibition of MERS-CoV cell interactions. The concept will be also expanded to other nanostructures (NDs, MoS2 nanosheets, etc.) to see if the concept is specific to CQDs or not.

Additionally, this will also allow answering questions concerning the importance of the size of these nanostructures in the inhibition process.

In the case of antibacterial nanostructures, further work will be devoted to understanding the mechanism of conjugates 4a-5b to ablate Gram-negative E.coli DSM 30083 and Gram-positive S.aureus ATCC 43300. In addition, no in vivo studies have been performed in this work, essential for establishing pharmacokinetic parameters of these nanostructures and elucidating their real potential for antibacterial applications. This is also true for the proposed antibacterial cryogels where testing on ex vivo infected skin will be one of the next steps. The healing of diabetic foot ulcers is targeted with these gels where the effect of heat and local antibacterial release might be of great advantage for treating such chronic wounds. The future of this interdisciplinary research that brings organic chemistry, nanotechnology and biology together is thus brighter than ever.

6.2.1.1.

Synthesis of H-RWRWRW-NH2: 1a

The peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure. HPLC (tR, min): 6.28.

MS (ESI

Synthesis of H-WRWRWR-NH2: 1b

The peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure.

MS (ESI

Synthesis of FcCO-WRWRW-NH2: 2a

The peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure.

MS (ESI

Synthesis of FcCO-RWRWR-NH2: 2b

The peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure.

MS (ESI

Synthesis of RcCO-WRWRW-NH2: 3a

The peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure.

MS (ESI

Synthesis of CHCCH2CH2C(O)-WRWRWR-NH2: 4b

The peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure.

MS (ESI

Synthesis of H-RWRWRWG(CH2CCH)-NH2: 5a

The peptide was synthesised by solid-phase peptide synthesis as described in general procedure

Synthesis of H-WRWRWRG(CH2CCH)-NH2: 5b

The peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure. HPLC (tR, min): 6.5. Intens.

MS (ESI

Synthesis of RcCO-WRWRWG(CH2CCH)-NH2: 7a

The peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure. Intens.

MS(ESI

Synthesis of RcCO-RWRWRG(CH2CCH)-NH2: 7b

The peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure. Intens. Intens. For the synthesis of (2), compound (1) (0.91 mmol) was dissolved in 1,2dimethoxyethane (8 mL). KOtBu (10 eq) and water were added, and the reaction mixture was stirred under reflux until the next day. The solution was diluted with 10 mL of water and left to cool down to room temperature. Et2O was added, and ruthenocenecarboxylate was extracted using 1 N NaOH. Aqueous layers were combined, acidified with 1 N HCl, and the desired carboxylic acid was extracted using Et2O. The organic phase was dried over Na2SO4 and concentrated under reduced pressure, resulting in the final product as a yellow solid (230 mg; 0.82 mmol; 91% yield). EDTA was added to the mixture prior to dialysis (SpectraPor 1, pore size: 1000 Da) against Milli-Q water for 48 h.

MS (ESI

CQDs-5

A suspension of CQDs-3 (1 mg mL -1 , 5 mL) was mixed with propargyl alcohol (2 mM), copper sulphate pentahydrate (200 µM) and ascorbic acid (300 µM). The reaction mixture was stirred for 24 h at room temperature. EDTA was added to the mixture prior to dialysis (SpectraPor 1, pore size: 1000 Da) against Milli-Q water for 48 h.

CQDs-6

CQDs-6 particles were prepared according to the protocol described recently by our group [5]. Phenylboronic acid (200 mg) was dissolved in water (20 mL), and the pH was adjusted to 9.0 with NaOH (1 M). The solution was degassed with nitrogen gas for 1 h in order to remove dissolved oxygen and heated in a Teflon-lined autoclave chamber (125 mLacid digestion vessel n°4748, Parr, France) for 8 h at 160 °C. After cooling to room temperature, the suspension was dialysed against Milli-Q water for 24 h, water being changed every 6 h (SpectraPor 1, pore size: 1000 Da). The formed functional CQDs-2 (200 µL) were dried and then weighed with a Sartorius microbalance (TG 209 F3 Tarsus®, Netzsch) to estimate the exact mass concentration. Particles were lyophilised and stored at 4 °C until use.

CQDs-7

CQDs-7 were prepared in a similar way to CQDs-6. In short, 4-aminophenylboronic acid (200 mg) was dissolved in Milli-Q water (20 mL), and the pH of the solution was adjusted to 9.0 with NaOH (1 M). The solution was degassed with nitrogen gas for 1 h in order to remove dissolved oxygen and heated in a Teflon-lined autoclave chamber (125 mLacid digestion vessel n°4748, Parr, France) for 8 h at 160 °C. After cooling to room temperature, the solution was centrifuged at 10,000 rpm for 30 min to remove large precipitates and dialysed against water for 24 h with Milli-Q water, water being changed every 6 h (SpectraPor 1, pore size: 1000 Da). The formed functional CQDs-7 (200 µL) were dried and then weighed with a Sartorius microbalance (TG 209 F3 Tarsus®, Netzsch) to estimate the exact mass concentration. The suspension was further lyophilised and stored at 4 °C.

CQDs-8

Particles were prepared in a similar manner to CQDs-6 and CQDs-7 by dissolving PEG600 (200 mg) in water (20 mL) and adjusting the pH to 9.0 by using NaOH (0.5 M). The solution was degassed with nitrogen gas for 1 h to remove dissolved oxygen and heated in a Teflon-lined autoclave chamber (125 mLacid digestion vessel n°4748, Parr, France) for 72 h at 120 °C. After cooling to room temperature, the solution was dialysed against Milli-Q water for 24 h, water being changed every 6 h (SpectraPor 1, pore size: 3500 Da).

Fluorescently labelled CQDs-7

CQDs-7 were dispersed in PBS buffer (pH 7.4) at the concentration of 2 mg mL -1 .

Fluorescein-NHS was dissolved in DMF (10 mg mL -1 ). The CQDs-7 dispersion was cooled down to 0 °C, and 10 µL of freshly prepared fluorescein was added. The reaction was stirred on ice for another 3 h. After that, a Sephadex G-25 PD-10 desalting column was used to remove the dye in excess. The formed fluorescent CQDs-7 were dried (200 µL) and then weighed with a Sartorius microbalance (TG 209 F3 Tarsus®, Netzsch) to estimate the exact mass concentration. Particles were stored as a dispersion. A suspension of CQDs-COOH was prepared in PBS buffer (pH 7.4) at the concentration of 2 mg in 1 mL (10 mg). To this suspension, a two-fold excess of EDC and a 2.5-fold excess of NHS (dissolved in the minimum amount of PBS) were added, and the mixture was stirred for 20 min in order to activate the carboxyl groups. The peptide (1a-3b) (5 mg, 1 eq), dissolved

in the minimum amount of Milli-Q water or DMSO, was added and the reaction was stirred overnight. 1 mL of the mixture was lyophilised to obtain powder and indirectly quantify the free peptide by quantifying the unbounded one. The rest of the mixture was dialysed against Milli-Q water for 24 h using a cellulose membrane (Spectra/Por 6 Standard RC Pre-wetted Dialysis Tubing, MWCO 3.5 kDa). The sample collected before dialysis was weighed on a microbalance (Mettler Toledo, XPR6UD5), dispersed in 1 mL Milli-Q water and dropped off on a size exclusion PD10 disposable column (GE Healthcare) in order to separate the free peptide from the particles. Some of the particles stayed at the top of the column, indicating the formation of aggregates. Therefore, this method was not used for purification purposes. The separated peptide was lyophilised and quantified by HPLC using the corresponding calibration curve.

6.2.3.1.11. CQDs-peptide conjugates: CQDs-3.4a; -3.4b; -3.5a; -3.6b; -3.7a; -3.7b

CQDs-N3 were dispersed in water at the concentration of 3 mg in 1 mL (9 mg). 5 eq of copper sulphate pentahydrate,10 eq of ascorbic acid and ca. 3 mg (1 eq) of the peptide (4a-7b)

were added. The reaction mixture was stirred for 24 h. 1 mL of the reaction mixture was collected in order to quantify the free peptide. To the rest of the solution, 1 mM solution of EDTA was added, and the mixture was dialysed against Milli-Q water for 24 h using a cellulose membrane (Spectra/Por 6 Standard RC Pre-wetted Dialysis Tubing, MWCO 3.5 kDa). The sample collected before dialysis was weighed on a microbalance (Mettler Toledo, XPR6UD5), dispersed in 1mL of Milli-Q water and dropped off on a size exclusion column (GE Healthcare, disposable PD10 columns) in order to separate the free peptide from the particles. Some of the particles stayed at the top of the column, indicating the formation of aggregates. Therefore, this method was not used for purification purposes. The separated peptide was lyophilised and quantified by HPLC using the adequate calibration curve.

6.2.3.2.

Nanodiamonds: ND

ND-3: ND-N3

ND-NH2 were suspended in dry ACN at a concentration of 5 mg in 1 mL (10 mL).

Particles were sonicated for 30 min before the addition of 4-azidobenzoic acid (0. ND-COOH (ND-1) were dispersed in acetonitrile at the concentration of 1 mg in 1 mL

(5 mg). The particles were sonicated for 30 min before addition of DIC (4 eq), HOBt (4 eq) and the peptide (1a-3b, approx. 3mg, 1 eq) dispersed in the minimum amount of water. The reaction was carried out for 12 h at room temperature. The particles were purified by repeated washing/centrifugation cycles at 12,500 rpm with ACN (2 times) and water (until the A) Buffer pH 5.5: 36 g of sodium acetate were dissolved in 6.9 mL of acetic acid and completed with Milli-Q water to a volume of 100 mL.

B) 5 % ninhydrin solution: 5 g of ninhydrin was dissolved in 100 mL of ethanol.

C) KCN solution: 2 mL of 0.03 M KCN solution was diluted to a final of the volume of 100 mL with pyridine. D) Phenol solution: 80 g phenol were dissolved in 20 mL of ethanol.

E) 60 % ethanol solution.

Photothermal release studies

Release experiments were performed into 1 mL PBS buffer. The cryogels were irradiated with laser in a continuous mode (Gbox model, Fournier Medical Solution) with an output light at 980 nm (1W cm -2 ) for various time intervals (1-60 min). Thermal images were captured by an Infrared Camera (Thermovision A40) and treated using ThermaCam Researcher Pro 2.9 software. The amount of released antibiotic was evaluated by HPLC. The quantity of N-(5-fluoresceinyl)maleimide released was evaluated by fluorescence spectroscopy (BioTek Instruments SAS, France).

6.11.

Biological assays 6.11.1.

Cytotoxicity assay -Huh-7 cells

The Huh-7 hepatocarcinoma cell line was cultured and maintained in Dulbecco's Modified Eagle's medium (DMEM, Gibco®) supplemented with 10% fetal bovine serum (FBS, Gibco®) and 1% penicillin-streptomycin (Gibco®) in a humidified incubator at 37 °C and 5% CO2. Cells were seeded at a density of 15×10 3 cells/well in a 96-well plate and grown for 24 h before assay. The culture medium was replaced with a fresh medium that contains increasing concentrations of CQDs for 2 h and 8 h from 1 to 100 µg mL -1 . Then, the old medium was aspirated, and the cells were washed with PBS. The cell viability was evaluated using resazurin cell viability assay. Briefly, 100 mL of the resazurin solution (11 µg mL -1 ) in DMEM/10% FBS were added to each well, and the plate was incubated for 4 h in the humidified incubator. The fluorescence emission of each well was measured at 593 nm (20-nm bandwidth) with an excitation at 554 nm (18-nm bandwidth) using a Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek Instruments SAS, France). Each condition was replicated three times, and the mean fluorescence value of non-exposed cells was taken as 100% cellular viability.

Cytotoxicity assay -HeLa cells

The HeLa cell line derived from the cervical carcinoma of a 31-year-old female [ATCC® CCL-2™, ECACC, Sigma Aldrich, Saint-Quentin Fallavier, France] were cultured and maintained in Dulbecco's Modified Eagle's medium (DMEM, Gibco®) supplemented with 10% fetal bovine serum (FBS, Gibco®) and 1% penicillin-streptomycin (Gibco®) in a humidified incubator at 37 C and 5% CO2. Cells were grown for 24 h before the assay to be treated with the cryogel for another 24 h. The cell viability was assessed by using CCK-8 APPENDIX 126 viability assay. Briefly, the cells were washed, CCK-8 was added, and cells were incubated for an additional 2 h at 37°C. The absorbance values were then measured at 450 nm. The results were expressed as a percentage compared to controls.

6.11.3.

Uptake mechanism: fluorescently labelled CQDs-7

Cells were seeded at a density of 15×10 4 cells/well in a 24-well plate with sterile coverslips at the bottom and grown for 24 h before assay. The culture medium was replaced with a fresh medium that contained 100 µg mL -1 of CQDs. After 1 h incubation at 4 °C and 37

°C, the Huh-7 cells were washed with PBS (three times), fixed with 4% para-formaldehyde for 10 min at room temperature and then stained with 10 µg mL -1 Hoechst 33342 in PBS for 10 min at room temperature in the dark. After washing with PBS, the coverslips were mounted on glass slides and recorded using a Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek Instruments SAS, France) equipped with a 40× objective (Plan Fluorite WD 2.7 NA 0.6). The fluorescence images were acquired with the same exposure using DAPI (377/447 nm) and GFP (469/525 nm) excitation/emission filter sets. All the images were processed using Gen5 Image+ software.

For cellular uptake, cells were seeded at a density of 15×10 4 cells/well in a 6-well plate and grown for 48 h before assay. The culture medium was replaced with a fresh medium that contained 100 µg mL -1 of CQDs. After 1 h incubation at 4 °C and 1, 3, and 6 h incubation at 37 °C, the Huh-7 cells were washed with PBS (three times) and collected by trypsinisation. The cells suspensions were resuspended in PBS/PFA 0.5% and analysed through a flow cytometer (BD LSR Fortessa) with FITC channel. The data were collected (10 4 cells per sample) and analysed using BD FACSDiva 8.0.1 software.

Antiviral assay: HCoV-229E-Luc

We used a modified HCoV-229E containing a renilla luciferase reporter gene HCoV- Germany). All the conditions were performed in triplicates.

Virus-nanoparticles interaction assay

Virus HCoV-229E-Luc was pre-incubated with CQDs at 10 µg mL -1 for 30 min at 37 °C. The mixture was diluted 10 times in culture medium leading to a final concentration of CQDs of 1 µg mL -1 and inoculated on Huh-7 cells for 1 h. In parallel, Huh-7 cells were inoculated with HCoV-229E-Luc in the presence of CQDs at 1 and 10 µg mL -1 for 1 h. Cells were lysed 7 h post-infection, and luciferase activity quantified. All the experiments were performed in triplicates.

Competitive assay with mannose

The suspensions of CQDs-4 and CQDs-7 were prepared at the concentration of 1mg mL -1 . pH was adjusted to 9 with 1N NaOH and excess of mannose (2:1) was added. The mixture was incubated overnight at 4 °C to be further dialysed against water for 24 h (SpectraPor 1, pore size: 1000 Da). As prepared CQDs were added to cells during the 1-h infection. Cells were lysed in 20 µL of renilla lysis buffer (Promega, Madison, USA) and luciferase activity quantified using a renilla luciferase assay System kit (Promega, Madison, USA) as recommended by the manufacturer and a Tristar LB 941 luminometer (Berthold Technologies, Bad Wildbad, Germany). All the experiments were performed in triplicates.

Susceptibility testing -MIC values determination

E. coli DSM 30083 and S. aureus ATCC 43300 (MRSA) were used. 3-5 morphologically similar colonies from a fresh agar plate were transferred into a sterile tube containing sterile broth. The bacteria were incubated overnight at 37 °C. The next day OD600 of the overnight culture was adjusted to reach OD600 0.1 and was left to continue growing until reaching the mid-log phase. After preparing the inoculum at the concentration of 5×10 5 CFU mL -1 , the bacterial suspension was used within 30 min to prevent changes in the cell number. Peptides and peptide-carbon conjugates were tested. MIC values were established in a microtiter plate assay according to CSLI guidelines [START_REF] Espitia | Chapter 34 -Zinc Oxide Nanoparticles for Food Packaging Applications[END_REF]. Briefly, the peptide and the peptide conjugate were dissolved in DMSO at the concentration of 10 mg mL -1 . Serial dilution was prepared in MH (Muller-Hinton) medium, inoculated with 5×10 5 CFU mL -1 and incubated for 16 h at 37 °C.

Inoculated medium without the sample served as a growth control and uninoculated medium as a sterility control. The lowest visible growth was reported as a MIC value. Visual counting of the number of colonies upon overnight incubation at 37 °C allowed reading out the initial and final concentrations of the bacteria strain in CFU mL -1 . All the experiments were performed in triplicates.

6.12. Instrumentation 6.12.1. NMR Spectroscopy B NMR spectra were recorded on a Bruker Avance II 400MHz. All the spectra were recorded in deuterated solvents.

Mass Spectrometry

ESI-MS spectra were measured on a Bruker Esquire 6000.

High-Performance Liquid Chromatography (HPLC)

Semi-preparative HPLC was performed on a Knauer instrument. A Macherey Nagel VP Nucleodur 125/10 100-5 C18ec reverse phase column was used at a flow rate of 5 mL min -1 .

As a mobile phase A (water:0.1% TFA); B (MeCN:0.1% TFA) was used.

Analytical HPLC was performed on a Knauer instrument. A Nagel EC 125/4 Nucleodur C18 pyramid, 5µm reverse phase column was used at a flow rate of 1 mL min -1 . As a mobile phase A (water:0.1% TFA); (MeCN:0.1% TFA) was used. With the linear gradient of buffer B (95% in 7min) from 95% buffer A with a total run time of 15min.

Calibration curves obtained for the loading and release studies were recorded on an HPLC system (Shimadzu, Tokyo, Japan) equipped with a 5 μm C4 QS Uptisphere ® 300 Å, 250 mm × 4.6 mm column (Interchim, Montluçon, France) heated to 40 °C at a flow rate 1 mL min -1 . As a mobile phase: A (water:0.1% TFA); B (MeCN:0.1% TFA) was used. With the linear gradient of buffer B (80% in 15min) from 100% buffer A with a total run time of 23min.

Calibration curves obtained for peptide quantification on the surface of nanoparticles were recorded on HPLC system (Shimadzu, Tokyo, Japan) equipped with a 5 μm C18AQ

Uptisphere ® column (Interchim, Montluçon, France) heated to 40 °C at a flow rate 1ml/min. As a mobile phase 100%A (water: 0.1%FA); 100B (MeCN: 0.1%FA) was used. With the linear gradient of buffer B (80% in 15min) from 100% buffer A with a total run time of 23min.

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RESUME

La résistance croissante aux antibiotiques et les limitations dans le développement de nouveaux médicaments nécessitent la recherche de stratégies alternatives afin d'éradiquer les infections bactériennes. Des problèmes semblables apparaissent dans le développement de thérapeutiques antivirales, en raison de l'émergence constante de nouveaux virus et leur capacité à contourner les thérapies par des mutations génétiques.

Ce travail de recherche examine la potentielle activité antibactérienne et/ou antivirale de nanostructures à base de carbone telles que les nanoparticules de diamant et les points quantiques carbonés (carbon quantum dots, CQDs), ainsi que l'oxyde de graphène réduit (reduced graphene oxide, rGO) combiné à des cryogels. Les CQDs produits par synthèse hydrothermale à partir de l'acide 4-aminophénylboronique comme précurseur carboné se sont montrés efficace en tant qu'inhibiteurs de l'attachement du coronavirus humain HCoV-229E-Luc aux cellules avec une EC50 de 5,2±0.7 µg mL -1 . Les études mécanistiques suggèrent que les CQDs agissent lors des tout premiers stades de l'infection virale ainsi que lors de l'étape de réplication du virus. En parallèle, nous avons tiré parti du caractère multivalent des CQDs et des nanodiamants pour les modifier en y fixant de courts peptides synthétiques antimicrobiens (antimicrobial peptides, AMPs). Ces nanostructures ont été testées contre des bactéries pathogènes à Gram positif Staphylococcus aureus et à Gram négatif Escherichia coli et ont montré une activité antibactérienne plus élevée que celle des AMPs seuls. Dans le cas du rGO combiné à des cryogels chargés en AMPs, l'éradication des bactéries a été réalisée efficacement et à la demande en utilisant une irradiation infrarouge comme activateur externe permettant le relargage des AMPs.

ABSTRACT

Increasing antibiotic resistance and limited development of new drugs necessitate the search for alternative strategies to eradicate bacterial infections. Similar problems are faced in the development of antiviral therapeutics, due to the constant emergence of new viruses and their ability to escape therapy by genetic mutations. This work investigates the potential antibacterial and/or antiviral activity of carbonbased nanostructures such as diamond nanoparticles and carbon quantum dots (CQDs) as well as reduced graphene oxide (rGO) in combination with cryogels. CQDs formed by hydrothermal synthesis from 4-aminophenylboronic acid as the carbon precursor showed to be efficient in the inhibition of the viral attachment of human coronavirus HCoV-229E-Luc to cells with an EC50 of 5.2±0.7 µg mL -1 . Mechanistic studies suggest that the CQDs are acting at the early stage of virus infection as well at the viral replication step. In parallel, we took advantage of the multivalent character of CQDs as well as nanodiamonds and modified them with short synthetic antimicrobial peptides (AMPs). Tests of these nanostructures against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli pathogens showed increased antibacterial activity when compared to AMPs alone. In the case of rGO combined with cryogels loaded with AMPs, bacterial eradication was achieved efficiently and on-demand using near-infrared light as external trigger to release AMPs.

Finally, I owe

  my deepest gratitude to my mother, grandmother and my two sisters for their unconditional love, encouragement and support in my decision for studying miles away from my hometown.Aleksandra ŁoczechinOctober 2019

Figure 1 . 1 :

 11 Figure 1.1: Design strategies and properties of nanoparticles for biomedical applications (image is taken from ref [1]).

Figure 1 . 2 :

 12 Figure 1.2: Different classes of the properties and functions of NPs utilised for biomedical applications (image is taken from ref [5]).

Figure 1 . 3 :

 13 Figure 1.3: Classification of nanomaterials commonly used in nanomedicine. Sketch inspired from ref [22].

Figure 1 . 4 :

 14 Figure 1.4: Schematic summary of bioconjugation strategies for inorganic nanoparticles [40].

Figure 1 . 5 :

 15 Figure 1.5: A) Schematic representation of quantum confinement effects (image is taken from ref [158]); B) Jablonski diagram (image is taken from ref [159]).

Figure 1 . 6 .

 16 Figure 1.6. Schematic illustration of the properties and applications of CQDs (image a is taken from [164]; image b is taken from [165]; c bio-distribution of CF-790-CQDs after intravenous injection (via tail) in five-week-old swiss nude mice; image d is taken from [166]; image e is taken from [167]; image f is taken from [168]; image g is taken from [169]).

Figure 1 . 7 :

 17 Figure 1.7: Covalent and non-covalent approaches for the surface functionalization of CQDs (image is taken from ref [173]).

Figure 1 . 8 :

 18 Figure 1.8: Illustration of the synthesis and post-synthetic treatment in the preparation of D-NDs (image is taken from ref [85]).

. 9 )

 9 . Non-covalent functionalisation includes mostly electrostatic (highly negatively charged surface of NDs) and hydrophobic interactions (adsorption onto the surface via apolar interactions) [179]. This approach found a successful application in drug delivery, where an adsorbed molecule can be released upon a specific stimulus, e.g. the pH [180, 181]. The group of Ho and co-workers applied non-covalent adsorption of doxorubicin (DOX) onto the surface of NDs. The composite led to a significant increase in cell apoptosis compared to conventional treatment [182, 183]. Another example of successful non-covalent adsorption is the adsorption of cell-penetrating peptide TAT onto NDs improving its delivery [184]. It is worth to notice that incorporating a peptide or protein can provide additional binding sites for other molecules to be linked covalently, leading to improved and more effective penetration and delivery abilities [185]. Main covalent modifications are similar to the one described for carbon dots. The presence of abundant carboxylic groups onto the surface allows condensation-type reactions between the NPs and the drug. Another popular response is the esterification reaction between the carboxylic groups on the particles and molecules bearing an alcohol moiety (or the other way around). Barras and co-workers created azido-or alkynyl-modified NDs by coupling 4azidobenzoic acid or 4-pentynoic acid with hydroxyl groups present at the ND surface [186].

  and coworkers reported the direct functionalisation of NDs-OH with dopamine derivatives bearing an azide function [188]. Such an approach creates new possibilities for preparing multifunctional particles. In addition, Turcheniuk and co-workers created pre-modified NDs with perfluorophenylazide ligands and linked them photochemically with unmodified polysaccharides to obtain NDs showing a pronounced affinity towards lectins in pathogenic UTI E. coli [189]. The group conducted further studies in the direction of biofilm disruption and the particles were tested against S. aureus and E. coli [190]. It has also been reported that NDs upon thiolation can be successfully conjugated with gold nanoparticles which provide a new platform for NDs applications [191].

Figure 1 . 9 :

 19 Figure 1.9: Common surface functionalization methods used in the chemistry of NDs: A) non-covalent electrostatic interactions; B) non-covalent hydrophobic interactions; C) covalent functionalization via carboxylic acid groups; D) covalent functionalization via hydroxyl groups. The image is taken from ref [192].

  exceptional properties [205-208]. Structurally graphene is considered the basic material with the ability to fold in a different way or aggregate thus resulting in carbon allotropes like 0Dfullerene, 1D-carbon nanotubes, 2D-graphene itself, 3D-graphite [209]. It exists as few layers of graphite with the crystal structure of a tightly packed honeycomb-like structure [210]. The family of graphene includes members such as graphene oxide (GO) and its reduced form reduced graphene oxide (rGO) (Figure 1.10).

Figure 1 . 10 :

 110 Figure 1.10: Schematic illustration of graphene (left), graphene oxide (middle), reduced graphene oxide (right).

  material enhances the antibacterial property of GO/rGO[215]. Non-covalent occurs without interrupting the extended π system of graphenic nanostructures. π-π stacking can be comparable to covalent interactions where two aromatic rings have overlapping π orbitals. They provide more stable alternatives to above mention interactions. One of the strategies incorporates modification of molecules with pyrene which is a π-orbital rich group[216]. Recently a group of Liu and co-workers synthesised thermoresponsive graphene/polymer by first preparing well defined thermoresponsive pyrene-poly(N-isopropylacrylamide) followed by the attachment onto the basal plane of graphene sheets[217]. In addition to that, Zhou et al. recently showed that the solubility of a very poor soluble drug could be increased by its interaction with GO surface. They incorporated hypocrellins onto the surface via π-π interaction/hydrogen bonds and the in vitro studies showed an effective uptake of the hybrid into the tumour, which subsequently was irradiated and damaged[218]. Another example of improving the solubility of the molecule is the incorporation of quercetin which resulted in stable hybrid in physiological solution with no toxicity in vitro[219]. Another example of successful surface loading of the drugs is doxorubicin[220]. Recently described by some of us, showed the potential of drug loading onto the surface of rGO with the combination of photothermal therapy. rGO was incorporated into the matrix of a hydrogel and further loaded with insulin. The release was achieved upon near-infrared irradiation thanks to the good photothermal properties of rGO.Another example developed by our group was the non-covalent functionalization of rGO with maleimide containing dopamine which further can undergo "click" reaction with sulphur containing molecules like the one used in the studies-c(RGDfC) peptide (cancer cell targeting peptide). It was shown that such a construct could work as a targeted drug delivery system for doxorubicin[221]

  and the limited development of new drugs make the search for alternative strategies to eradicate bacterial infections imperative. Although the last class of antibiotics was introduced on the market in 2000 (Figure2.1), its discovery dates from the 1970s, which shows that no significant progress has been made in the development of new classes of antibacterial agents. Certainly, a lot of "new" products approved for clinical use have been released as 2 nd , 3 rd , 4 th generation antibiotics, but they are mostly derivatives of existing compounds, and their mode of action remains the same. The shortage of newly developed drugs in the past years, together with overusing and abusing current antibacterial agents, led to the increasing development of resistance in pathogenic bacteria. This continued evolution of antimicrobial resistance brings a real threat to human health by seriously compromising our ability to treat infections[1]. The annual deaths worldwide caused by pathogenic bacteria are estimated to reach 10 million by 2050[2]. Thus, the development of new antibacterial agents becomes an urgent and crucial task to overcome this growing problem.

Figure 2 . 1 :

 21 Figure 2.1: History of antimicrobial agent development vs subsequent acquisition of resistance by microorganisms [3].

  photocatalysed production of reactive oxygen species (Figure2.2)[4,[8][9][10][11].

Figure 2 . 2 :

 22 Figure 2.2: Various antimicrobial mechanisms due to nanomaterials. The form was inspired by the sketch in [3].

Figure 2 . 3 :

 23 Figure 2.3: Structure of the synthesised synAMPs being an arginine-and tryptophan-rich hexapeptide.

. 4 )

 4 . First, to enable the incorporation of peptide amides, amine groups were reacted with ClCH2COONa under strong alkaline conditions resulting in carboxyl capped CQDs (CQDs-2). The second modification includes grafting 2-azidoacetic acid onto the surface in the presence of EDC/NHS (CQDs-3). In case of nanodiamonds, for the coupling of peptide amides, we used commercially available NDs-COOH, and for the "click" reactions ND-NH2 was directly modified with 4-azidobenzoic acid in the presence of N, N'dicyclohexylcarbodiimide (DCC) and a catalytic amount of 4-dimethyl aminopyridine (DMAP) (ND-3) (Figure2.4).

Figure 2 . 4 :

 24 Figure 2.4: Schematic illustration of synthesis and surface functionalization used for carbon nanoparticles.

Figure 2 . 5

 25 shows transmission electron microscopy (TEM) of as-prepared material confirming their spherical shape with an average diameter of 4.5 ±0.2 nm. From the image, most of the observed particles are of a crystalline character possessing well-resolved lattice fringes.

Figure 2 . 5 :Figure 2 . 6 :

 2526 Figure 2.5: TEM image, magnified TEM, FFT images and size distribution histograms for CQDs-1, CQDs-2, and CQDs-3.

Figure 2 . 7 :

 27 Figure 2.7: Characterisation of CQDs-1: (A)XPS survey of CQDs-1 with inset of high resolution C1s spectrum; (B) N1s spectrum of CQDs-1.

Figure 2 . 8 :

 28 Figure 2.8: Characterisation of CQDs: FTIR spectra of as-prepared CQDs-1 (black), CQDs-2 (blue), CQDs-3 (red).

1 :Figure 2 . 9 :Table2. 2 :Figure 2 . 10 :Figure 2 .

 12922102 Figure 2.9: (A) FTIR of ND-2, ND-3; (B) High resolution N1s XPS spectrum of CQDs-3 (up) and ND-3 (down).

Figure 3 . 1 :

 31 Figure 3.1: Schematic illustration of different furan-based cryogels loaded non-covalently as well as covalently via Diels-Alder reaction with an antimicrobial agent for the killing of bacteria triggered by near-infrared laser.

Figure 3 . 2 :

 32 Figure 3.2: (A) Hydrogel samples prepared using a FuMA/PEDMEM= 20/80 with (left) and without (right) embedded rGO; (B) SEM picture of furan-containing cryogels with (up) and without (down) embedded rGO (scale bar: 100nm); (C) Schematic illustration of the fabrication of furan containing cryogels.

Figure 3 . 3 :Figure 3 .Figure 3 . 4 :

 33334 Figure 3.3: Swelling behaviour of the different cryogels: Water uptake of different cryogels at 24 o C without and with rGO (1 weight %) embedded into the cryogels.

Figure 3 . 5 :

 35 Figure 3.5: (A) Furan-maleimide chemistry; (B) Loading capacity of CG-rGO-40 (3 mg) for ampicillin using different initial concentrations; (C) Release behaviour of ampicillin from CG-rGO-40 loaded with 114 µg mL -1 of antibiotic under 10 min laser activation (1 W cm -2) and passively at 24 o C; (D) Comparison of the action of free ampicillin (2 µg mL -1 , negative control), ampicillin (10 µg mL -1 ; positive control) and released ampicillin after 10 min upon activation and passively when incubated with E. coli (110 8 CFU mL -1 ).

Figure 3 . 6 :

 36 Figure 3.6: (A) Schematic representation of dye loading and microscopy images of CG-rGO-0(control) and CG-rGO-40 (CG-Furan) after treatment with maleimide dye; (B) Capacity of furan free CG-rGO-0 (control) and furfuryl-containing CG-rGO-40 cryogels for the integration N-(5-fluoresceinyl)maleimide via Diels-Alder cycloaddition; (C) Release of fluorescein maleimide dye via retro Diels Alder from CG-rGO in % using a laser (active) and at 37 o C (passive).

Figure 3 . 7 :

 37 Figure 3.7: (A) Synthesis of maleimide modified peptide via Thiol Michael addition reaction between a PEG bismaleimide and the cysteine end and Schematic representation of loading and release of maleimide containing molecules based on Diels Alder based linkage and retro Diels Alder based cleavage at Temperature > 42 °C; (B) Loading capacity of CG-rGO-40 cryogel with time from a stock solution of maleimide-modified peptide of 600 µg mL -1 ; (C) Release of maleimide containing peptide via retro Diels Alder from cryogel at 37 o C (passive) and 70°C (active); (D) Photothermal heating curves of CG-40 via Kpaton/rGO and CG-rGO-40 when immersed in PBS (0.1 M, pH 7.4) and exposed to laser light of 980 nm for 10 min at 1W cm -2 ; (E) Activity of maleimidemodified peptide released from cryogel under different conditions against S. aureus ATCC4300.

  the irradiation (Figure3.7D) but affects the release efficiency of the loaded molecules. As shown in (Figure3.7C) the peptide release was almost 100%

Figure 3 . 8 :

 38 Figure 3.8: HeLa cell viability of after 24h incubation with different cryogels with and without incorporated rGO.

Figure 4 . 1 :

 41 Figure 4.1: Influence of CQDs, prepared by hydrothermal carbonisation, on the binding of HCoV-229E virus to cells: (a) inhibition of protein S receptor interaction, (b) inhibition of viral RNA genome replication.

4. 2 . 1 .CHAPTER 4 3 1 (Figure 4 .Figure 4 CHAPTER 4 Figure 4 . 3 :

 2143144443 photographs in Figure 4.2F.

1 h incubation at 4

 4 °C and 37 °C. Then nuclei were stained with Hoechst 33342, a fluorescent dye for labelling DNA in fluorescence microscopy (Figure 4.4B). The green fluorescence, attributed to CQDs-4, is homogeneously distributed in the cytoplasm after 1 h when incubated at 37 °C, which confirms the internalisation of CQDs-4 in the cells. The reduction of green fluorescence, observed in the cytoplasm after 1 h incubation at 4 °C, suggests that the active internalisation mechanism may be partially blocked and a small portion of CQDs was internalised by passive penetration.

and 6 hFigure 4 . 4 :

 644 Figure 4.4: Characterization of post-functionalized CQDs: (A) Viability of Huh-7 cells treated with the different CQDs: Huh-7 cells were grown in 96-well plates (15×10 3 cells/well) with 100 µL of culture medium containing increasing concentrations of CQDs for 8 h (left) and 24 h (right). The results, expressed as the percentage of viability, are the mean value of two independent experiments with each treatment performed in triplicate. Negative control: without CQDs; (B) Fluorescence microscopy of Huh-7 cells treated with 100 µg mL - 1 of CQDs-4 for 1 h at 4 °C (upper image) and 37 °C (lower image). The blue signal corresponds to the nuclei stained with Hoechst 33342, while the green signal is attributed to CQDs-4. Scale bars = 50 µm. (C) Flow cytometry analysis of cellular uptake of CQDs in Huh-7 cells treated with 100 µg mL -1 of CQDs-4 for 1 h at 4 °C and 1 h, 3 h and 6 h at 37 °C.

Figure 4 . 5 .

 45 Figure 4.5. Viral infection inhibition in the presence of CQDs: (A) Viral inhibition using CQDs at various concentrations. Huh-7 cells (1.5×10 4 cells/well) were inoculated with HCoV-229E-Luc for 1 h (in an atmosphere with 5% CO2 at 37 °C) in the presence or absence of different CQDs in medium without FBS for 1 h. Afterwards, the inoculum was removed and replaced with DMEM with FBS for 6 h. Cells were lysed, and luciferase activity quantified. The results expressed as the percentage of infection normalised to the control without CQDs, which is expressed as 100% infection. Data are means of two independent experiments with each treatment performed in triplicate. (B) Determination of EC50 for CQDs-4 and CQDs-5, and the effect of viral inhibition using CQDs-4 after incubation with mannose (2:1) overnight at 4 °C.

  g mL -1 system, in accordance with the literature data [25]. The CQDs exhibit different fluorescence quantum yields (QY) of 0.03 (CQDs-6), 0.05 (CQDs-7), and 0.09 (CQDs-8) (Figure 4.8C). The wavelength-dependent fluorescence emission properties of the CQDs are comparable (Figure 4.8A). The zeta potential and hydrodynamic size of CQDs-6, -7 and -8 are summarised in Table 4.2.

Figure 4 . 7 : 6 , - 7 , - 8 .

 47678 Figure 4.7: TEM, magnified TEM and size distribution histograms of CQDs-6, -7, -8.

CHAPTER 4 Figure 4 . 8 :

 448 Figure 4.8: (A) Wavelength-dependent fluorescence emission properties of CQDs-6, -7, -8; (B) UV-Vis absorption spectra of 100 µg mL -1 suspensions of CQDs-6, -7, -8; (C) Fluorescence spectra of CQDs-6, -7, -8 upon excitation at 350 nm.

- 6 , - 7 , - 8

 678 exhibited a negative zeta potential in water (pH 7.4) at room temperature and showed excellent long-term stability even in a biological medium such as Dulbecco's Modified Eagle's medium (Figure 4.9D). The cytotoxicity of CQDs 5-7 (Figure 4.10A) is comparable to the CQDs discussed before (Figure 4.4A), with CQDs-7 being slightly more toxic at concentrations >25 µg mL -1 after 24

Figure 4 .Figure 4 . 9 :

 449 Figure 4.9: (A) FTIR spectra of CQDs-6, -7, -8; (B) N1s high resolution XPS spectrum of CQDs-7; (C) C1s high resolution XPS spectrum of CQDs-6, -7, -8; (D) Photographs of CQDs CQDs-6, -7, -8 suspensions (1mg mL -1 ) after 1 month in Dulbecco's Modified Eagle's medium.

Figure 4 . 12 :

 412 Figure 4.12: 11 B NMR spectra of CQDs-6 and CQDs-7 prepared by hydrothermal method from phenylboronic acid (PBA) and 4-aminophenylboronic acid (4-APBA) precursors respectively, and 11 B NMR spectra of 4aminophenylboronic acid (4-APBA) and phenylboronic acid (PBA) starting materials.

infection (Figure 4 .Figure 4 . 13 :

 4413 Figure 4.13: Time-of-addition assay of CQDs-4 and -7 during HCoV-229E infection. (A) CQDs at 10 µg mL - 1 were added at different time points during infection of Huh-7 cells with HCoV-229E-Luc as shown below the graph. Cells were lysed, and luciferase activity quantified. Results are representative of 3 experiments performed in triplicate. Error bars represent SD of three independent values; (B) Virus HCoV-229E-Luc was pre-incubated with CQDs at 10 µg mL -1 for 30 min at 37 °C. The mixture was diluted 10 times in culture medium leading to a final concentration of CQDs of 1 µg mL -1 and inoculated on Huh-7 cells for 1 h. In parallel, Huh-7 cells were inoculated with HCoV-229E-Luc in the presence of CQDs at 1 and 10 µg mL -1 for 1 h. Cells were lysed 7 h postinfection, and luciferase activity quantified. Results are means of 3 experiments performed in triplicate. Error bars represent means of three independent values. Statistic evaluation (confidence interval of 95%), ns (p>0.99); * (p<0.1);** (p<0.01).

  , m/z): 1044.79 (calc. 1044.24 for [M+H] + ).

  , m/z): 1044.75 (calc. 1044.24 for [M+H] + ). HPLC (tR, min): 6.36.

  , m/z): 1100.53 (calc. 1100.08 for [M+H] + ). HPLC (tR, min): 7.4.

  , m/z): 1070.75 (calc. 1070.06 for [M+H] + ). HPLC (tR, min): 6.63.

  , m/z): 1146.04 (calc. 1145.31 for [M+H] + ). HPLC (tR, min): 7.43.

  , m/z): 1124.80 (calc. 1124.33 for [M+H] + ). HPLC (tR, min): 6.68.

  for SPPS. MS (ESI, m/z): 1139.86 (calc. 1139.34 for [M+H] + ). HPLC (tR, min): 6.36.

  , m/z): 1139.93 (calc. 1139.34 for [M+H] + ).

11 . 12 .

 1112 Synthesis of FcCO-WRWRWG(CH2CCH)-NH2: 6aThe peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure.MS (ESI, m/z): 1195.68 (calc 1195.18 for [M+H] + ).HPLC (tR, min): 7Synthesis of FcCO-RWRWRG(CH2CCH)-NH2: 6bThe peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure.MS (ESI, m/z): 1165.98 (calc. 1165.16 for [M+H] + ). HPLC (tR, min): 6.83.

  , m/z): 1240.9 (calc. 1240.41 for [M+H] + ). HPLC (tR, min): 7.51.

  , m/z): 1211.01 (calc. 1210.39 for [M+H] + ). HPLC (tR, min): 7.41.

15 .

 15 Synthesis of H-WRWRWRC-NH2: 8The peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure.MS (ESI, m/z): 1147.86 (calc. 1147.38 for [M+H] + ). HPLC (tR, min): 7.06.

Figure 6 . 1 :

 61 Figure 6.1: UV-Vis calibration curve of benzylamine.

229E-

  Luc. The viral stocks were produced in Huh-7 cells. Huh-7 cells were infected with a prestock of HCoV-229E-Luc in flasks. After 5 days, the supernatants of flasks were collected. For the infection assay, Huh-7 cells, 15,000/well seeded in 96-well plate, were inoculated with HCoV-229E-Luc at a multiplicity of infection (MOI) of 1 for 1 h at 37 °C in DMEM without serum, then the inoculum was removed and cells were incubated in complete culture medium for 6 h at 37 °C. CQDs were added to cells during the 1-h infection. Cells were lysed in 20 µL of renilla lysis buffer (Promega, Madison, USA) and luciferase activity quantified using a renilla luciferase assay system kit (Promega, Madison, USA) as recommended by the manufacturer and a Tristar LB 941 luminometer (Berthold Technologies, Bad Wildbad, Germany). To measure EC50, the dose-response experiment was performed with CQDs added at different concentrations during the inoculation step and post-inoculation step. For time-ofaddition assays, CQDs were added at different time points at 10 µg mL -1 . For all experiments, water was used as a control because CQDs are suspended in water. were infected with a pre-stock of HCoV-229E-Luc in flasks. After 5 days, the supernatants of flasks were collected. For the infection assay, Huh-7 cells, 15,000/well seeded in 96-well plate, were inoculated with HCoV-229E-Luc at a multiplicity of infection (MOI) of 1 for 1 h at 37 °C in DMEM without serum, then the inoculum was removed and cells were incubated in complete culture medium for 6 h at 37 °C. CQDs were added to cells at a different time during the infection of Huh-7 with HCoV-229E-Luc. Cells were lysed in 20 µL of renilla lysis buffer (Promega, Madison, USA) and luciferase activity quantified using a renilla luciferase assay System kit (Promega, Madison, USA) as recommended by the manufacturer and a Tristar LB 941 luminometer (Berthold Technologies, Bad Wildbad,

1 H

 1 NMR and13 C NMR spectra were recorded at room temperature on a Bruker DPX 200 ( 1 H: 200 MHz,13 C: 50 MHz) spectrometer. Chemical shifts are given in ppm relative to tetramethylsilane (TMS). Coupling constant J are reported in Hz. Multiplicities are noted as singlet (s), doublet (d), triplet (t), multiplet (m).

11

 11

••

  Loczechin A, Seron K, Barras A, Giovanelli E, Belouzard S, Chen YT, Metzler-Nolte N, Boukherroub R, Debuisson J, Szunerits S; (2019) Functional Carbon Quantum Dots as medical Countermeasures to Human Coronavirus (HCoV). ACS Applied Materials & Interfaces (accepted) DOI: 10.1021/acsami.9b15032 RESEARCH VISITS April 2017-October 2017; May 2018-August 2018; October 2018-April 2019-three PhD mobilities at Ruhr University Bochum, Germany. MEMBER GRANTS Pathogen and Graphene (September 2016-December 2019); funding from the European Union's Horizon 2020 Research and Innovation Staff Exchange (RISE) Marie Skłodowska-Curie Actions under grant agreement No 69083

TABLE OF CONTENTS i TABLE OF CONTENTS ACRONYMS a

 OF

Table 1 . 1 :

 11 Clinically approved intravenous nanoparticle therapies and diagnostics.[21]

	Name	Particle type	Approved application	Year of approval
	Doxil/Caelyx	Liposomal doxorubicin (PEGyleated)	Cancer treatment	1995 (FDA); 1996 (EMA)
	Abraxane	Albumin-particle bound paclitaxel	Cancer treatment	2005 (FDA); 2008 (EMA)
	Onivyde MM-398 Liposomal irinotecan (PEGylated)	Cancer treatment	2015 (FDA)
	Injectafter	Iron carboxymaltose colloid	Iron-replacement nanoparticle therapies	2013 (FDA)
	Definity	Perflutren lipid microspheres	Imaging agents	2001 (FDA)
	Epaxal	Liposome with hepatitis A virus	Nano vaccines	Some of Europe(Discontinued)
	Inflexal V	Liposome with trivalent-influenza	Nano vaccines	Some of Europe (Discontinued)
	AmBisome	Liposomal amphotericin B	Fungal treatment	1997 (FDA); Most of Europe
	Diprivan	Liposomal propofol	anaesthetics	1989 (FDA)

Table 1 . 2 :

 12 Examples of antimicrobial inorganic nanoparticles and their mechanism of action.

	Nanoparticles	Targeted microorganism	Mechanism of action	Ref.
		E. coli, B. subtilis, S. aureus	Release of Ag + ions	[87]
	Ag	C. albicans	Cell membrane disruption and electron transport, DNA damage	[88]
		E. coli, B. megaterium	Production of reactive oxygen species Cell membrane and cell wall damage	[89]
	TiO2	C. albicans	Generation of electron-hole pairs by visible light excitation with low recombination rate Intracellular accumulation of NPs	[90]
		E. coli, S. aureus B. cinerea, P. expansum	Release of Zn 2+ ions Cell membrane damage H2O2 production	[91-93]
		P. aeruginosa, E. coli	Sequestration of Mg 2+ or Ca 2+ ions to disrupt bacterial cell membrane	[94]
			Changed the membrane potential and reduced
	Au	E. coli, K.pneumonia,	adenosine triphosphate (ATP) synthase activities	[95]
		S. aureus, and B.subtilis	Declined the subunit of the ribosome for tRNA
			binding
	QDs	E.coli	Increased antibacterial activity, higher therapeutic indices	[96]

Table 1.3. Table 1.3: Organic

  nanoparticle-based systems for drug delivery.

	Micelles and dendrimers are another class of organic nanoparticles which are used as
	drug delivery vehicles. Micelles are polymeric structures that consist of monomer joined
	together; it is the bipolarity of the monomer molecules that causes the formation of micelles.
	Dendrimers, on the other hand, are highly branched molecules that combine the properties of
	polymers as well as small discrete entities. In comparison to micelles, their structure does not
	have a completely hollow core, but the polymeric network of repeating units is expanding
	inside-out. Drugs can be incorporated into the dendrimer structure by three different ways:
	i) covalent linkage to outer boundary ii) ionically joined to functional groups on the periphery
	iii) encapsulated inside the dendrimer. Some examples of different drug delivery systems
	together with delivered substance and targeted microorganism are collected in the NPs Drug Targeted microorganism Activity and features	Ref.
		-	S.epidermidis	Inhibition of biofilm formation	[103]
		Vancomycin	S.aureus	Inhibition of biofilm formation	[104]
		Amikacin	P.aeruginosa	Inhibition of biofilm formation	[105]
		Tobramycin	P.aeruginosa	Liposome fusion with bacterial cells	[106]
		Vancomycin/ rifampin	S.epidermidis	Enhanced biofilm eradication	[107]
				Significantly increased efficacy as
	Liposomes	Gentamycin	S.aureus	compared to liposomal gentamicin; complete sterilization of bone tissues;	[108]
				prolonged drug release
		Diclofenac sodium	-	Improved drug delivery through skin	[109]
		Peptides	Cancer cells	Improved pharmacokinetics and pharmacodynamics of the drug	[110, 111]
		Dox	Cancer cells	Enhanced antitumor efficacy	[112, 113]
		Vancomycin	P.aeruginosa	Decrease bacterial colonization	[114]
		Piperacillin/ Tazobactam	P.aeruginosa	Enhanced inhibition of biofilm increase the antibacterial activity formation	[115]
		Hypocrellin A	S.aureus	Enhanced antibacterial activity for non-resistant bacterial strain	[116]
	Rifampicin Solid lipid nanoparticles (SLNs) consists of the lipid solid core such as glycerides, which can Mycobacterium Enhanced antibacterial activity [117] tuberculosis decrease solubilize lipophilic molecules. SLNs are physiologically well tolerated, protect labile drugs Enhanced accumulation in tumor Micelles PTX Cancer cells tissue and significant tumor weight [118]
	from chemical degradation, control the release of the drug due to the solid state of the lipid Dox/ cRGDfK Cancer cells (human Kaposi's sarcoma) Enhanced internalization in tumor [119] endothelial cells matrix. Nanostructured lipid carriers consist of a matrix composed of solid and liquid lipids Increased therapeutic activity
	stabilized by a surfactant solution. Other than that, we can distinguish lipid, protein and tumor metastasis activity in vivo polysaccharides based systems that belong to the group called "generally recognized as safe Dox Cancer cells better cytotoxicity in vitro and low systemic toxicity with superior anti-[120]
	(GRAS) nanoparticles" [102]. These systems are often made from natural products and
	therefore are much safer than their chemically synthesized counterpart.

different molecules with different solubility

[101]

.

Another NPs that consists of the lipid-based formulation are nanoparticles/ nanoemulsions, solid-lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs).

Table 1 .3: Continued NPs Drug Targeted microorganism Activity and features Ref.

 1

			1-hexadecyl-	E.coli
			azoniabicylo [2.2.2] octane	B.cereus P.aeruginosa S.aureus	Increased antibacterial activity against gram positive bacteria	[121]
			(C16-DABCO)	S.oralis
		Dendrimers	PAMAM
			dendrimer/ nadifloxacin and	E.coli	Increased solubility of quinolones and antibacterial activity	[122]
			prulifloxacin
			PEI/silver NPs	-	Used as imaging agent, antimicrobial agent	[123, 124]
			Hyaluronic acid/PTX	Cervical breast tumor	Sustained drug release profiles, increased tumor drug concentration	[125]
			Floxuridine	Cancer cells	Higher efficacy on cancer cells	[126]
			Curcumin	Skin disorders	Controlled drug delivery	[127]
					squalene-based adjuvants with
					excellent biocompatibility and
		SLNs	Squalane	Yiest vaccines	provided immune stimulating properties comparable to Freund's
					adjuvant.
			Tretinoin	Propionibacterium acnes, S. aureus	Improved drug loading capacity Greater stability	[128]
			Tobramycin	P.aeruginosa	Increased drug bioavailability	[129]
			Atazanavir	HIV	Improved drug loading capacity	[130]
			penciclovir	-	Improved targeting	[131]
					Greater stability
		SLN/NLC	clotrimazole	fungi	Improved drug loading capacity Improved drug penetration through	[132]
					stratum corneum
	1.4.	Carbon nanoparticles
		In the field of biomedicine, carbon-based materials are becoming more and more
	attractive [133-137]. The existence of different carbon allotropes, from well-known and
	characterised allotropic phases such as amorphous carbon, graphite and diamonds to more lately
	discovered carbon nanotubes, graphene (and graphene oxide) or carbon dots, has recently
	attracted a lot of attention. Each of these structures exhibits distinctive features and has been
	extensively exploited in diverse biological applications [138, 139]. NPs from this group show
	unique mechanical, electrical and structural characteristics which find their application in
	sensing, medical diagnosis, and disease treatment [140, 141].
	1.4

.1. Carbon quantum dots-CQDs

	can exist as amorphous or crystalline nanostructures commonly with an sp 2 carbon
	hybridisation, even if cases of sp 3 hybridisation have also been reported [144]. The large
	number of oxygen-based functional groups they bear is responsible for their excellent water
	dispersibility and fluorescent properties [143]. In addition, the presence of these different
	reactive groups on their surface makes further functionalization possible. The diversity of
	surface functional groups on CQDs is related to the carbon precursor used for the synthesis as
	well as to the synthesis method itself. In general, the synthetic methods for the development of
	CQDs can be classified into two main categories commonly found in all nanotechnological
	approaches, namely top-down and bottom-up. Top-down processes are based on breaking down
	larger carbon structures while bottom-up processes rely on synthesis from small molecular
	precursors. Table 1.4 shows different methods corresponding to each approach. Depending on
	the synthesis route chosen for the preparation of the carbon nanomaterial, their properties may
	differ significantly.
	Carbon quantum dots (CQDs) have attracted significant attention since their accidental
	discovery in 2004 [142]. They show an overall size diameter below 10 nm and with [143] and

Table 1 .4: Top

 1 -down and bottom-up methods for the synthesis of CQDs.

	Top-down	Bottom-up
	Laser ablation [145]	Hydrothermal/Solvothermal [146, 147]
	Ultrasonic synthesis [148]	Microwave-hydrothermal
	Electrochemical/chemical oxidation [149]	Plasma hydrothermal [150]
	Arc discharge [142]	Chemical Vapor Deposition [151]
	Ball milling [152]	Pyrolysis [153]

does not include any metals. Comprehensive studies of in vitro cytotoxicity have already been conducted by many research groups [161]. As for in vivo studies, Figure 1.6c shows

	a
	biodistribution of CQDs used in this thesis labelled with a NIR-fluorescent dye. It shows that
	the fluorescence signal can be observed in the bladder after already 2.5 h after injection,
	indicating that the CQDs are removed from the body of mice through an excretion pathway (via
	urine) which is in accordance to other studies carried out in this direction [162, 163]. Additional
	tests of hemolytic activity of such nanostructures still need to be performed as there is only a
	little information available up to date. These properties of make CQDs promising candidates in
	the biomedical field (Figure 1.6).

der Waals forces have been reported [170]. Figure 1.7 shows some of the most common

	provide energy or emissive trap states for a photoexcited electron capture. As a consequence, it
	can potentially alter the electronic structure of CQDs. Another kind of reaction that is becoming
	increasingly attractive for biological purposes is the copper(I) azide-alkyne cycloaddition
	reaction. It relies on the reaction between an azide function which can be grafted onto the
	surface of the NPs by EDC/NHS chemistry and an alkyne group born by the molecule to be
	linked to the CQDs. An example, recently described by us, utilisation of the click reaction for
	the inhibition of viral infection [171]. Carbon dots rich in amino groups prepared from citric
	acid and ethylenediamine were first functionalized with an azide moiety and further reacted
	with the boronic acid molecule, as prepared conjugate show a moderate activity towards Human
	coronavirus-229E (HCoV-229E). A group of Huang [172] reported the synthesis of carboxyl
	rich CQDs and further amidation with propargylamine, which in consequence, was further
	reacted with azido beacon DNA, as prepared conjugate was then bonded to gold nanoparticles
	and applied to the recognition of single-base-mismatched target. The advantage of such a
	functionalization approaches used in biomedicine for CQDs. The majority of CQDs contain
	abundant carboxyl or amino groups on their surface, which allow grafting the molecules via
	condensation reactions either via an amine-or a carboxy-group-containing agent. Condensation
	reactions (mainly peptide-coupling-like reactions) usually use EDC/NHS chemistry due to the
	high solubility of the material in aqueous media. Next to carboxylic and amino functions,
	hydroxyl groups allow modifications by silylation which is the reaction between silane and
	active hydrogen at the surface. The resulting alkoxysilane derivative is a biocompatible and
	nontoxic material which can act as a protective silicon shell, encapsulating the CQDs inside.
	Other classes of widely used covalent modifications are the esterification and sulfonylation
	reactions. The second reaction introduces a sulphur atom close to the surface which may

subjected to an intense oxidative purification process [175]. During the pre- treatment/purification process, the surface of particles covers itself with various functional groups which are further oxidised and eventually lead to hydroxyl, anhydride or carboxylic groups. Such NDs subjected to strong acid oxidation exhibit a significant affinity for proteins, easily

	core. Bombarding them with high-energy
	ions implants defects inside the diamond lattice [174]. These defects are commonly called
	"coloured centres" because the creation of vacancies in the lattice of the material leads to
	fluorescence properties. This makes the corresponding NDs especially suitable for bioimaging.
	Like previously mentioned for CQDs, nanodiamonds can be produced by a variety of
	bottom-up and top-down methods [175]. Bottom-up processes include the formation of
	detonated NDs: there, the carbon lattice is built from a molecular precursor. During this process,
	a mixture of 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitroperhydro-1,3,5-triazine (hexogen) is
	exploded, and nanodroplets of liquid carbon are converted into D-NDs by fast cooling
	(Figure 1.8). Detonated NDs contain metal impurities and sp 2 carbon; therefore, the material is

form stable conjugates under different conditions through adsorption [176].

  160. Havrdova, M., et al., Toxicity of carbon dots -Effect of surface functionalization on the cell viability, reactive oxygen species generation and cell cycle. Carbon, 2016. 99: p. 238-248. 161. Yan, J., et al., The effect of surface charge on the cytotoxicity and uptake of carbon quantum dots in human umbilical cord derived mesenchymal stem cells. Colloids and Surfaces B: Biointerfaces, 2018. 171: p. 241-249. 162. Yang, S.-T., et al., Carbon dots for optical imaging in vivo. Journal of the American Chemical Society, 2009. 131(32): p. 11308-11309. 163. Choi, H.S., et al., Renal clearance of quantum dots. Nature biotechnology, 2007. 25(10): p. 1165-1170. 164. Wang, Q., et al., Hollow luminescent carbon dots for drug delivery. Carbon, 2013. 59: p. 192-199. 165. Ge, J., et al., Red-Emissive Carbon Dots for Fluorescent, Photoacoustic, and Thermal Theranostics in Living Mice. Advanced Materials, 2015. 27(28): p. 4169-4177. 166. Zheng, M., et al., Integrating Oxaliplatin with Highly Luminescent Carbon Dots: An

Unprecedented Theranostic Agent for Personalized Medicine. Advanced Materials, 2014. 26(21): p. 3554-3560. 167. Weng, C.-I., et al., One-step synthesis of biofunctional carbon quantum dots for bacterial labeling. Biosensors and Bioelectronics, 2015. 68: p. 1-6. 168. Chandra, S., et al., One-step synthesis of amikacin modified fluorescent carbon dots for the detection of Gram-negative bacteria like Escherichia coli. RSC Adv., 2016. 6. 169. Zhong, D., et al., Employing carbon dots modified with vancomycin for assaying Gram-positive bacteria like Staphylococcus aureus. Biosensors and Bioelectronics, 2015. 74: p. 546-553. 170. Yan, F., et al., Surface modification and chemical functionalization of carbon dots: a review. Microchimica Acta, 2018. 185(9): p. 424. 171. Loczechin, A., et al., Functional Carbon Quantum Dots as Medical Countermeasures to Human Coronavirus (HCoV). ACS Applied Materials & Interfaces, 2019. 172. Gao, M.X., et al., "Click" on Alkynylated Carbon Quantum Dots: An Efficient Surface Functionalization for Specific Biosensing and Bioimaging. Chemistry -A European Journal, 2017. 23(9): p. 2171-2178. 173. Nekoueian, K., et al., Carbon-based quantum particles: an electroanalytical and biomedical perspective. Chemical Society Reviews, 2019. 48(15): p. 4281-4316. 174. Chipaux, M., et al., Nanodiamonds and Their Applications in Cells. Small, 2018. 14(24): p. 1704263. 175. Turcheniuk, K. and V.N. Mochalin, Biomedical applications of nanodiamond (Review). Nanotechnology, 2017. 28(25): p. 252001. 176. Kong, X.L., et al., High-Affinity Capture of Proteins by Diamond Nanoparticles for Mass Spectrometric Analysis. Analytical Chemistry, 2005. 77(1): p. 259-265. 177. Krüger, A., et al., Unusually tight aggregation in detonation nanodiamond: Identification and disintegration. Carbon, 2005. 43(8): p. 1722-1730. 178. Neburkova, J., J. Vavra, and P. Cigler, Coating nanodiamonds with biocompatible shells for applications in biology and medicine. Current Opinion in Solid State and Materials Science, 2017. 21(1): p. 43-53. 179. Shuai, C., et al., Surface modification of nanodiamond: Toward the dispersion of reinforced phase in poly-l-lactic acid scaffolds. International Journal of Biological Macromolecules, 2019. 126: p. 1116-1124. 180. Wang, P., W. Su, and X. Ding, Control of nanodiamond-doxorubicin drug loading and elution through optimized compositions and release environments. Diamond and Related Materials, 2018. 88: p. 43-50.

Table 2 . 1 :

 21 Summary of HPLC retention times (tR), ESI-MS data and MIC values for the peptides 1a-7b

	Molecular mass	MIC [µg/ml]

Table 2 . 2

 22

		: Physicochemical properties of nanostructures
	Particles  (mV) 1	Size (nm)	Hydrodyna	PDI	C1s	O1s	N1s	Surface
				mic size					loading
				(nm) 2					mmol/g
									Keiser test
	CQDs-1	-9.9 ± 3.4 4.5±0.2	11 ± 0.1	0.22±0.11	72.62 14.94 12.43 0.144
	CQDs-2	-27 ±2.1	5.75±0.78	17± 2	0.357±0.2	68.89 26.87 4.23	0.0445
	CQDs-3	7.14±3	5.5 ± 0.3	12 ± 0.1	0.23 ± 0.11	68.8	13.9	17.3	0.055

Table 2 . 3 :

 23 Physicochemical properties of carbon-conjugates

							CQDs-2		ND-1
	a.u.						CQDs-2.2a	a.u.	ND-1.1a
	/								/
	Transmittance						CQDs-2.1a	Transmittance	ND-1.2a ND-1.3a
							CQDs-2.3a
															1135
						1530									1534	802
															1200
	4000	3500	3000	2500	2000	1500	1000	500	4000	3500	3000	2500	2000	1500	1000	500
			Wavenumber / cm -1						Wavenumber / cm -1
	Particles		 (mV) 1	Hydrodynamic size (nm)		PDI		Loading µg/mg	QY (%)
	CQDs-2		-27 ±2		68±2.1			0.24±0.13		-			7
	CQDs-2.1a		2±0.4		141±2.5			0.21±0.09		134		6.54
	CQDs-2.1b		2±0.5		137±1.7			0.17±0.10		115		6.72
	CQDs-2.2a		-2±0.3		153±2.4			0.14±0.04		98			6.1
	CQDs-2.2b		0.5±0.3		157±3.6			0.51±0.09		103			6.3
	CQDs-2.3a		-2±0.8		149±3.2			0.42±0.02		95		6.41
	CQDs-2.3b		-0.4±0.7		147±4.6			0.33±0.41		97		6.44
		ND-1		-45±2		78±3			0.19±0.02		-			-
	ND-1.1a		-25±2		154±2			0.14±0,02		284			-
	ND-1.1b		-17±3		147±3.5			0.16±0.03		292			-
	ND-1.2a		-19±3		161±4,1			0.17±0.02		266			-
	ND-1.2b		-15±3		167±3			0.17±0.02		268			-
	ND-1.3a		-20±2		159±2			0.18±0.02		275			-
	ND-1.3b		-15±3		160±2.7			0.18±0.02		267			-

1

 : zeta potential, PDI Polydispersity index;

Table 2 . 4 :

 24 Physicochemical properties of carbon conjugates: "clicked" nanoparticles

	Particles	 (mV) 1	Hydrodynamic size (nm)	PDI	Loading µg/mg	QY (%)
	CQDs-3	7±3	71±4	0.24±0.13	-	34
	CQDs-3.4a	21±2	181±2	0.28±0.09	94	10
	CQDs-3.4b	26±1	175±3	0.22±0.08	95	13
	CQDs-3.5a	23±1	179±4	0.19±0.05	101	14
	CQDs-3.5b	25±2	158±2	0.20±0.10	103	11
	CQDs-3.6a	19±2	198±3	0.34±0.09	100	9.8
	CQDs-3.6b	22±2	191±4	0.32±0.13	97	10
	CQDs-3.7a	21±3	193±3	0.31±0.11	99	15
	CQDs-3.7b	24±1	190±3	0.34±0.15	96	14.7
	ND-2	18±3	134±7	0.35±0.02	-	-
	ND-3	24±3.	183±5	0.34±0.10	-	-
	ND-3.4a	34±3	213±4	0.36±0.02	243	-
	ND-3.4b	33±3	234±6	0.36±0.02	239	-
	ND-3.5a	33±3	219±2	0.34±0.04	257	-
	ND-3.5b	29±3	229±3	0.39±0.10	256	-
	ND-3.6a	28±3	261±5	0.41±0.09	261	-
	ND-3.6b	29±3	267±7	0.40±0.07	273	-
	ND-3.7a	31±2	253±6	0.39±0.15	269	-
	ND-3.7b	30±2	249±8	0.4±0.11	258	-

1

: zeta potential, PDI Polydispersity index;

Table 2 .

 2

	6: Antibacterial properties of carbon nanostructures (CQDs-3, ND-3) and carbon nanoconjugates prepared
	by Cu(I) catalysed azide-alkyne cycloaddition (CuAAC) (CQDs-3.4a-3.7b; ND-3.4a-ND-3.7b). MIC value for the
	conjugates: [µg/ml] NP // [µg/ml] of the peptide conjugated on the surface
			MIC [µg/ml]
	Compound	Sequence	Gram-negative E.coli	Gram-positive S.aureus (MRSA)
			DSM 30083	ATCC 43300
	CQDs-3	-	n.a.	n.a.
	4a CQDs-3.4a	CHCCH2CH2C(O)-RWRWRW-NH2	32 64 // 6.02	16 32 // 3.01
	4b CQDs-3.4b	CHCCH2CH2C(O)-WRWRWR-NH2	32 64 // 6.08	16 32 // 3.04
	5a CQDs-3.5a	H-RWRWRWG(CH 2 CCH) -NH 2	32 64 // 6.46	16 32 // 3.23
	5b CQDs-3.5b	H-WRWRWRG(CH2CCH) -NH2	32 64 / /6.59	16 32 // 3.3
	6a CQDs-3.6a	FcCO-WRWRWG(CH2CCH) -NH2	64 512 // 51.2>	32 128 // 12.8
	6b CQDs-3.6b	FcCO-RWRWRG(CH2CCH) -NH2	128 512 // 49.7>	32 128 // 12.4
	7a CQDs-3.7a	RcCO-WRWRWG(CH2CCH) -NH2	128 512 // 50.07>	8 64 // 6.34
	7b CQDs-3.7b	RcCO-RWRWRG(CH2CCH) -NH2	128 512 // 50.07>	32 128 // 12.7
	ND-3	-	n.a.	n.a.
	4a ND-3.4a	CHCCH2CH2C(O)-RWRWRW-NH2	32 32 // 7.78	16 16 // 3.89
	4b ND-3.4b	CHCCH2CH2C(O)-WRWRWR-NH2	32 32 // 7.65	16 16 // 3.82
	5a ND-3.5a	H-RWRWRWG(CH2CCH) -NH2	32 32 // 8.22	16 16 // 4.11
	5b ND-3.5b	H-WRWRWRG(CH2CCH) -NH2	32 32 // 8.19	16 16 // 4.1
	6a ND-3.6a	FcCO-WRWRWG(CH2CCH) -NH2	64 512 // 134>	32 128 // 33.4
	6b ND-3.6b	FcCO-RWRWRG(CH2CCH) -NH2	128 512 // 140>	32 64 // 17.5
	7a ND-3.7a	RcCO-WRWRWG(CH2CCH) -NH2	128 512 // 138>	8 64 // 17.2
	7b ND-3.7b	RcCO-RWRWRG(CH2CCH) -NH2	128 512 // 132>	32 64 // 16.5

Indicated in orange: alkyne group necessary for the click reaction; Indicated in blue: conjugates that showed increased antibacterial activity.

Table 3 . 1 :

 31 Composition, pore size and swelling capacity of cryogels fabricated by photo-crosslinking

	Cryogel No embedded rGO	FuMA/PEGMEMA	Pore size / µm	Swelling capacity
	CG-0	0/100	36.4 ± 9.6	1123 ± 18
	CG-10	10/90	26 ± 10.8	992 ± 30
	CG-20	20/80	23.9 ± 5.1	934 ± 28
	CG-30	30/70	16.2 ± 4.0	808 ± 33
	CG-40	40/60	13.5 ± 2.7	759 ± 38
	Cryogel rGO embedded	FuMA/PEGMEMA	Pore size / µm	Swelling capacity
	CG-rGO-0	0/100	13.1 ± 1.6	468 ± 7
	CG-rGO-10	10/90	10.3 ± 2.9	428 ± 5
	CG-rGO-20	20/80	9.5 ± 2.6	317 ± 5
	CG-rGO-30	30/70	7.7 ± 1.9	303 ± 7
	CG-rGO-40	40/60	6.5 ± 1.6

Table 4 .

 4

	CQDs	 (mV) 1	Size (nm)	Hydrodynamic size (nm) 2	PDI 3	C1s 4 (at. %)	O1s 4 (at. %)	N1s 4 (at. %)	B1s 4 (at. %)
	CQDs-6 -20.0 ± 5.5 7.6 ± 0.2	13 ± 1.8	0.14±0.09	77.4	21.7	-	0.9
	CQDs-7 -41.2 ± 1.0 9.2 ± 0.3	12 ± 0.2	0.11± 0.06	69.4	21.5	7.4	1.7
	CQDs-8 -39.2 ±1.5 8.0 ± 0.2	13 ± 3.1	0.28± 0.34	60.8

2: Physico-chemical characteristics of CQDs-6, -7, and -8.

Intensity / a.u. CQDs-8 CQDs-7 Binding energy / eV C1s CQDs-6

								CQDs-7
									N1s
				CQDs-8
	a.u.							a.u.
	/							/
	Intensity			CQDs-7			Intensity
				CQDs-6
	4000	3500	3000	2500	2000	1500	1000	500
				Wavelength / nm			408	406	404	402	400	398	396
									Binding energy / eV
	294	292	290	288	286	284	282	280

  11 B NMR spectra of CQDs-6 and CQDs-7 display, on the other hand, peaks at 13 ppm (CQDs-6) and a band at 10 ppm with a shoulder at 12 ppm for CQDs-7. This means that, during the hydrothermal reaction, boron was incorporated through doping rather than in the form of a surface function. Indeed, one-pot solvothermal synthesis using aminophenylboronic acid as a precursor was reported by Wang et al. to result in N and B co-doped CQDs.[31] They reported the presence of 0.7 at.% B measured by XPS, which is comparable to the amount obtained in the present work (Table4.2).

Table 4 . 3 :

 43 Summary of the main features of CQDs-1 to -8.

	CQDs	Size (nm)	charge	functions	antiviral EC50 / µg mL -1
	CQDs-1 4.5 ± 0.2	-9.9	NH2, COO -	-
	CQDs-3 5.5 ± 0.3	-7.9	N3	-
	CQDs-4 6.3 ± 0.4	-15.9	triazole, R-B(OH)2	++	52 ± 8
	CQDs-5 6.5 ± 0.4	-15.9	triazole, OH	+	n.d.
	CQDs-6	7.6± 0.2	-20.0	R-B(OH) 2	+++	11.6 ± 1.1
	CQDs-7 9.2 ± 0.3	-41.2	R-B(OH)2, NH2	++++	5.2 ± 0.7
	CQDs-8 8.0 ± 0.3	-39.2	PEG	-
	n.d. not determinable
	4.6.

Table 6 .

 6

		2: Surface loading of different CQDs
	(mmol/g) obtain by modified Keiser method
	Sample	Sample mass (mg)	Absorbance at 570nm	Surface loading (mmol/g)
	CQDs-1 0.1375	0.343	0.144
	CQDs-3	0.056	0.068	0.0445
	CQDs-2	0.099	0.116	0.055

CHAPTER 3

On-demand antimicrobial releasing platform triggered by nearinfrared light

The content below was done in collaboration work with Laura Chambre from Prof. Amitav Sanyal group (Boğaziçi University, Istanbul, Turkey). Cryogels were fabricated by Laura Chambre. The author performed or coordinated most of the experiments including the biological assays.

We decided to investigate the possibility of loading a short synthetic antimicrobial peptide (AMP) onto the cryogel. The AMP chosen is a maleimide-modified cationic peptide containing three arginine units serving as a positive charge donor and three tryptophan serving as a bulky lipophilic part-necessary to obtain the optimum antibacterial activity (Figure 3.7A).

This small peptide targets the bacterial membrane and inhibits cellular respiration and cell wall synthesis [28,29] and was shown to be effective against Gram-positive bacteria including methicillin-resistance S. aureus while being moderately active against Gram-negative bacteria.

MIC values obtained for the peptide and the peptide-maleimide conjugate remain unchanged, indicating that the modification did not alter the antibacterial activity of AMP. : zeta potential; 2 The hydrodynamic size was recorded at 37 °C; 3 PDI Polydispersity index; 4 XPS was used to determine the respective atomic percentages of the indicated elements. All the solvents used for solid-phase peptide synthesis were peptide grade solvents.

The formation of

Ultrapure water (Milli-Q, 18 MWcm) was used for all the experiments. All the chemicals were used as received unless otherwise noted.

Synthesis

General procedure for solid-phase peptide synthesis (SPPS)

Solid-phase peptide synthesis (SPPS) was performed manually using the Fmoc protection strategy on a Rink amid resin. The resin was left to swell for 30 min in dichloromethane (DCM) before use. Removal of Fmoc protection was achieved using 20% (v/v) piperidine in dimethylformamide (DMF), deprotection was performed in duplicate in the course of 5 min. For each coupling step, a mixture of Fmoc-aa-OH (4 eq); TBTU (4 eq); HOBt (4 eq); DIPEA (8 eq) in DMF was used (metallocenes were attached using [Ru(Cp)(C5H4-COOH)] or [Fe(Cp)(C5H4-COOH)] (2 eq); TBTU (2 eq); HOBt (2 eq); DIPEA (4 eq)). Each coupling reaction was carried out 2 times for 60 min. In case of unsuccessful coupling, the reagents were changed to Fmoc-aa-OH (4 eq); DIC (4 eq); HOBt (4 eq). After attaching the last aa, the Fmoc group was removed, the resin was washed and dried under vacuum. Sideprotecting groups were removed along with the cleavage of the peptide from the resin in the course of 3 h with a treatment of:

• TFA:water:TIS (95:2.5:2.5) for peptides 1a, 1b, 3a-5b, 7a, 7b.

• TFA:phenol:TIS (95:2.5:2.5) for ferrocene-containing peptides 2a, 2b, 6a, 6b.

• TFA:TIS:EDT (95:2.5:2.5) for peptide 8.

The cleavage cocktail was filtered, and the crude peptide was precipitated and washed with ice-cold Et2O/hexane (v/v). Peptides were dissolved in a water/acetonitrile mixture and lyophilised to be further purified by RP-HPLC. APPENDIX 104 6.2.1.6.

Synthesis of RcCO-RWRWR-NH2: 3b

The peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure.

MS(ESI

Synthesis of CHCCH2CH2C(O)-RWRWRW-NH2: 4a

The peptide was synthesised by solid-phase peptide synthesis as described in the general SPPS procedure. Intens. Peptide 8 (0.871 µmol, 1 eq) was added to the solution. The reaction mixture was stirred overnight at room temperature. The next day, the solution was precipitated in ice-cold diethyl ether and centrifuged at 13,000 rpm for 5min, resulting in the desired maleimide-modified peptide (yield 73%).

MS (ESI

Ruthenocenecarboxylic acid

Ruthenocenecarboxylic acid was prepared by Friedel-Crafts acylation (1) followed by hydrolysis of the ketone, resulting in the desired carboxylic acid (2). The synthesis was performed according to the literature with minor modifications [1].

CQDs-1: NH2-rich CQDs

Carbon dots were prepared following a method reported previously by Zhu et al. [3] with some modifications. Citric acid (2.1014 g) was dissolved in Milli-Q water (20 mL), and ethylenediamine was added (670 µL). The mixture was transferred into a Teflon-lined autoclave (125 mL acid digestion vessel n° 4748, Parr, France) and heated at 250 °C for 5 h. The resulting product was dialysed against Milli-Q water for 24 h (SpectraPor, pore size 1 kDa). To obtain the powder, carbon dots were lyophilised and stored at 4 °C.

CQDs-2: CQDs-COOH

To introduce carboxyl groups at the surface of CQDs-1, a simple method was used similar to the one reported to Zu et al. [4]. Briefly, 100 mg of CQDs-1 were dispersed in 50 mL of Milli-Q water containing 2.5 g NaOH and 2.5 g ClCH2COONa. The mixture was sonicated for several hours, left shaking overnight and neutralised with 1 N HCl to obtain CQDs-COOH.

The suspension was dialysed against Milli-Q water using a cellulose ester dialysis membrane (Biotech CE N°131093, pore size 500-1000 Da) for 2 days to remove all ions.

CQDs-3: CQDs-N3

Azide-terminated carbon dots were prepared from CQDs-1 using carbodiimide chemistry. To a solution of 2-azidoacetic acid (1 mg in 1 mL, 0.1 X PBS) were added equimolar amounts of EDC•HCl and NHS. The solution was stirred for 20 min in order to activate the carboxyl groups. To this solution, CQDs-1 (1 mg in mL, 0.1X PBS) were added in a 1:2 ratio (v/v). The reaction was carried out for 5 h at room temperature, and the resulting suspension was then dialysed against Milli-Q water using a cellulose ester dialysis membrane for 24 h (Biotech CE N°131093, pore size 500-1000 Da) to remove all unreacted material.

CQDs-4: CQDs-BA

A dispersion of CQDs-3 (1 mg mL -1 , 5 mL) was mixed with previously prepared 4-[(1oxo-4-pentyn-1-yl)amino]phenylboronic acid (2 mM), copper sulphate pentahydrate (200 µM) and ascorbic acid (300 µM). The reaction mixture was stirred for 24 h at room temperature.

absorbance at 214 nm = 0). The supernatants collected from the washing steps were lyophilised, and the amount of the free peptide was quantified by HPLC (calibration curve).

6.2.3.2.3.

Nanodiamonds-peptide conjugates: ND-3.4a; -3.4b; -3.5a; -3.5b; -3.6a; -3.6b; -3.7a; -3.7b

ND-N3 (ND-3) were dispersed in Milli-Q water at the concentration of 2.5 mg in 1 mL

(5 mg). The particles were sonicated for 1 h before the addition of 5 eq of copper sulphate pentahydrate, 10 eq of ascorbic acid and 3 mg (1 eq) of a peptide. The reaction mixture was stirred for 24 h at room temperature, and the particles were purified by several washing/centrifugation cycles at 12,500 rpm with water (until the absorbance at 214 nm = 0) and EDTA (3 times). Supernatants collected from the washing steps in water were lyophilised, and the amount of the free peptide was quantified by HPLC using a calibration curve.

Reduced graphene oxide: rGO

A GO aqueous suspension (150 mg, 3 mg mL -1 ) was prepared by sonication and hydrazine hydrate (50 µL, 1.03 mmol) was added to this suspension. The mixture was heated in an oil bath at 100 °C for 24 h over which reduced GO gradually precipitated out the suspension. The product was isolated by filtration over a PVDF membrane with a 0.45 μm pore size, washed copiously with water (5 × 20 mL) and methanol (5 × 20 mL), and dried in an oven.

Preparation of flexible patch photothermal heaters

Kapton foils (10 × 10 mm 2 ) were cleaned with acetone in an ultrasonic water bath for 30 min, followed by isopropanol for 10 min and then dried under a nitrogen flow. The cleaned Kapton foils were modified with rGO using a drop-casting procedure (100 μL) repeated three times and dried at room temperature for several hours.

Fabrication of furfuryl-containing cryogels

A mixture of all monomers and crosslinkers (FuMA, PEGMEMA, PEGDMA, DMPA) at the appropriate ratios were prepared in 1,4-dioxane. Gelation was undertaken at -13 °C under UV irradiation exposure (365 nm). After 1 h, cryogels were thawed at room temperature, washed with 1,4-dioxane and rinsed with water to remove unreacted monomers. The cryogels were dried under vacuum to yield spongy materials. APPENDIX 121 6.2.6.

Synthesis of rGO embedded furfuryl containing cryogels

rGO was introduced into the reaction mixture, and the synthesis remains the same. Resulting in a black crosslinked material after drying in vacuo 6.3.

Swelling of cryogels

Swelling studies were conducted by sampling a circular piece of a dried hydrogel in a vial containing distilled/deionised water at room temperature. After removing the hydrogel from water and absorbing excess surface adhered water with a tissue paper, the mass increase of the cryogel sample was recorded at periodic time points. The water uptake percentage was obtained using the following equation:

Percentage of swelling (%) = (Wwet -Wdry)/Wwet ×100

where Wwet and Wdry are the weights of the hydrogels in their swollen and dried states, respectively.

Drug loading 6.4.1. Non-covalent interaction with ampicillin

The cryogels (30 mg) were loaded with ampicillin (ampicillin, 1 mL, 800 µg mL -1 ) under shaking for 12 h at 4 °C. The concentration of ampicillin loaded onto the gel was determined using HPLC analysis.

Conjugation of maleimide-functionalized antibacterial peptides

Maleimide-modified peptides (1 µL, 600 µg mL -1 ) were loaded into CG-40 (30 mg) for 24 h at room temperature. Any unbound peptide was washed with water. Washing solution was analysed by UV-Vis to ensure the total removal of the free peptide.

6.5.

High-Performance Liquid Chromatography for quantification 6.5.1.

AMP loading and release

The concentration of AMP loaded onto CG-GrGO-40 was determined by an HPLC system (Shimadzu, Tokyo, Japan) equipped with a 5 μm C4 QS Uptisphere ® 300 Å, 250 mm × as above to determine the amount and % of released AMP.

Quantification of peptides incorporated into the nanostructures of carbon nanoparticles

The concentration of AMP loaded onto CQDs and NDs was determined by HPLC.

Calibration curves were recorded on HPLC system (Shimadzu, Tokyo, Japan) equipped with a 5 μm C18AQ Uptisphere ® column (Interchim, Montluçon, France) heated to 40 °C at a flow rate 1 mL min -1 . As a mobile phase: 100% A (water: 0.1% FA); 100% B (MeCN: 0.1% FA) was used.

Fluorescent plate reader for quantification of dye loading and release

The concentration of N-(5-fluoresceinyl)maleimide loaded onto CG-rGO-40 was determined using a plate reader (BioTek Instruments SAS, France). First, a calibration curve of a series of dye solutions of different concentrations was generated. The concentrations of dye APPENDIX 123 remaining in the supernatant used for loading and washing solutions were measured, allowing the determination of the dye concentration in CG-GrGO. The concentration of an aliquot removed at different time intervals during the photothermal treatment and passive release was measured using the same parameters as above to determine the amount and % of released dye.

6.7.

Purification and purity assessment of peptides: 1a-8

For purification purposes, semi-preparative HPLC was performed on a Knauer instrument. A Macherey Nagel Nucleodur 100-5 C18 reverse phase column was used at a flow rate of 5 mL min -1 . The gradient used was changed as a function of the impurities of the crude product. Chromatograms were recorded at λ = 214 nm and λ = 254 nm.

Purity was assessed by analytical HPLC, performed on Knauer instrument. Macherey Nagel Nucleodur 100-5 Pyramid C18 reverse phase column was used at a flow rate of 1 mL min -1 . With the linear gradient of buffer B (95% in 7min) from 95% buffer A with a total run time of 15min (Table 6.1). Chromatograms were recorded at λ = 214 nm and λ = 254 nm.

Quantification of the amino groups by a modified Keiser test

The primary amino groups onto the surface of CQDs were quantified using a modified photometric assay based on the Kaiser test reported by [START_REF] Mahamuni-Badiger | Biofilm formation to inhibition: Role of zinc oxidebased nanoparticles[END_REF]. CQDs carrying amino groups (0.1-0.5 mg of powder) were dispersed in 1 mL Milli-Q water. To this suspension, 1 mL of the buffer solution (A) was added, followed by 15 min sonication. 1 mL of solution (C) and 1 mL of solution (D) were added, and the dispersion was heated at 120 °C for 10 min; 1 mL of solution (B) was added, and the mixture was heated for another 10 min. After cooling to room temperature, 5 mL of the ethanol solution (E) were added. As a reference sample, benzylamine was used at different concentrations in aqueous solution. The calibration curve is shown in (Figure 6.1).

Fourier Transform Infrared (FTIR) Spectroscopy

FTIR spectra were recorded using a ThermoScientific FTIR instrument (Nicolet 8700) in the 550 -4000 cm -1 range at a spectral resolution of 6 cm -1 . 1 mg of dried sample was mixed with 200 mg of KBr powder in an agate mortar. The mixture was pressed into a pellet under 7 tons of load for 2-4 min, and the spectrum was recorded immediately. A total of 64 accumulative scans were collected. The signal from a pure KBr pellet was subtracted as the background.

6.12.5.

X-ray Photoelectron Spectroscopy (XPS)

XPS measurements were carried out using a ESCALAB 220 XL spectrometer from Vacuum Generators featuring a monochromatic Al Kα X-ray source (1486.6 eV) and a spherical energy analyser operated in the CAE (constant analyser energy) mode (CAE = 100 eV for survey spectra and CAE = 40 eV for high-resolution spectra), using the electromagnetic lens mode. The angle between the incident X-rays and the analyser was 58°, and the detection angle of the photoelectrons was 30°.

6.12.6.

Raman Spectroscopy

Raman spectroscopy measurements were performed on a LabRam HR Micro-Raman system (Horiba Jobin Yvon) using a 473-nm laser diode as an excitation source. Visible light is focused by a 100× objective. The scattered light is collected by the same objective in backscattering configuration, dispersed by an 1800 mm focal length monochromator and detected by a CCD camera.

UV-Vis Spectroscopy

UV-Vis measurements were performed with a Perkin-Elmer Lambda 950 dual-beam spectrophotometer (resolution of 1 nm) in a 1-cm quartz cuvette. Scanning electron microscope (SEM) images were obtained using an ESEM-FEG/EDAX Philips XL-30 (Philips, Eindhoven, The Netherlands) instrument operated at an accelerating voltage of 10 kV.

Emission fluorescence Spectroscopy

Emission fluorescence spectra were recorded between 340 and 800 nm using a Cary Eclipse spectrometer (Agilent, France). Samples were excited at 350 nm, and different wavelengths (excitation and emission slit: 10 nm, scan rate: 600 nm min -1 ).

Particle Size and Zeta Potential Measurements

The average hydrodynamic diameter, the polydispersity index (PDI), and the zetapotential were recorded on a Zetasizer® Nano ZS (Malvern Instruments S.A., Worcestershire, UK). All the batches were diluted to 100 µg mL -1 in water and analysed in triplicate. The size of suspended nanoparticles was measured at 25 °C or 37 °C in the 173° scattering geometry, and zeta potentials were measured using the electrophoretic mode.

Photothermal Effect Measurements

All irradiations were performed in 12 or 6 well plates. The temperature changes were captured by an infrared camera (ThermoVision A40) and treated using ThermaCam Researcher Pro 2.9 software. APPENDIX 132 6.13.