Bright shining single-chain nanoparticles: Advanced applications in photocatalysis and photodynamic therapy

Bright Shining Single-Chain Nanoparticles: Advanced Applications in Photocatalysis and Photodynamic Therapy by Davide Arena Supervised by Prof. José A. Pomposo and Dr. M. Ester Verde Donostia - San Sebastián, 2024 (cc) 2024 Davide Arena (cc by-nc-nd 4.0) Acknowledgements Firstly, I sincerely acknowledge my thesis directors Prof. José A. Pomposo and Dr. M. Ester Verde. With wise guidance and daily support, you both made me grow as a researcher and as a person. I gratefully acknowledge the Material Physics Center and the Polymers, Soft Matter & Sustainable Materials Group for giving me the opportunity to carry out my doctoral studies in a scientifically stimulating environment. I would like to especially acknowledge Prof. Arantxa Arbe and Dr. Amaia Iturrospe for the continue dedication and support with the SAXS characterization. I would like express deep gratitude to Dr. Magali Gary-Bobo, not only for giving me the opportunity to work on an essential part of this work at the Université de Montpellier, but also for the kindest welcoming and continuous support. I would also like to extend my most sincere gratitude to Dr. Christophe Nguyen, your teachings are, in a word, irreplaceable. Thank you to all the Glyco and Nanovectors for Therapeutic Targeting Group, Lamiaa, Laure, Mariana, Nadir, Alain, Denis, Kamel, Marie, Khaled, Melanie, for transforming my stay in such a meaningful and enriching experience. I would also like to gratefully acknowledge Prof. Fabienne Dumoulin, Prof. Zoraida Freixa, Dr. Ane Izaskun Aranburu Leiva and Dr. Iván Rivilla, for their crucially important contributions and support to the work which is now condensed in this thesis. I would like to thank my family and Daniel, for understanding and always being there, believing in me, bringing inextinguishable light to my life. This project would have not been possible without you. Finally, thank to my brother, this thesis is dedicated to you, for the time that, with my absence, I stole you. 1 Summary Keywords: Single-Chain Nanoparticles, Photochemistry, Photocatalysis, Photodynamic Therapy, Functional Polymers. With the work which we present in this thesis, we aimed to establish a link between single-chain nanoparticle (SCNP) technology and photocatalysis, finding novel and advanced applications of next-generation, light-harvesting SCNPs. In particular, we focused on employing the opportunities offered by SCNP unique topology to enable advanced applications of photocatalysis in aqueous and complex environments such as for organic photocatalysis and photodynamic therapy (PDT) of cancer. The present thesis’ structure comprises two first Chapters, in which basic concepts and recent literature on both SCNPs technology and organic photocatalysis are briefly reviewed, opening the discussion for the experimental results disclosed in Chapter III and IV. Specifically, in Chapter I the definition of SCNPs is given and the relevant synthetic aspects for their preparation are illustrated. Namely, the synthesis of the polymeric precursors via reversible-addition fragmentation reaction (RAFT) polymerizations and the main strategies for chain folding / collapse reported in literature are discussed. In conclusion to the first Chapter we delineated the main aims of the present work, and finally outlined some major contributions to the fields of catalysis and nanomedicine involving SCNPs-based systems. 2 In Chapter II, we introduce the definitions, the fundamental concepts of photocatalysis, with a special focus on organic photocatalyzed reactions and the major constraints presented by the use of water as solvent to carry out such kind of valuable transformations. Apart from first, seminal reports at the infancy of Organic Chemistry, in which water was commonly used as reaction medium, the aqueous environment quickly disappeared amongst the common practice of synthetic organic chemists over the course of the past century. Considering the growing demand for increasingly lowimpact processes, both from an economic and ecological point of view, the use of water to replace flammable, often toxic and expensive organic solvents in industrially relevant processes, e.g. the valuable small organic molecules preparation, recently gained back interest amongst the scientific community. For this reason, in Chapter II we put special attention on how supramolecular approaches have been recently applied to address the issues associated with the use of water as solvent for photo-induced organic transformations. In Chapter III, we continue the discussion reporting the preparation of a novel class of versatile SCNP capable of efficiently carry out photocatalytic organic reactions in water. We designed an amphiphilic polymeric precursor of defined molecular weight and dispersity exploiting the amphiphilic polymeric scaffold of the Poly[(olygoethylene glycol)monomethylether methacrylate]-r-Poly(acetoacetoxyethyl methacrylate), Poly(OEGMA)-r-Poly(AEMA), which was prepared by RAFT copolymerization of the commercially available monomers OEGMA and AEMA. The obtained copolymeric precursor was then functionalized and given of photocatalytic activity by decoration with an iridium(III)-based cyclometalated complex though a mild post-polymerization functionalization approach, exploiting the rich β-ketoester chemistry of the hydrophobic comonomer AEMA. The prepared photoactive amphiphile copolymer resulted to efficiently self-assemble in aqueous solution by folding / collapse into a SCNP structure as revealed by dynamic light scattering (DLS) techniques and as confirmed by UV-Visible spectrophotometry. Interestingly, the water soluble, iridium(III)-containing SCNPs, which we called artificial photosynthase (APS) allowed the observation of an enhancement in the photoluminescence (PL) in water with respect of the organic solvent solutions, suggesting the arising of 3 aggregation-induced emission phenomena arising from the locally compact hydrophobic pockets of the SCNP. With the APS in hand, we subsequently tested their ability to perform a variety of organic reactions. Specifically, we were able to observe efficient visible-light induced photocatalytic activity for two unprecedently reported organic reactions in water, namely the photo[2+2]cycloaddition of vinyl arenes and the α-arylation of arylamines, as well as the oxidation of 9-substituted anthracenes and the βsulfonylation of styrene-like compounds. Due to the similarities of these APS to enzymes, kinetics data of the photo[2+2]cycloaddition of vinyl arenes “in water” photocatalyzed by APS were analyzed in terms of the traditional Michaelis-Menten model. The apparent values of kcat and KM obtained were 2.6 s-1 and 4.6 × 10-2 M, respectively, values which are comparatively and significantly close to what reported for some biotic enzymes (Chymotrypsin shows kcat = 0.14 s-1 and KM = 1.5 × 10-2 M, Pepsin kcat = 0.50 s-1 and KM = 3.0 × 10-4 M, and tRNA synthetase kcat = 7.6 s-1 and KM = 9.0 × 10-4 M). In summary, in Chapter III we describe and report a first generation of APS, broadening the possibilities for performing challenging “in water” organic transformations via APS-mediated visible-light photocatalysis. In Chapter IV, we report the design and synthesis of a polymeric precursors to enhance the PDT efficiency of a novel, long-wavelength absorbing zinc(II)- phthalocyanine (ZnPc). For this, we took advantage of the well-known self-assembly capability of anthracene molecules, we prepared anthracene-based amphiphilic copolymers from the commercially available hydrophobic monomer 9anthracenylmethyl methacrylate (AnMA) and the hydrophilic OEGMA via RAFT copolymerization. The prepared Poly(AnMA)-co-Poly(OEGMA)s resulted to be both capable of self-assembly in water and of efficiently encapsulating the far-redresponsive complex ZnPc, yielding stable, water soluble, red-light reactive SCNPs, which we called artificial photo-oxidases (APO), mainly due to their ability to induce oxidative stress in cell upon exposure to light and for their ultra-small dimensions (< 20 nm). The nano-objects were characterized both by DLS and SAXS, which revealed 4 the effect of the ZnPc encapsulation on the steric hindrance of the SCNPs core through the measurement of the radius of gyration in presence and / or absence of ZnPc. Interestingly, the readily prepared nano-assemblies showed different photoluminescent properties in the red region depending on the overall anthracene molar fraction in the polymeric precursor. In particular, the extent of either broadening or quenching of Qband transitions of ZnPc increased upon decreasing the anthracene molar fraction in the nanocarrier. Analogously, a pronounced quenching (λexc = 650 nm) of the ZnPc emission in the far red is observed upon decreasing the anthracene content, though allowing the tunability of the degree of aggregation within the hydrophobic core of the nanocarrier. We finally tested APO-ZnPc against human breast cancer cell MDA-MB-231 lines to assess their PDT efficiency. Having observed outstanding performance for one of the selected formulations, we finally proved their PDT activity in zebrafish embryo xenografts as a more accurate human cancer model. In conclusion, in the present thesis, the development of novel systems based on SCNPs for advanced applications in photocatalyzed organic reactions and photodynamic therapy has been studied and carried out, demonstrating that the technology of single-polymeric chains folding can be exploited for the fabrication of artificial nano-objects with protein-resembling structure of tailored photocatalytic activity. 5 Resumen Palabras clave: Single-Chain Nanoparticles, Fotoquímica, Fotocatálisis, Fototerapia Dinámica, Polímeros Funcionales. Mediante el presente trabajo, se pretende establecer una conexión entre la tecnología de nanopartículas poliméricas unimoleculares (SCNP, del inglés singlechain nanoparticles) y la fotocatálisis, encontrando aplicaciones novedosas y avanzadas para las SCNP de nueva generación capaces de utilizar la luz como fuente de energía. En concreto, se ha tratado de aprovechar las características topológicas de las SCNP para implementar aplicaciones avanzadas de fotocatálisis en ambientes acuosos y complejos, con el objetivo de emplearlas en casos como la fotocatálisis orgánica y la terapia fotodinámica (PDT, del inglés photodynamic therapy) del cáncer. La estructura de la presente tesis consta de dos primeros capítulos, en los que se revisan brevemente los conceptos básicos y la bibliografía reciente tanto de la tecnología de las SCNPs como de la fotocatálisis orgánica, abriendo la discusión para los resultados experimentales expuestos en los capítulos III y IV. Concretamente, en el Capítulo I se da la definición de SCNPs y se ilustran los principales aspectos sintéticos para su preparación. En concreto, se discute la síntesis de los precursores poliméricos mediante polimerizaciones por adición, fragmentación y transferencia reversible (RAFT, del inglés reversible addition-fragmentation transfer) y las principales estrategias para el plegamiento / colapso de cadenas descritas 12 2.2.4. Soft Polymeric Materials for Aqueous Organic Photocatalysis 77 2.3. Conclusions 79 2.4. References 81 3. Chapter III 89 3.1. Introduction 89 3.2. Main Aims 90 3.3. Results and Discussion 91 3.3.1. Preparation of the photoactive polymers 91 3.3.2. Self-assembly in aqueous solution 94 3.3.3. Photocatalytic activity “in water” 96 3.3.4. Effect of APS type on conversion 103 3.3.5. Kinetic aspects 104 3.3.6. Recyclability of APS 108 3.4. Experimental Techniques 110 3.4.1. Solvents and reagents 110 3.4.2. Analytical methods and techniques 111 3.4.3. Synthesis and characterization of compounds 114 3.4.3.1. Synthesis of the polymeric precursor Poly(OEGMA300-coAEMA) (P1) 114 3.4.3.2. Synthesis of the Hydroxo-Bridged Iridium(III) Dimer Tetrakis(2-phenylpyridinato-N,C2)(m-dihydroxy)diiridium(III) ([Ir(ppy)2OH]2) (C1) 115 3.4.3.3. Synthesis of the Cyclometalated Complex Bis[2-(2-pyridinylN)phenyl-C](methyl acetoacetato)iridium(III) (C2) 116 3.4.3.4. Synthesis of Iridium(III)-Decorated Copolymers at Different Iridium(III) Loadings (P1-Ir40, P1-Ir23 and P1-Ir10) 117 3.4.3.5. Preparation of Artificial Photo-Synthases based on Iridium(III)- Decorated Single Chain Nanoparticles in Aqueous Solutions (APS-Ir40, APSIr23, APS-Ir10) 118 3.4.4. “In Water” [2+2] Photocycloaddition of Vinyl Arenes 119 13 3.4.5. “In Water” Oxidation of 9-Substituted Anthracenes 122 3.4.6. “In Water” α-Arylation of Arylamines 123 3.4.7. “In water” β-Hydroxysulfonylation of α-Methyl Styrene 126 3.5. Conclusions 128 3.6. References 129 4. Chapter IV 137 4.1. Introduction 137 4.1.1. Photodynamic Therapy 138 4.1.2. Traditional Photosensitizers for PDT 141 4.1.3. Nanostructures in PDT 143 4.2. Objectives 148 5.3. Results and Discussion 152 4.3.1. Preparation of π-π self-assembled amphiphilic SCNPs containing Zn(II)-phthalocyanine ZnPc 152 4.3.1.1. Preparation of the nanocarriers 152 4.3.1.2. Preparation of the photosensitizer ZnPc 156 4.3.1.3. Encapsulation of the photosensitizer ZnPc 158 4.3.2. In vitro Imaging and PDT with Amphiphilic SCNPs Containing ZnPc Molecules and Anthracene Moieties 161 4.3.3. π-π self-assembled amphiphilic SCNPs containing Zn(II)- phthalocyanine Pc as imaging and far-red photo-killing agents for PDT in Zebrafish embryo xenografts 168 4.4. Experimental Techniques 171 4.4.1. Materials 171 4.4.2. Synthesis of the photosensitizer ZnPc 171 4.4.3. Synthesis of P1 171 4.4.4. Synthesis of P2 172 4.4.5. Synthesis of P3 172 4.4.6. Preparation of APOx-Pcy 173 4.4.7. Cell culture conditions for in vitro experiments 173 14 4.4.8. Cell viability assay 174 4.4.9. In vitro dark cytotoxicity 174 4.4.10. In vitro phototoxicity assay 174 4.4.11. Reactive oxygen species (ROS) production 175 4.4.12. Danio Rerio embryos handling for in Zebrafish experiments 175 4.4.13. Injection, irradiation, and imaging of MDA-MB-231 in Zebrafish embryos 176 4.5. Conclusions 177 4.6. References 178 5. Conclusions 187 Appendix to Chapter III 193 A.III.1. SEC chromatograms 193 A.III.2. DLS data 196 A.III.3. Supplementary UV-Vis spectra 202 A.III.4 NMR spectra 203 Appendix to Chapter IV 236 A.IV.1. preparation of copolymers with higher anthracene content 236 A.IV.1.1. Synthesis of the copolymer P4 236 A.IV.1.2. Synthesis of the copolymer P5 236 A.IV.2. Room-light PDT experiment 237 A.IV.3. SEC chromatograms 238 A.IV.4. NMR spectra 241 A.IV.5. Supplementary spectroscopic data 245 15 List of Abbreviations [Ir(ppy)2OH]2 Dihydroxotetrakis[2-(2-pyridinyl)phenyl]diiridium-(III) dimer °C Celsius degree / degrees µL Microliter / microliters µmol Micromole / micromoles 1Sn Singlets excited states 2-CNIPB 2-chloro-N,N-diisopropylbenzamide Å Angstrom / angstroms AA α-Arylation of arylamines ABA 4-acetoxybenzaldehyde AcOEt Ethyl acetate AEE Aggregation enhanced emission AEMA 4-acetoacetoxyethyl methacrylate AFM Atom-force microscopy AgCl Silver chloride AgOTf Silver(I) trifluoromethansulfonate AIBN Azobisisobutyronitrile APS Artificial photosynthase ATRP Atom-transfer radical polymerization AuNP Gold nanoparticle BHT Butylated hydroxytoluene BODIPY 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes c% Conversion CA [2+2] Cycloaddition of vinyl arenes cal Calory / calories CDCl3 Deuterated chloroform CH2Cl2 Methylene chloride CHCl3 Chloroform cm Centimeter / centimeters 16 COSY Homonuclear correlation spectroscopy CPADB 4-cyano-4-(thiobenzoylthio)pentanoic acid CRP Controlled radical polymerizations CTA Chain-transfer agent CTAB Cetrimmonium bromide d intra-particle photocatalytic unit density Đ Dispersity Da Daltons (u.m.a.) DCB 1,4-dicyanobenzene DEPT Distortionless enhancement by polarization transfer Dh Hydrodynamic diameter DLS Dynamic light scattering DMA Dimethylacetamide DMEM Dulbecco’s Modified Eagle’s Medium DMF N,N-dimethylformammide DMSO Dimethyl sulfoxide DP Degree of polymerization DPBF 1,3-diphenylisobenzofuran DRI Differential refractive index E*ox Excited state’s oxidation potential E*red Excited state’s reduction potential Eox Oxidation potential EPR Enhanced permeability and retention Ered Reduction potential ET Energy transfer Et2O Diethyl ether EtOH Ethanol FRET Förster resonance energy transfer FT-IR Fourier transform infrared GPC Gel permeation chromatography h Hour / hours HPAM N-(2-hydroxypropyl)acrylamide HPLC High pressure liquid chromatography HS α-Styrene β-hydroxysulfonylation HSQC Heteronuclear single quantum coherence spectroscopy IONP Iron oxide nanoparticle IR Infrared Ir(ppy)2(acac) Bis(2-phenylpyridine)-(acetylacetonate)iridium(III) Ir(ppy)2(meacac) bis[2-(2-pyridinyl-N)phenyl-C](methyl acetoacetato)iridium(III) 17 IrCl3 Iridium(III) trichloride Irsppy fac-tris[2-(5’-sulfonatophenyl)pyridine]iridate(III) ISC Inter-system crossing IUPAC International Union of Pure and Applied Chemistry J Joule / joules JNPs Janus nanoparticles K2CO3 Potassium carbonate kcat,app Apparent catalytic constant kDa Kilodalton / kilodaltons KM,app Apparent Michaelis-Menten constant kV Kilovolt / kilovolts L Liter / liters LAM Less activated monomer LED Light-emitting diodes LIr iridium loading M Molarity / molar m Meter / meters MA Methyl acrylate mA Milliampere / milliamperes MALDI Mass analysis laser desorption ionization MALS Multi-angle laser light scattering MAM More activated monomer MDO 2-methylene-1,3-dioxepane MeOH Methanol mg Milligram / milligrams MgSO4 Magnesium sulfate MHz Megahertz min Minute / minutes mL Milliliter / milliliters mm Millimeter / millimeters MMA Methyl methacrylate Mn Number average molecular weight mol Mole / moles MPEG Poly(ethylene glycol ether) methyl ether MTT 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Mw Weighted average molecular weight mW Milliwatt / milliwatts Na2HPO4ꞏnH2O Sodium phosphate dibasic n-hydrate NaCl Sodium chloride NaOAc Sodium acetate 18 NaOH Sodium hydroxide NBD Nitrobenzoxadiole NHSMA N-hydroxysuccinimide ester nm Nanometer / nanometers NMP Nitroxide-mediated polymerization NMR Nuclear magnetic resonance NVC N-vinyl carbazole NVP N-vinyl pyrrolidone OA Oxidation of 9-substituted anthracenes OD Optical density OEGMA (Oligoethylene glycol monomethyl ether) methacrylate PC Photocatalyst PC* Photocatalyst excited state PC-SCNP Photocatalytic single chain nanoparticles PDI Polydispersion index PDMEAMA Poly(dimethylaminoethylmethacrylate) PDT Photodynamic therapy PEG Polyethylene glycol PL Photoluminescence PMAA Poly(methacrylic acid) PN Phenoxazine PNIPAM Poly(N-isopropylacrylamide) ppm Parts per million PS Photosensitizer PVBC Poly(vinylbenzylchloride) PVP Polyvinylpirrolidone r.t. Room temperature RAFT Reversible-addition-fragmentation chain-transfer RDRP Reversible-deactivation radical polymerization Rg Radius of gyration Rg Radius of gyration Rh Hydrodynamic radius ROS Reactive-oxygen species rROP Radical ring opening polymerization SAPC Supramolecular assembly photocatalyst SAXS Small-angle X-ray scattering SCE Saturated calomel electrode SCNP Single-chain nanoparticles SDS Sodium dodecyl sulfate SEC Size exclusion chromatography 19 SET Single-electron transfer SLA Star-like aggregates Sty Styrene TEA Triethylamine THF Tetrahydrofuran TLC Thin layer chromatography TMS Tetramethylsilane TOF Time of flight UPy 2-ureidopyrimidinone UV Ultraviolet V Volt / volts VAc Vinyl acetate WA Wenreib amide Zn(OAc)2 Zinc diacetate λ exc max Maximum excitation wavelenght λmax Maximum wavelength ν Size scaling exponent 20 21 1. Chapter I 1.1. Introduction Despite the nanometer (nm) being an extremely small unit of length – thousand million (10-9) of a meter – the manipulation of matter with dimensions sized from 1 to 100 nanometers is a deeply investigated branch of the technology we now dispose of, namely, the nanotechnology. Covering from medicine, molecular biology, energy storage, chemistry, semiconductor physics, just to cite a few, nanotechnology includes all kinds of scientific research, having possibly gained place at the center of the attention of the scientific community worldwide, in the nanotechnology era it has already been demonstrated how powerfully our daily life can be facilitated by its application. The remarkably fascinating pictures presented in Figure 1.1 gives us a bite of how extensively the nanotechnology is infusing our scientific culture and life. 28 Scheme 1.2. Equilibria between radicals and dormant species in the controlled radical polymerizations. At the present day, several CRP techniques are available, each relying on different chemistries, most famously Atom Transfer Radical Polymerization (ATRP),5 Nitroxide-Mediated Polymerization (NMP)6 and Reversible-Addition Fragmentation Chain-Transfer Polymerization.7 While of extreme importance for synthetic applications, to exhaustively report all the mechanistic aspects of CRPs is far beyond the scope of the present work, for this, in the following discussion only the main features RAFT polymerization will be mentioned, being this latter the more extensively CRP technique employed for the works that constitute this thesis, as it will be disclosed in the next chapters, for the fabrication of next-generation, functional SCNPs. As anticipated above, RAFT polymerization is one of the most versatile and powerful tools for the construction of polymeric materials of complex architecture, defined molecular weights and low dispersity.8 RAFT is a robust technique, showing great versatility to a wide range of monomers and tolerance towards many functional groups. Notably RAFT does not require the use of metals, which is very desirable in terms of overall process economy, purification protocols and end-use in biology and electronic applications, allowing to get closer to milder reaction conditions which are more typical of uncontrolled radical polymerizations.4,9 RAFT polymerization requires the presence of a radical initiator and a chain transfer agent (CTA), commonly a thiocarbonyl-based compound (Figure 1.4). 29 Figure 1.4. Effect of the substituent R and Z of commonly employed CTAs on the polymerization of commercially available monomers. Continuous arrow indicates high to good compatibility, dotted arrow low compatibility and no line scarce to no compatibility.4 See text for monomers complete names (MMA, HPAM, Sty etc.). As mentioned above, the RAFT process offers a wide tolerability towards different polymerizable monomers and, for that to be achieved, CTAs should be chosen in relation to experimental conditions,10 as the nature of the Z and R groups of the RAFT agent (CTA) is fundamental and can be varied accordingly to the type of the selected monomers. In Figure 1.4, a schematized selection guide for CTA-monomer pairing is presented.4 The monomer species that can be used in this type of polymerization are conventionally divided into more activated monomers (MAMs) and less activated monomers (LAMs). The first group includes monomers with vinyl groups conjugated to aromatic rings (as styrene, Sty) double bonds, carbonyl or nitrile groups such as acrylates and methacrylates (e.g. methyl methacrylate, MMA; and methyl acrylate, MA) dienes, or methacrylamides (e.g. N-(2hydroxypropyl)acrylamide HPAM), which can be controlled using dithioesters / 30 dithiobenzoates or trithiocarbonates as CTAs. On the other hand, commonly employed LAMs are vinyl acetates (VAc), vinyl chlorides and 1-alkenes which have double bonds adjacent to atoms of oxygen, nitrogen, halogens or saturated carbons (e.g. Nvinyl pyrrolidone, NVP; N-vinyl carbazole, NVC). As illustrated in Scheme 1.3, in the RAFT reaction mechanism an initiator generates a radical species that is readily added to a monomer M, thus forming a Pn polymer chain. Scheme 1.3. RAFT polymerization mechanism. This is followed by the addition-fragmentation stage in which the propagating chains are added to the CTA, releasing a new radical species R·, capable of initiating a new chain (re-initiation process). Then, in a typically short time with respect to the decomposition of the initiator, an equilibrated exchange is established between dormant species, which are illustrated as S=CS-Pn and contribute to the controlled character of the RAFT process, and active chains. In an analogy to what illustrated in Scheme 2, the intermediate radical, Pn-SĊ(Z)S-Pm, can split in both directions, 31 providing all polymer chains with the same chance to grow. Termination occurs, as with any radical polymerization, through coupling and disproportion mechanisms. Is worth noting that the radical species that are formed remain constant throughout the course of the reaction. This results in a uniform distribution of molecular weights and a Ð close to unity and, for this to occur, the propagation speed should always be maintained much lower than that of the addition-fragmentation stage, ensuring that a single monomeric unit is incorporated, on average, after few activation cycles, thus generating chains of similar polymerization degree. The small percentage of termination reactions, which leads to the formation of dead chains, is related to the number of radicals generated in the initial stage according to Equation 1.1.9 %𝑑𝑒𝑎𝑑 𝑐ℎ𝑎𝑖𝑛 =1 [𝐶𝑇𝐴] 2𝑓[𝐼]0(1 − 𝑒−𝑘𝑡)+ 1 102 Equation 1.1 with [CTA] equal to the concentration of the chain transfer agent in solution, [I]0 equal to the concentration of initiator present in solution at the beginning of the reaction, k kinetic constant of the initiator decomposition rate in the selected experimental conditions and f is the decomposition rate of the initiator. Notably, at the end of the process, each RAFT (non-dead) chain will have a carbonylthioylthio (Z-C(C=S)S) group and an R group at the polymer chain extremities, which is an essential feature for the fabrication of block copolymers and for site-specific post-polymerization modifications.11 Despite the robustness of the RAFT technique, it should be emphasized that thiocarbonyl moieties are subject to undesirable reactions in the presence of nucleophilic species such amine impurities, which must be strictly absent in the reaction environment or, similarly, it is necessary to protect any amino functions if present in the monomers used, as polar chemical reactions as aminolysis of the Z-C(C=S)S group irreversibly terminate the chain polymerization. The ratio between the concentration of CTA and radical initiator [CTA] / [I]0 is therefore an important parameter to consider for controlling the final properties of 32 the synthesized material. It is usually greater than one, so that the number of molecules of the RAFT agent in solution is always maintained greater than the free radical species. While the concentration of free radicals in the system is related to the rate of decomposition of the initiator, the number of chains is, indeed, determined by the amount of CTA.12 An increase in the concentration of RAFT agent leads to the formation of polymers with lower molecular weight and lower Đ. Conversely, decreasing the [CTA]/[I] ratio leads to a higher conversion rate but worse control over the final molecular weight, increasing the likelihood of irreversible termination reactions. For this reason, while optimizing different reaction conditions for RAFT polymerizations, it is commonly a good practice to maintain a low [CTA] / [I] to achieve better control over the final molecular weight distributions of the product. It is worth noting that, to achieve a certain desired molecular weight is possible also by stopping the polymerization reaction at defined reaction times. The degree of polymerization DPth, in fact, is given by the following equation. 𝐷𝑃𝑡ℎ = [𝑀]0− [𝑀]𝑡 [𝐶𝑇𝐴]0+𝑑𝑓([𝐼]0− [𝐼]𝑡) Equation 1.2 where [M]0 and [M]t are the monomer concentrations at the beginning of the reaction and at time t, respectively; [CTA]0 is the initial concentration of chain transfer agent, [I]0 is equal to the concentration of initiator present in solution at the beginning of the reaction, [I]t is equal to the concentration of initiator present in solution at the time t, d is the fraction of chains resulting from coupling reactions, and f is the efficiency of the initiator. As a first approximation, with almost unitary efficiency of the initiator and a low incidence of termination reactions, the equation can be simplified to the following. 𝐷𝑃𝑡ℎ = [𝑀]0− [𝑀]𝑡 [𝐶𝑇𝐴]0 Equation 1.3 33 This control allows the synthesis of virtually any desired polymer architecture: multi-block, star, graft, statistical, alternating, and gradient copolymers, just to cite a few (Figure 1.5).9 Figure 1.5. Examples of different architectures which can be achieved by RAFT copolymerization. 1.3.2. Strategies for SCNP Compaction To facilitate the intramolecular crosslinking, a wide range of functional groups has been explored, utilizing for example photochemistry, metal-complexation or noncovalent interactions, as highlighted by several review articles in which vast collections of techniques for SCNPs folding have been reported.13 Herein, three main strategies available for polymer single-chain compaction will be briefly mentioned, namely (i) covalent cross-linking, (ii) metal-induced cross-linking and (iii) non-covalent crosslinking, by retracing some representative and recent literature examples in the field. (i) Covalent cross-linking. Thanks to the high dissociation energies of the covalent range of chemical bonds, folding a polymer molecule into a single-chain nanoparticle via covalent chemistry is usually a way to ensure durability, morphology stability and compactness 34 of the final product. SCNPs which are obtained by means of covalent chemistries are more stable than their non-covalent counterparts, therefore facilitating their analysis via common chromatography techniques and reducing irreversible aggregation phenomena upon crowding. Starting from the thermal dimerization of benzocyclobutene units of a singlepolymeric chain,14 milder and more controlled methodologies of fabrication of covalent SCNPs have been investigated, as amide15 and urea formation,16 as well as thiolMichael utilizing diacrylate moieties in the lateral polymeric chain as electrophiles for the cross-linking, which was successfully carried out both in organic solvents and in water for different functional copolymers.17-19 1,3-dipolar cycloadditions have also been utilized for the formation of SCNPs, either exploiting two complementary functional monomers within the same polymeric precursor or with the introduction of a crosslinker.20-23 Several strategies have already been reported in this sense,24-27 amongst which the photochemically induced collapse reported by Thanneeru et al. is worth mentioning.28 In their work on tadpole-shaped SCNPs, the authors demonstrated the use of intermolecular photo-crosslinking of amphiphilic polyacrylates copolymethacrylates block copolymers, which were efficiently converted into tadpoleshaped SCNPs of various morphologies thanks to the photo[2+2]cycloaddition of the cinnamoyl moieties present in the lateral chains of the precursors. Interestingly, different morphologies were achieved depending both on the solvent used for the photo cross-linking reaction and the position of the cinnamoyl moieties in the main chain, within the hydrophilic block or in the hydrophobic section (Figure 1.6). 35 Figure 1.6. a) Tadpole-shaped, amphiphilic SCNPs of different morphologies upon switching precursors’ cross-linkable units position, obtained via photo[2+2]cycloaddition of cinnamoyl moieties.28 b) FRET-SCNPs synthesisez by Maag et al. via reaction with the dibromo bimane cross-linker.29 c) Reaction scheme of the reaction of diazaylide bond formation for the preparation of the ultra-stable “Staudinger” SCNPs.30 In an analogous work, Zhang et al. report the folding of a linear polymer via photodimerization of cyanostilbene moieties pendants, favored by self-organization of the polymeric precursor via the non-covalent π-π stacking forces,31 displays how the concomitant use of intramolecular non-covalent interactions and photochemically induced covalent bond formation can lead to reliable SCNPs of defined morphology. 36 The formation of amide and ester bonds for the chain collapse reaction in the fabrication of SCNPs is another reliable strategy which have been recently employed, as demonstrated by Maag et al., the collapsed, highly packed polymeric structure exhibiting Förster resonance energy transfer was achieved by reacting a linear polymeric precursor bearing nitrobenzoxadiole (NBD) units and free carboxylic moieties with the fluorescent reactant dibromobimane (Figure 5).29 In the work published by Jackson et al., stimuli-degradable SCNPs were obtained through folding of a polymeric precursor, which was obtained via radical ringopening polymerization (rROP) of 2-methylene-1,3-dioxepane (MDO) and methacrylic acid N-hydroxysuccinimide ester (NHSMA) and subsequently crosslinked by condensation reaction with a diamino-cross-linker.17 As anticipated, covalently cross-linked SCNPs are especially desirable when stability, either thermal or chemical, is required for a specific application, for this, the preparation of novel systems as the ultra-robust “Staudinger” SCNPs was recently reported, for which the authors took advantage of a pentafluorophenyl-bearing styrenic copolymer, which was azidated as showed in Figure 1.6 and subsequently covalently cross-linked by reaction with a diphosphine cross-linker, to give the stable azaylide bonds, as showed in Figure 1.6.30 (ii) Metal-mediated folding / collapse. Metal-organic compounds present a rich chemistry, which is often present in naturally occurring proteins and exploited by enzymes not only for the ultimate catalytic purposes, but with structural functions as well, the “zinc finger” is a representative phenomenon, being a recurrent structural motif in a plenty of abundant proteins of living organisms.32 Analogously, the formation of metal-organic complexes have been employed for the fabrication of SCNPs, as in the case of the preparation of the heterobimetallic SCNPs reported by Knöfel et al.13 The authors exploited a bifunctional terpolymer precursor, synthesized by NMP, containing two orthogonal ligand moieties, phosphines and phosphine oxide which allowed the facile and selective incorporation of the two metals Pt(II) and Eu(III) respectively, as showed in Figure 1.7. 37 Figure 1.7. a) Orthogonal complexation modalities employed for heterobimetallic metal-induced SCNP collapse.33 b) Sequential and orthogonal metal insertions within the structure of a metal-folded SCNP.34 c) SCNPs embedded with a ferrocene-unit, allowing the subsequent introduction of Pd(II) ions within the nanoparticle.35 In an analogous work, heterobimetallic Au(I) / Y(III)-SCNPs were prepared taking advantage of the orthogonal chemistries of triphenylphosphines and carboxylate anions.34 In this latter work, the introduction of the first metal, Au(I), though complexation with phosphines in the lateral chains of a functionalized polystyrene (Figure 1.7) did not involve the collapse of the polymeric chains, which was instead induced only by introduction of the second metal, Y(III), via complexation with three carboxylate anion pendants of the same polymeric chain. A third example of heterobimetallic SCNPs is provided by the work of Reith et al., in which a method of folding / collapse of the polymeric chains to SCNPs based 44 The tumor targeting capability and the clearance studies of SCNPs have been evaluated in xenograft mouse models for different kinds of human tumors, and zebrafish embryos.60,61 SCNPs have also been recently tested for targeting for specific cells, a crucial challenge in biomedical applications to step forward drug selectivity for various diseases treatments. E.g. Kröger et al. glucose-based SCNPs which, after anchoring on nanodiamonds scaffolds, showed efficient selective macrophages imaging capability in vitro.62 Specific receptor targeting was also utilized by Baij et al. for cell labeling and imaging of human breast cancer (SK-BR-3) cells.63 Another opportunity provided by SCNPs for nanomedicine applications is the encapsulation of active compounds, both for imaging and therapeutic purposes. The encapsulation abilities of SCNPs have been investigated for different systems, both covalently and non-covalently formed SCNPs. Kröger et al. reported a SCNP system that is able to encapsulate both hydrophobic and hydrophilic small drug molecules.64 To conclude, SCNPs have shown promise in in vitro studies and in preliminary in vivo observations. The investigated systems usually maintain good cell viabilities and the facile access to different functional groups allowed the preparation of selective, ultra-small nano-objects which presented good malignant cell targeting behavior. Despite the encouraging results, systematic investigations of biocompatibility, biodistribution and behavior of the SCNPs in their biological applications are required to unravel the full potential of SCNPs for nanomedical purposes. 1.5. Main Aims and Structure of the Thesis The principal objective of this work consisted in building a bridge, both conceptual and empirical, between SCNP-based technology and photocatalysis. In particular, the present thesis focused on employing the opportunities offered by SCNP topology to enable photocatalysis in aqueous environments. In Chapter II, we aimed at introducing the definitions, fundamental concepts and major constraints presented by organic photocatalyzed processes in aqueous solutions, with special attention on how supramolecular approaches have been recently 45 applied to address the issues associated with the use of water as solvent for photoinduced organic transformations. These concepts put the basis for what we subsequently reported in Chapter III, in which we aimed to prepare a novel class of versatile SCNP capable of efficiently carry out photocatalytic organic reactions in water. The solvent selected by Nature for performing biochemical transformation is, indeed, water, a strategy which often relies on the exploitation of the hydrophobic interactions between the nano-catalysts, the enzymes, and substrates. For this, we designed an amphiphilic polymeric precursor of ultra-high molecular weight and low dispersity, which was embedded with photocatalytic activity by decoration with an iridium(III)-based cyclometalated complex though a post-polymerization functionalization approach. The readily prepared photoactive amphiphile, which resulted to efficiently self-assemble in aqueous solution by folding / collapse into a SCNP structure, was subsequently tested for its capability of performing two unprecedently reported organic reactions in water, namely the photo[2+2]cycloaddition of vinyl arenes and the α-arylation of arylamines, as well as the oxidation of 9-substituted anthracenes and the β-sulfonylation of αstyrene. In Chapter IV we decided to further exploit the opportunities provided by SCNP towards photocatalytic applications, proving their suitability as efficient photosensitizer nanocarriers for photodynamic therapy (PDT) applications. For this, we developed a facile protocol for the encapsulation of a synthesized, long-wavelength absorbing zinc(II)-phthalocyanine (ZnPc) within the core of a novel, self-aggregating generation of amphiphilic copolymers. Taking advantage of the well-known selfassembly capability of anthracene molecules, we prepared anthracene-based amphiphilic copolymers both capable of self-assembly in water and of efficiently encapsulating the far infrared-responsive complex ZnPc, yielding stable, water soluble, red-light reactive SCNPs, as confirmed by SAXS characterization. We then tested this long-wavelength reactive amphiphilic SCNPs against human breast cancer cell MDAMB-231 lines to assess their PDT efficiency. 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Dis. 2017, 3, 237–248. 53 60 In presence of a third substance, the photocatalyst (PC), capable to absorb light and to take place in a certain process without being chemically altered, photochemical transformation can occur even between reactants which either tend not to react or do not react at all thermally. In this case, the process is said to be photocatalyzed by PC. From the thermodynamic point of view, a photocatalyzed transformation can occur via two different modalities, which are schematized in Figure 2.3: in the case b, is described a photosensitized process, while the case c is referred to the conversion of light into chemical energy. In a photosensitized process, a thermodynamically allowed, yet slow, reaction between A and B to yield C and D, is sped up by the presence of PC, whose electronic excited state generated upon absorption of light augment the total free energy of the system. In the case of the conversion of light into chemical energy, an initially thermodynamically impossible reaction becomes allowed by the presence of the photoexcited species PC*. Both modes of action are the resultant of the contribution of the electronic excited state of PC to the total free Gibbs energy of the system.15,16 It is worth noting that all photocatalyzed reactions can be grouped into two main classes, according to the mechanism with which the PC enables the catalytic cycle: (i) energy transfer and (ii) photoredox catalysis. In the reaction d is schematized the main process of photosensitized reactions by energy transfer. (d) In energy transfer catalysis, a triplet excited state T1 of a molecule (3PC* for the photocatalyst) is deactivated to a lower energetic state by transferring energy to a second molecule (the substrate A, the energy acceptor), which is thereby promoted to a higher energetic level, usually its triplet excited state 3A*, with this latter, being capable of decaying into products (Figure 2.4). 61 Figure 2.4. Schematic representation (Jablonski diagram) of the main electronic transitions involved in energy transfer photocatalytic processes.17 The fine vibronic structures of the singlets excited states 1Sn with internal relaxation thereof were omitted for pictorial clarity. The reactive triplet state of the acceptor substrate 3A*, the crucial intermediate in the mechanistic pathway to products, is highlighted in yellow. The spectroscopic triplet energies ET and the associated transitions are represented in grey. 2.1.2. Photocatalysis for Organic Reactions. As anticipated in the previous paragraphs, to access common organic substrates’ triplet states through direct excitation would generally require both short (280-315 nm) irradiation wavelengths and high light intensities, which result in unselective absorption and undesired side reactions. Efficient photocatalysts, must then exhibit high extinction coefficients and rapid inter system crossing (ISC), which in conjunction with a long-living triplet state (>100 ns), explains the prevalence of heavyatom Ru/Ir complexes and carbonyl compounds as efficient triplet sensitizers.18 Photocycloadditions, heterolytic bond dissociations, carbon-carbon double bonds photoisomerizations and reactions involving the formation of photosensitized singlet 62 oxygen are the four most commonly used classes of reactions in which the energy transfer mechanism is involved. In Figure 2.5 are shown some exemplificative applications of the abovementioned transformations. Figure 2.5. On the left, four examples of recently reported photocatalyzed reactions via energy transfer mechanism: the heterolytic bond dissociation of carbazides,19 the regioselective photo Diels-Alder cycloadditions of anthracenes to olefins,20 the singlet oxygen cycloaddition to dienes,21 and the isomerization of carbon-carbon double bonds.22 On the right, the structure of commonly used photosensitizers with their respective ET (in parentheses) reported in kcal mol-1.18 Regarding the second mode of action of a photocatalyzed reaction, the photoredox catalysis, the reaction mechanism involves the electron transfer between the excited photocatalyst and substrates, facilitated by the principle discussed above by which the excited states of a photocatalyst are usually both better reductants and better oxidizers.15 If PC’s excited state transfers an electron to one substrate to initiate the catalytic cycle, the mechanistic route is called reductive quenching cycle (Scheme 2.1), which is usually completed by the reduction of the oxidized intermediate of the photocatalyst via electron transfer form a second, electron donating reagent. In some cases, the redox potentials of the species involved in the cycle can undergo the opposite process, an oxidative quenching cycle, in which the photoexcited state of PC abstract an electron from a donor to initiate the catalysis (Scheme 2.1). 63 Scheme 2.1. Possible mechanisms cycles allowed for photoredox catalysis. On the right the photocatalyst PC is reduced by a donor reactant D via single-electron transfer (SET) after being excited upon light absorption, the reduced form of PC is oxidized by an acceptor reactant A to finally yield the regenerated PC. On the left, after being promoted to the reactive excited state upon light absorption, PC* transfers an electron via SET to an accepting reactant A. The ground state photocatalyst is then regenerated by a second SET step in which a donating reactant D is oxidized by the intermediate oxidized PC. In the case of thermally-equilibrated excited states and reversible electron transfer, the redox potentials of the reactive excited state of PC (PC*) can be calculated according to Equations 1 and 2. Eredox(PC+/PC*) = Eredox(PC+/PC) – Eoo(PC*/PC) Eq. (1) Eredox(PC*/PC-) = Eredox(PC/PC-) + Eoo(PC*/PC) Eq. (2) Where Eoo(PC*/PC) is the one-electron potential corresponding to the zerozero spectroscopic energy of the excited state and Eredox(PC/PC-) and Eredox(PC+/PC) the ground state redox potentials measured for PC.23 As a general rule, it is possible to tune 64 the oxidation potentials of the photocatalyst to switch from one mechanism to another, opening new synthetically valuable possibilities.24 Photoredox catalysis, and visible-light photoredox catalysis in particular, is an undoubtedly powerful, yet still flourishing approach to the preparation of organic molecules. First pioneering reports of photoredox catalysis were only published starting form 1979 by Kellogg,25 followed by Fukuzumi and Tanaka,26 Pac and Deronzier,27 all concerning the capability of ruthenium polypyridyl complexes to accelerate a variety of organic reaction, in Figure 2.6 some significative examples are shown. Figure 2.6. Three representative examples of historically relevant photoredox organic reaction. From the top: the light-induced acceleration of the reductive desulfuration of trialkyl sulfonium salts in presence of catalytic amounts of [Ru(bpy)3]Cl2, reported by Kellogg in 1979.25 The light-dependent reductive coupling of benzyl bromide in presence of 1-benzyl-1,4-dihydronicotinamide and catalytic amount of [Ru(bpy)3]Cl2.26 The photooxidation of benzylic alcohols to the corresponding aldehydes, mediated by polypyridyl Ru(II) complexes and using diazonium salts as sacrificial electron donors.27 By the beginning of this century, photoredox catalysis started its blossoming season with the works of the Nobel Laureate MacMillan and Yoon’s groups on the 65 effect of visible light on organic reactions in presence of metal complexes.28 From then on, photoredox catalysis now counts with a plethora of different applications and a variety of reactions, often carried out in milder conditions than their thermal analogues, have been discovered and optimized.15 Due to the possibility to introduce structurally complex moieties in mild conditions, an increasing number of authoritative industries decide to introduce photoredox catalysis-based reactions in the synthesis and late-stage functionalization of highly-valuable molecules.29 Regardless the application, mostly all photochemistry and photocatalysisbased technologies have exponentially grown during the course of the past decade, a rise in development which can be easily correlated to the availability of novel, more stable and powerful light sources. Especially, related to experimental set-ups for visible-light photocatalysis practice, which traditionally relied on the use of either the sunlight or household light bulbs, the commercialization of standardized light emitting diodes (LED) permitted a net gain in terms of reaction repeatability (LEDs sources usually present narrow spectral emission) and overall performance.30 It is also important to take in mind that light is always a proper reactant in photocatalyzed processes, which is stoichiometrically consumed, though taking part neatly in the final yield of a process and making it strictly dependent to the light intensity of the source employed.30,31 The tendency can be visually appreciated in Figure 2.7, in which a graphic of the estimation of the number of publications with time in the fields of visiblelight photocatalysis and photochemistry is reported and superimposed with three of the milestone optoelectronic inventions, which undoubtedly contributed to the revolution of the scientific community in the past decade.32 66 Figure 2.7. Graphic of the tendencies, given by the number of publications containing the expressions visible-light photocatalysis, photochem and photocatalysis per quinquennia, starting from 1910s to 2020s and derived from the digital search portal Google Scholar (14th February 2024). 2.2. Discussion The following discussion will mainly focus on recent developments of softmatter based photocatalytic systems, capable of carrying out organic reactions in water. The use of supramolecular scaffolds is one of the possible strategies explored by recent development in the field of photocatalysis to address, not only the major constraints of scarce solubility of catalysts and products, but which is capable of outperform natural systems in many cases. In supramolecular systems, discrete molecules interact and come together to form assembled entities. This approach, generally inspired by biological reactions which often occur in the confined pockets of enzymes, allow lowering the transition state energies of reactant molecules, which are hosted in the supramolecular assemblies’ nanocavities and are facilitated to react in the microenvironment provided by the confinement.33,34 A variety of supramolecular hosts have been developed with well-defined nanocavities to mimic enzyme activity, scientific literature is plenty of brilliant examples of how supramolecular chemists are 67 capable to employ weak, noncovalent interactions to build defined and discrete molecular architectures for catalysis purposes35-39 and, notably, supramolecular hosts are, in some cases, superior to molecular catalysts in terms of higher yields and better selectivity.40-42 Nevertheless, the application of supramolecular systems for visiblelight photocatalysis of organic transformations in water still is in its infancy and, although some representative examples reported in literature already disclosed the great potential of this approach, questions and unsolved major issues remain abundant. 2.2.1. Water as Solvent in Organic Photocatalysis. One could easily and reasonably affirm that, when thinking about photocatalysis, scientists are someway taking or took inspiration from Nature. Considering the central role that the photocatalytic process plays in the ecological homeostasis of life, the planet earth itself can be considered as a big, almost indefinitely complex photoreactor. In 1772, Joseph Priestley reported the first experiment demonstrating the production of oxygen gas by plants, while five years later Jan IngenHousz documented that the production of oxygen gas in plants leaves was a lightdependent process.43 From then on, countless of scientists started the race towards the comprehension of the phenomenon, which remained covered in mystery up until the advent of cell biochemistry and modern biomolecular techniques. In this scenario, first reports of photocatalyzed reactions in water appeared. Probably aiming to simulate and unveil the processes behind plants’ photosynthesis, Barker reported in 1921 the photoinduced in water synthesis of formaldehyde and carbohydrates from carbon dioxide in presence of uranium salts and colloidal iron oxides.44 Apart from some more seminal reports concerning zinc and titanium oxides photobleaching properties in aerobic environment,45 photocatalysis in aqueous media largely remained an uncharted challenge until the last decade. Nevertheless, Wohler’s synthesis of urea, performed by heating an aqueous solution of ammonium cyanate, was developed by the first mid of the 19th century and is commonly considered the beginning of synthetic organic chemistry. As well as for 68 many other transformations reported in the same period, the use of water as solvent for reaction optimization was not uncommon up until the second decade of the 20th century,46 a tendency which have been surely inverted with the advent of Grignard reactants and the rise of petrochemical industry.28,47 It should be pointed out that replacing hazardous solvents with safer, renewable alternatives is receiving increasing attention both in academia and industries. Almost all organic solvents, except chlorinated solvents, are flammable, chlorinated and aromatic solvents are carcinogenic, ether and chloroform have narcotic properties, high vapor pressures and form smog, just to cite few of their very undesirable characteristics.48 Making a process greener, therefore decreasing the environmental impact and risks associated with handling and residues management, is undoubtedly linked to the use of greener solvents alternatives. The fact that water fulfills all the criteria for being an excellently green solvent is commonly agreed, as it is not only allowing the minimization of health and environmental risks, but also presents unique opportunity for producing polaritytunable reaction media.49,50 In addition, when water is used as solvent, different and unexpected reactivities can be disclosed. Due to this renewed attention to sustainability, it seems logical to merge the intrinsically green features of photocatalysis to the benefits that the use of water as reaction media could provide. As highlighted by recent reviews on the topic, the same can be said for photocatalysis, still little have been reported on purely in water photocatalyzed organic reactions,28,51 as the scarce solubility of the most potent photocatalysts and reactants is of major concern. In Figure 2.8, some significative examples of photocatalytic synthesis of organic molecules in which water is used as the sole solvent medium are schematized. 69 Figure 2.8. Six representative examples of light-induced photocatalytic organic reactions allowed in water: a) Arylation of N-heteroarenes with aryldiazonium salts, first reported in 2014 by Xue et al.52 b) Visible-light-promoted oxidative radical cyclization of NN-biarylglycines, reported by Natarajan et al. in 2019.53 c) In water, visible light-induced acylative epoxidation of vinylarenes, reported by Salles et al. in 2018.54 d) Aromative dehydrogenation of cyclic amines, first reported by Balaraman’s group in 2019.53 e) Visible- 76 2.2.3. Supramolecular Nanocapsules. While exploring the different possibilities offered by supramolecular interactions for photocatalysis in water, it is worth reporting the use of tailor-made soft nanocapsules for the stabilization of hydrophobic photocatalyst in water. This strategy was remarkably employed by Akita et al. by preparing V-shaped aromatic amphiphiles, whose structure is shown in Figure 2.12, which are capable to spontaneously selfassemble in water and generate hydrophobic nanocavities, subsequently exploited to host the phenoxazine photoredox catalyst PN.71 The authors reported that, the resulting supramolecular assembly (SAPC, Figure 2.12), composed of organic PC enclosed in the V-shaped aromatic amphiphile, resulted to efficiently catalyze the metal-free pinacol coupling in water, using blue LED light as the only external energy source. The same concept was successfully applied by the same group, which exploited the same organic V-shaped amphiphile to trap three different photocatalyst PC (Scheme 2.3), enabling a demethoxylative reductive cleavage of N-O bonds in Wenreib amides (WA, Scheme 2.3) in water, mild conditions and good yields on a reasonably large substrate pool (62 to 88% yield).72 Figure 2.12. Pictorial representation of the in water self-assembly of the nanocavities prepared by Akita et al. The V-shaped organic amphiphile spontaneously aggregate in aqueous environment, generating hydrophobic nanocavities in which lipophilic photocatalyst as PC can be hosted.71 77 Scheme 2.3. On the left, structures of the three different organic photoredox catalysts selected by Akita et al. for the incorporation within the supramolecular assembly (SAPC). On the right, reaction scheme of the in water reductive cleavage of N-O bond of WA.72 2.2.4. Soft Polymeric Materials for Aqueous Organic Photocatalysis. As anticipated above, in all these works in the field of photocatalysis of organic transformations in water, we observed the extensive use of supramolecular assembly to simultaneously shield the photocatalytic species from the aqueous media and to provide a confined environment in which the substrates can be hosted and undergo the desired process. While polymeric scaffold has been already widely employed for catalyst supporting, demonstrating their superiority in terms of robustness, catalyst recyclability, product selectivity enhancement and restricted catalyst leaching, still little have been described on their application in photocatalysis. Polyvinylpyrrolidone (PVP)-stabilized colloidal platinum nanoparticles in combination with a water-soluble zinc porphyrin were exploited to promote the visible-light reduction of the substrate pyruvate to lactate, enabling a photoredox-based, mild and green procedure for the preparation of a valuable raw material for the preparation of biodegradable polymeric compounds.73 Palmans et al. reported two seminal works in which water-soluble photoactive SCNPs were proven to be capable of efficiently catalyze the metal-free reduction and aromatic C-C cross-coupling reactions in water and without the use of any cosolvent.74,75 In their reports, the authors introduce a systematic methodology for the preparation of stable, self-assembled photocatalytic SCNPs of defined structure and chemical composition. In their first work, a high-molecular weight, poly(pentafluoro phenyl) acrylate precursor was modified through a post-functionalization approach 78 with four different functionalities including phenothiazine pendants, ensuring both the homogeneity of the molecular weight of the polymeric precursors and to confer the photoredox properties to the material (Figure 2.13). The obtained amphiphilic copolymers resulted to spontaneously self-assemble in water to yield well-defined noncovalent SCNPs, which were exploited to perform the UV-light induced reduction of haloarenes and the cross-coupling of 2-cyanoiodobenzene and N-methyl pyrrole in water and in presence triethylamine (Scheme 2.4).74 Figure 2.13. On top, the formation of non-covalent, photocatalytic SCNP upon spontaneous folding/collapse in water is depicted. The unimolecular polymeric aggregate enables the photocatalytic process to occur in its lipophilic, confined core. On the bottom, the synthetic route explored by Palmans et al. for the preparation of the amphiphilic photoredox SCNP polymeric precursor is shown.74 79 Scheme 2.4. Reaction schemes of the “in water”, light-driven transformation explored by Palmans’ group in their works on photocatalytic SCNPs (PC-SCNPs).74,75 On top, the reduction of C-halogen bonds of aromatic substrates, on the bottom the crosscoupling of N-methyl pyrrolidone and iodoarenes. Barner-Kowollik et al. recently reported a SCNPs-based photoreactor capable of performing photooxidation of fatty acid in water and under visible-light irradiation.76 Notably, through the precise control of the folding/collapse process of the polymeric chains by means of the use of a rose Bengal derivative crosslinker, the authors were able to tune the degree of hydrophobicity of the lipophilic pockets within the core of the single-chain nanoreactor. Interestingly, the striking augment in efficiency (up to three-times) of the photocatalytic SCNP toward the photooxidation of fatty acids in comparison to the free-photosensitizer was interpreted, and supported by MD simulations, in terms of augmented polarity and high confinement in the active pockets provided by the SCNP.76 2.3. Conclusions. To summarize, despite the great interest emerged amongst pure organic chemists towards visible-light photocatalysis in the last decades and considering the quick last century rise of photochemistry, the implementation of photocatalysis and 80 soft-matter based systems for performing valuable and challenging organic reaction still is in its infancy. With this brief overview, is in the author’s hope that the importance, both academically and ecologically speaking, have been stressed out. 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Sci. 2018, 9, 5052-5056. (58) Von Lier, R. C. W.; de Brujin, A. D.; Roelfes, G. A water-soluble iridium photocatalyst for chemical modification of dehydroalanine in peptides and proteins. Chem. Eur. J. 2021, 27, 1430-1437. (59) Nguyen, T.-T. H.; O’Brien, C. J.; Minh, L. N. T.; Olson, S. H.; Settineri, N. S.; Prusiner, S. B.; Paras, N. A.; Conrad, J. Water soluble iridium 92 We then prepared Ir(III)-containing copolymers at three iridium loading (LIr) regimes, P1−Ir10, P1−Ir23, and P1−Ir40 (LIr = 10, 23, and 40 mol % with respect to AEMA units, respectively), by decorating the copolymer P1 through reaction with the C1 under very mild conditions, at room temperature and without the use of any additives (see Experimental Techniques for procedure’s details). Table 3.1. Properties of neat (P1) and functionalized copolymers (P1-Irx) synthesized in this work Sample Mw (kDa)a Đb AEMA (mol%)c LIr (mol%)d P1 169.8 1.06 20 - P1-Ir10 178.6 1.10 18 10 P1-Ir23 174.5 1.13 15.4 23 P1-Ir40 211.7 1.03 12 40 a) Weight-average molecular weight. b) Dispersity. c) Molar content of AEMA units. d) Iridium content. A second cyclometalated complex, bis[2-(2-pyridinyl-N)phenyl-C](methyl acetoacetato)iridium(III) Ir(ppy)2(meacac) C2, was then prepared following a wellknown literature procedure,22 to be used as reference compound both for analytic purposes and catalysis performance comparations, as it will be discussed later. After determining the molar extinction coefficient in chloroform of the complex C2 (Figure 3.1), which was selected as model compound, it was possible to measure by UV-Vis 93 spectrophotometry of polymeric solutions in chloroform ([polymer] = 1 mg mL-1) the quantity of incorporated iridium(III) in the lateral chain of the polymer, by means of its conversion into the desired species shown in Table 1. Figure 3.1. On the left, superimposed absorption spectra of the model compound C2 in chloroform 6.5 10-5 < [C2] < 3.7 10-4 M in chloroform. On the right, superimposed absorption spectra in chloroform of the three copolymers P1-Irx at polymer concentration of 1 mg mL-1, used for incorporated iridium LIr quantification. 94 3.3.2. Self-assembly in aqueous solution Thanks to their amphiphilic nature, high molecular weight (>150 kDa), and finely tuned composition, stable, self-assembled SCNPs23,24 that we denoted as SCNPP1, APS-Ir10, APS-Ir23, and APS-Ir40 were obtained upon simple dissolution of P1, P1−Ir10, P1−Ir23, and P1−Ir40, respectively, in water. Self-assembly was ascertained by measuring via dynamic light scattering (DLS) the difference ΔDh between hydrodynamic diameters Dh of P1, P1−Ir10, P1−Ir23, and P1−Ir40 in tetrahydrofuran, THF, (good solvent for AEMA and OEGMA300) and those of SCNP-P1, APS-Ir10, APS-Ir23, and APS-Ir40 measured in water (selective solvent for OEGMA300). iridiumfunctionalized copolymers all presented a positive ΔDh (Table 3.2) because of the formation of a self-collapsed architecture (see Figure 3.2 and the Appendix: Section III). Table 3.2. Hydrodynamic radii measured in THF (good solvent) and water (selective solvent) Sample DhTHF (nm) Sample Dhwater (nm) P1 9.9 SCNP-P1 8.7 P1-Ir10 12.3 APS-Ir10 10.4 P1-Ir23 11.8 APS-Ir23 9.6 P1-Ir40 10.4 APS-Ir40 9.1 95 Figure 3.2. Illustration of the reduction in hydrodynamic size due to self-assembly of P1-Ir23 (linear architecture) to APS-Ir23 (SCNP architecture) on changing from THF (good solvent) to water (selective solvent). Additional evidence of the formation of a self-assembled conformation in APS-Ir10, APS-Ir23, and APS-Ir40 was obtained through photoluminescence (PL) experiments. As illustrated in Figure 3.3a, we measured the PL of eight solutions of the model compound, bis(2-phenylpyridine)(methyl acetoacetonate)iridium(III) C2 (see the Appendix: Sect III), in THF / water mixtures upon increase of the water content (from 0% to 70%). We observed a decrease in the PL intensity of C2 at high water content, which is consistent with literature data reported for the analogous complex bis(2-phenylpyridine)- (acetylacetonate)iridium(III), Ir(ppy)2(acac).25,26 Conversely, APS-Ir10, APS-Ir23, and APS-Ir40 in water displayed significant PL intensity enhancement with respect to P1−Ir10, P1−Ir23, and P1−Ir40 recorded in two different nonselective solvents (chloroform and THF) (see Figure 3.3b). 96 Figure 3.3. a) PL emission of model compound bis(2-phenylpyridine)(methyl acetoacetonate)iridium(III) C2 in THF / water mixtures (from 0% to 70%) ([Ir(III)] = 3 μM, λexc = 450 nm). b) Illustration of the aggregation-enhanced emission (AEE) of APS-Ir40 in water vs P1–Ir40 in CHCl3 and THF under LED illumination ([Ir(III)] = 3 μM, λexc = 450 nm, λem max = 521 nm). These findings suggest that that confinement of the hydrophobic photocatalyst in the limited space of the self-assembled APS-Ir10, APS-Ir23, and APS-Ir40 induces significant aggregation-enhanced emission (AEE). 3.3.3. Photocatalytic activity “in water” With the self-assembled SCNPs in hand, we tested their suitability as artificial photosynthases (APS) for in water photocatalysis of a variety of organic transformations, which were selected from among the plethora of iridium(III) cyclometalated complex-mediated reactions in organic solvents.27−29 Visible light has 97 been recently applied successfully as an energy source for [2 + 2] cycloadditions in organic solvents;30 herein, we report an unprecedented procedure that employs water as the sole reaction medium. For instance, an APS-Ir40 solution was prepared by dissolving 2 mg of P1−Ir40 in 1 mL of deionized water, which was then charged with 58 μmol of the vinylic compound 1a, and the resulting mixture was left stirring at room temperature and under LED illumination (λmax = 450 nm). After this time, the extracted crude product was analyzed via quantitative 1H NMR (see the Appendix: Sect III) for conversion (c%) determination. Multiplets at 3.54 and 3.98 ppm (see Figure 3.4) indicate the formation of the 1,2-bis-substituted cyclobutane product 2a, which was then isolated in 90% yield as a mixture of cis and trans diastereomers. Figure 3.4. 1H NMR of the crude of “in water” [2+2] photocycloaddition reaction of 1a in the presence of APS-Ir40 (see text). To our delight, we observed similar results when exploring a variety of substrates, as illustrated in Table 3.3. Considering typical redox potentials of vinyl arenes (e.g., Eox = 1.97 V vs SCE, Ered = −2.53 V vs SCE)31,32 and photocatalyst excitedstate potentials (e.g., E*ox = 0.43 V vs SCE and E*red = −2.57 V vs SCE),27 it seems difficult to imagine the activation of olefins 1a−1f by typical single-electron transfer (SET).33 We hypothesized that, helped by the locally hydrophobic packed environment 98 of the APS, these unlike processes could be allowed via an energy transfer (ET) mechanism because of the similar triplet energies of the involved species (e.g., ET = ∼60 kcal mol−1 for vinyl arenes, ET = ∼55 kcal mol−1 for C2).34 Table 3. Photocatalyzed “in water” [2+2] cycloaddition of vinyl arenes (CA reaction)a Entry PC Transformation c%b trans:cisc 1 APS-Ir40 1a → 2a 96(90)d 1:0.3 2 APS-Ir23 1a → 2a 94 1:0.3 3 APS-Ir10 1a → 2a 96 1:0.3 4 APS-Ir40 1b → 2b 97 1:0.2 5 APS-Ir40 1c → 2c 96 1:0.3 6 APS-Ir40 1d → 2d 90 1:0.3 7 APS-Ir40 1e → 2e 60 1:0.3 8 APS-Ir40 1f → 2f 79 1:0.3 9 APS-Ir40 1g → 2g n.p.e - 10 APS-Ir40 1h → 2h n.p e - 11 No PC 1a → 2a n.p.e - 12 C2 1a → 2a 63 1:0.3 aSee Experimetal Techniques for details. bConversion from 1H NMR. cDetermined by 1H NMR. dIsolated yield in parenthesis. eNo product. 99 Byproducts formed in trace amounts, among which we assigned35 the structure of 4-acetoxybenzaldehyde, ABA, to be the major constituent in agreement with the reactivity of electron-rich vinyl arenes with reactive oxygen species that may be generated during irradiation.36 Performance of the same reaction using the model photocatalyst C2 dropped conversion to 63% (see the Appendix to Chapter III) with the remaining components of the crude being the reactant and byproducts. No conversion was observed by employing water-soluble substrates (1g, 1h, Table 3.3), probably because of differences in the ET values. Interestingly, when 9-substituted anthracenes were employed as the substrate, we observed the formation of anthraquinone 4. Since singlet oxygen and other reactive oxygen species (ROS) are generated upon excitation of C2 in organic solvents under aerobic conditions,37 we surmised that APS-Ir10, APS-Ir23, and APS-Ir40 could be efficient photo-catalysts for the “in water” oxidation of 9-substituted anthracenes 3 to anthraquinone 4 via reaction with ROS species38 (see Table 3.4). The formation of 4 that was confirmed by 1H NMR, which is consistent with literature data,39 resulted in being completely neglected when the complex is not confined within the SCNP (see the Appendix to Chapter III). 100 Table 3.4. Photocatalyzed “in water” oxidation of 9-substituted anthracenes (OA reaction)a Entry PC Substrate c%b 1 APS-Ir40 3a 62 (58)c 2 APS-Ir23 3a 21 3 APS-Ir10 3a 19 4 APS-Ir40 3b 59 5 No PC 3a n.p.d 6 C2 3a n.p.d aSee Experimental Techinques for details. bConversion from 1H NMR. cIsolated yield. dNo product. Next, we evaluated the capability of APS-Ir10, APS-Ir23, and APS-Ir40 to catalyze visible-light-induced α-arylation of arylamines that, since its discovery reported by McMillan et al.,40,41 have never been reported in water. Table 3.5, entries 1−3 show the procedure in organic solvents. To enable the photocatalytic cycle to occur in water, we prepared APS-Ir40 by dissolving 2 mg of P1−Ir40 in 1 mL of degassed deionized water. We then charged the APS aqueous solution with 29 μmol of 6 and with a large excess of sodium acetate, NaOAc, (84 equiv.). The resulting mixture was deoxygenated by three consecutive vacuum / argon backfill cycles, finally 87 μmol of 5 were added and left stirring at room temperature and under blue LED (λmax = 450 nm) irradiation for 12 h. Under these conditions, product 7 was obtained by using APS-Ir40 as photocatalyst in 57% conversion and 48% purified yield (Table 3.5, entry 5). 101 Table 3.5. Photocatalyzed “in water” α-arylation of arylamines (AA reaction)a Entry PC mol% PC [NaOAc] Mb Solventb c%c 1 C2 0.5 0.06 DMA >95(95)d 2 P1-Ir40 0.5 0.06 DMA >95 3 C2 0.5 0.06 MeOH 8 4 APS-Ir40 2 0.06 H2O n.p.e 5 APS-Ir40 2 2.4 H2O 57(48)d 6 APS-Ir23 2 2.4 H2O 22 7 APS-Ir10 2 2.4 H2O 21 8 C2 2 2.4 H2O n.p.e 9 APS-Ir40 2 2.4 H2O+ DMA 33 10 APS-Ir40 2 sat.f H2O 24 11 No PC - 2.4 H2O n.p.e aSee Experimental Techinques for details. bNaOAc = sodium acetate; DMA = dimethylacetamide; MeOH = methanol. cConversion of 7 from 1H NMR. dIsolated yield of 7 in parenthesis. eNo product. fSaturated in NaOAc. Conversion was found to decrease upon reduction of the iridium loading in the APS photocatalyst (Table 3.5, entries 6 and 7). We attributed the striking conversion drop upon either lowering or increasing the base concentration (Table 3.5, entries 4 and 10) to the altered acidities of the reactants and additives in aqueous media with respect to the reaction conditions reported in literature for organic solvent.40,42 108 Figure 3.9. Michaelis-Menten plot of the “in water” [2+2] photocycloaddition of 1a catalyzed by APS-Ir40. An apparent catalytic constant kcat,app of 2.6 s-1 was obtained from Equation 3.3: 𝑘𝑐𝑎𝑡,𝑎𝑝𝑝(𝑠−1)=𝑉 𝑚𝑎𝑥 𝑁𝑇 Equation 3.3 where Vmax is the maximum measured velocity of conversion of 1a into the title product 2a and NT is the nanoparticle concentration employed for the catalytic reaction, calculated taking the Mw (SEC) as molecular weight. The value of the apparent Michaelis-Menten constant obtained was KM,app = 4.6 × 10-2 M. 3.3.6. Recyclability of APS To assess the reusability of APSs, we performed a recyclability experiment against the “in water” [2+2] photocycloaddition of vinyl arenes in the best conditions in terms of product yields. After a 1st “in water” [2+2] photocycloaddition reaction of 109 1a with APS-Ir40 as catalyst, the organic products were extracted by three consecutive times with 3 mL of diethyl ether each. The collected organic fractions were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The extracted crudes were then directly diluted with 0.5 mL of CDCl3 for quantitative NMR analysis. The aqueous phase was then collected and centrifuged at 4°C, the pale-yellow sediment was re-dispersed in 1 mL of deionized water by means of continuous stirring at r.t. and in the dark for 1 h. For the 2nd catalytic cycle, the resulting clear, faded-yellow solution was subsequently transferred to a 4 mL reaction vessel, charged with 58 µmol of vinyl arene 1a and left open and stirring at r.t. and under LED illumination (λmax = 450 nm) for 12 hours. After this time, the organic products were extracted by three consecutive times with 3 mL of diethyl ether each. The collected organic fractions were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The extracted crudes were then directly diluted with 0.5 mL of CDCl3 for quantitative NMR analysis. Next 3rd and 4th catalytic cycles were carried out following the same procedure as described for the 2nd one. Figure 3.10 illustrates the results of the recyclability of APS-Ir40 in 4 consecutive cycles. Figure 3.10. Results of the recyclability experiment of APS-Ir40 as catalyst of the “in water” [2+2] photocycloaddition of 1a. 0 20 40 60 80 100 1 2 3 4 2a : 1a (mol/mol) Nº cycle 110 3.4. Experimental Techniques 3.4.1. Solvents and reagents Unless otherwise noted, all reagents and solvents were used as received from vendors. (Oligoethylene glycol monomethyl ether) methacrylate (OEGMA300, average molecular weight = 300 Da) (>99%) and 4-acetoacetoxyethyl methacrylate (AEMA) (>95%) were purchased from TCI Europe N.V. and were filtered over basic alumina before use. n-Hexane (96%), acetone (>99%), methylene chloride (CH2Cl2) (>99%, +0.2 % EtOH), n-pentane (99%), ethyl acetate (AcOEt) (>99.8) and diethyl ether (Et2O) (>99%, +7 ppm BHT) were purchased from Scharlab. Tetrahydrofuran (THF) (>99 %, +0.025% BHT) and methanol (MeOH) (>99%) were purchased from Fisher Scientific, inhibitor-free THF was obtained by filtration over basic alumina. 1,4Dioxane (>99%), methyl acetoacetate (99%), 2-ethoxyethanol (≥99%), sodium hydroxide (NaOH) (≥98%), chloroform (>99%, +100-200 ppm amylene), α-methyl styrene (99%), 2-naphtalenesulfonyl fluoride (95%), sodium acetate (NaOAc) (>99%), anhydrous magnesium sulfate (MgSO4) (>99%), sodium chloride (NaCl) (>99%), silver(I) trifluoromethansulfonate (AgOTf) (≥98%), 4-cyano-4- (thiobenzoylthio)pentanoic acid (CPADB), 1,4-dicyanobenzene (DCB) (98%), triethylamine (TEA) (>99%), linalool (97%) and dimethylacetamide (DMA) (≥99%) were purchased from Sigma-Aldrich. p-Toluenesulfonyl chloride (99%) and ptoluenesulfonyl bromide (98%) were purchased from Sigma-Aldrich and handled under argon atmosphere. 4-Acetoxystyrene (96%), 4-chlorostyrene (99%), 3bromostyrene (97%), 4trifluoromethylstyrene (99%), 4-vinylpyridine (≥95%) and 1vinyl-1,2,4-triazole (≥97%) were purchased from Sigma-Aldrich and used after filtering over basic alumina. Bis(μ-chloro)tetrakis(2phenyl-pyridinato)diiridium(III) (98%), iridium(III) chloride trihydrate (98%), 2-phenylpyridine (>99%), 4carboxystyrene (>99%) and 4-methoxystyrene (>99%) were purchased from BLD Pharma. Azobisisobutyronitrile (AIBN) (98%) was purchased from Fluka and recrystallized from MeOH prior use. Silica gel for column chromatography (0.035-0.07 nm 60 A) was purchased from Acros Organics. Basic alumina (0.063-0.2 mm) was purchased from Merck. N-phenylpyrrolidine (>98%) was purchased from Alpha Aesar. Deuterated chloroform (CDCl3, 99.8% D, + 0.03% tretramethylsilane) for NMR 111 analysis was purchased from Eurisotop. Deionized water was obtained from a Thermoscientific Barnstead TII System. 3.4.2. Analytical methods and techniques Nuclear Magnetic Resonance (NMR) Spectroscopy: 1H and 13C NMR spectra were recorded at room temperature (r.t.) on a Bruker spectrometer operating at 400 MHz, using CDCl3 as solvent. - Hydrophobic monomer content in the copolymer, %AEMA (mol%), was calculated according to Equation 3.4: %𝐴𝐸𝑀𝐴 (𝑚𝑜𝑙%)= 𝑆𝐴𝐸𝑀𝐴 𝑂𝑀𝑒 𝑆𝑂𝐸𝐺𝑀𝐴 𝑂𝑀𝑒 + 𝑆𝐴𝐸𝑀𝐴 𝑂𝑀𝑒 ×100 Equation 3.4 where SOMeAEMA is the integrated area of the signal corresponding to the methoxylic protons of the AEMA units within the copolymer (2.29 ppm) and SOMeOEGMA is the integrated area of the signal corresponding to the methoxylic protons of OEGMA units (3.37 ppm). - NMR conversion of the photo [2+2] cycloaddition reaction, c% (mol%), was calculated according to Equation 3.5: 𝑐% (𝑚𝑜𝑙%)= 𝛴𝑃 𝑆𝑅+ 𝛴𝑃×100 Equation 3.5 where ΣP is the sum of the normalized integrated area signals of the alkylic protons of all the isomers of the product (3.56 ppm and 4.02 ppm) and SR is the normalized area of the signal of the vinylic proton of the reactant (5.74-5.69 ppm). The diastereomeric ratio d.r. was calculated using the integrated arylic proton signals of the product (7.03-7.01 ppm for the trans isomer and 6.86-6.83 ppm for the cis isomer). - NMR conversion of the α-arylation of arylamines, c% (mol%), was calculated according to Equation 3.6 or Equation 3.7 when an internal standard was used: 112 𝑐% (𝑚𝑜𝑙%)= 𝑆𝑃 𝑆𝐷𝐶𝐵 𝐴𝑟 + 𝑆𝑃×100 Equation 3.6 𝑐% (𝑚𝑜𝑙%)= 𝑆𝑃𝑛𝑆𝐼 𝑆𝑆𝐼 + 𝑛0 𝑃×100 Equation 3.7 where SP is the normalized area of the arylic signals of the product (7.60-7.58 ppm), SArDCB is the normalized area of the aromatic signal of the reactant 1,4dicyanobenzene (7.79 ppm), nSI is the internal standard (linalool) number of moles corresponding to the weighted amount in the tube, SSI is the normalized intensity of the signal of the internal standard (5.95-5.89 ppm) and nP0 is the number of moles of the reactant 1,4-dicyanobenzene corresponding to the weighted amount before the reaction. - NMR conversion of the oxidation of 9-substituted anthracenes, c% (mol%), was calculated according to Equation 3.8: 𝑐% (𝑚𝑜𝑙%)= 𝑆𝑃 𝑆𝑅+ 𝑆𝑃×100 Equation 3.8 where SP is the normalized area of the arylic signals of the anthraquinone product (7.81-7.79 ppm), SR is the normalized area of the signal of the reactant (e.g,. signal at 8.04-8.01 ppm for the 9-hydroxymethyl anthracene). - NMR conversion of the β-hydroxysulfonylation reaction, c% (mol%), was calculated according to Equation 3.9: 𝑐% (𝑚𝑜𝑙%)= 𝑆𝑃𝑛𝑆𝐼 𝑆𝑆𝐼 + 𝑛0 𝑃×100 Equation 3.9 where SP is the normalized area of the arylic signals of the product (7.49-7.48 ppm), nSI is the internal standard (1,4-dicyanobenzene) number of moles corresponding to the weighted amount in the tube, SSI is the normalized intensity of the signal of the internal standard (7.79 ppm) and nP0 is the number of moles of the reactant α-methyl styrene corresponding to the amount before the reaction. 113 Size-Exclusion Chromatography (SEC): SEC measurements were performed at 30 °C on an Agilent 1200 system equipped with PLgel 5m Guard and PLgel 5m MIXED-C columns, and triple detection: a differential refractive index (dRI) detector (Optilab Rex, Wyatt), a multi-angle laser light scattering (MALLS) detector (MiniDawn Treos, Wyatt), and a viscosimetric (VIS) detector (ViscoStar-II, Wyatt). Data analysis was performed with ASTRA Software (version 6.1) provided by Wyatt. THF was used as eluent at a flow rate of 1 mL min-1. A value of dn/dc = 0.115 mLg-1 was used for copolymer P1 and derivatives thereof. Dynamic Light Scattering (DLS): DLS measurements were carried out at r.t. on a Malvern Zetasizer Nano ZS apparatus, using high precision quartz cells, light path 10×10 mm, provided by Hellma Analytics. Data are given as by Number as an average of at least four measurements. All the solvents for sample preparation were filtered with 2 µM Teflon filters prior use. UV-Vis Spectroscopy (UV-Vis): UV-Vis spectra were recorded at 25 °C in an Agilent 8453A apparatus with Peltier thermostatic cell holder, T-controller 89090A, using high precision quartz cells, light path 10×10 mm, provided by Hellma Analytics. Photoluminescence (PL) Spectroscopy: PL spectra were recorded at r.t. on an Agilent Cary Eclipse spectrometer at an excitation wavelength of 365 nm, using high precision quartz cells, light path 10×10 mm, provided by Hellma Analytics. Samples were degassed by purging argon gas for 5 consecutive minutes before each measurement. Photocatalytic Reactions, Irradiation Set-Up: Photoreactions were carried out using a Penn PhD Photoreactor, equipped with a 450 nm LED source, purchased from Merck. LED intensity was set at 100%, and a 4 mL vial-holder was used (10 cm light path to the reaction vessel and form the light source). 114 3.4.3. Synthesis and characterization of compounds 3.4.3.1. Synthesis of the Polymeric Precursor Poly(OEGMA300-co-AEMA) (P1) In an oven dried Schlenk flask equipped of a magnetic stir bar, 1.92 g (6.4 mmol) of OEGMA300, 343 mg (1.6 mmol) of AEMA, 10.6 mg (38 µmol) of 4-cyano4-(thiobenzoylthio)pentanoic acid (CPADB), 1.29 mg (7.7 µmol) of AIBN and 3.4 mL of 1,4-dioxane were added in this order. The flask was then sealed with a rubber septum and, after purging the solution with argon flow for 20 min., the reaction mixture was left stirring at 70ºC under argon atmosphere for 19 h. After this time, the reaction was quenched submerging the tube in liquid nitrogen. The crude was then precipitated in a large excess of n-hexane for three consecutive times, yielding the desired polymeric product P1. Mw (kDa) = 169.8, PDI = 1.06, %AEMA (mol%) = 20, 1H NMR (400 MHz, CDCl3): δ (ppm) = 4.33 (m., CH3COCH2CO), 4.14 (m., CH2CO2CH2CH2), 4.07 (m., CH2CO2C), 3.65-3.54 (m., OCH2CH2O), 3.37 (COCH3), 2.29 (s., CH3COCH2), 2.091.17 (m., CH2CCH3), 1.01-0.86 (m., CH2CCH3). 13C NMR (100 MHz, CDCl3): δ (ppm) = 30.42, 44.90, 45.26, 49.75, 59.12, 64.03, 68.58, 70.69, 72.05. 115 3.4.3.2. Synthesis of the Hydroxo-Bridged Iridium(III) Dimer Tetrakis(2phenylpyridinato-N,C2)(m-dihydroxy)diiridium(III) ([Ir(ppy)2OH]2) (C1) The dimeric complex C1 was synthesized according to a procedure reported in literature.21 A mixture of 257 mg (0.73 mmol) of IrCl3 3H2O, 276 mg (1.71 mmol) of 2-phenyl-pyridine in 12 ml of a solution of 2-ethoxy-ethanol/water (3:1) was refluxed under argon for 4.5 h. After this time, an excess of NaOH (1.25 g, 30 mmol) dissolved in 12.5 mL of H2O was added and the resulting mixture was left stirring under reflux for 2 h. After cooling to r.t., 25 mL of H2O was added. An orange-brown precipitate was filtered off, dissolved in 15 mL methylene chloride and, subsequently, filtered. The filtrate was treated with a NaOH solution (1.68 g, 0.04 mol in 4.5 mL H2O) at reflux for 6 h. Afterwards, the organic solvent (CH2Cl2) was evaporated, and H2O (125 mL) was added. The crude product was filtered off and washed with n-pentane (10 mL) and diethyl ether (10 mL). Further purification was carried out by precipitation (CH2Cl2 solution) in n-pentane, yielding [Ir(ppy)2OH]2 (C1) as a brown powder (295 mg, 78%). 1H NMR (400 MHz, CDCl3): δ (ppm) = 9.4-9.23 (m, 1H, Hk), 8.69-8.47 (m, 1H, Hh), 7.9-7.45 (m, 2H, He,i), 6.84-6.51 (m, 3H, Hc,d,j), 6.02-5.83 (m, 1H, Hb), –1.54 (s, 1H, Hl). 116 3.4.3.3. Synthesis of the Cyclometalated Complex Bis[2-(2-pyridinylN)phenyl-C](methyl acetoacetato)iridium(III) (C2) The complex C2 was synthesized as follows and according to a procedure for the preparation of the analogue bis[2-(2-pyridinyl-N)phenylC](acetylacetonato)iridium(III), which is well described in literature.22 Specifically, 117.9 mg (0.11 mmol) of [Ir(ppy)2Cl]2 and 84 mg (0.32 mmol) AgOTf were dissolved, in this order, in 8 mL of degassed acetone and refluxed at 55ºC under nitrogen atmosphere and continuous stirring for 2 h. The solution was cooled to r.t. and filtered to remove AgCl. The filtrate was refluxed under nitrogen atmosphere for 1 h and added under inert atmosphere to a 1 h refluxed solution of methyl acetoacetate (46 µl, 0.43 mmol) and triethylamine (113 µl, 0.81 mmol) dissolved in degassed acetone (4 ml), The resulting bright-brown solution was refluxed overnight under nitrogen atmosphere. The crude was then cooled to r.t. and filtered on cotton to eliminate last residues of AgCl. The volatiles were removed and the solid was sonicated in deionized water and centrifuged for three consecutive times. The obtained solid was then dried under vacuum at r.t. for three days, affording the desired product C2. Yield % (weight %): 85 %. 1H NMR (400 MHz, CDCl3): δ (ppm) = 8.64-8.53 (m, 2H, Hk), 7.90-7.84 (m, 2H, Hh), 7.79-7.73 (m, 2H, Hi), 7.58-7.53 (m, 2H, He), 7.21-7.15 (m, 2H, Hj), 6.86-6.80 (m, 2H, Hd), 6.73-6.86 (m, 2H, Hc), 6.29-6.25 (m, 2H, Hb), 4.72 (s, 1H, Hm), 3.40 (s, 3H, Hl), 1.83 (s, 3H, Hn). 13C NMR (100 MHz, CDCl3): δ (ppm) = 28.73, 46.67, 51.28, 83.19, 99.95, 118.10, 118.39, 119.18, 120.60, 120.69, 121.42, 121.52, 123.49, 123.87, 128.78, 132.92, 133.10, 136.75, 136.96, 148.62, 148.80, 168.73, 186.37. 117 3.4.3.4. Synthesis of Iridium(III)-Decorated Copolymers at Different Iridium(III) Loadings (P1-Ir40, P1-Ir23 and P1-Ir10) 50 mg (0.707 mmol of AEMA) of polymer precursor P1 and 18.6 mg (17.7 µmol) of C1 were dissolved in 2 mL of anhydrous chloroform. The mixture was then degassed for 10 minutes and left stirring at r.t. under inert atmosphere and in the dark for 2 days. After this time, the crude was filtered and the volatiles were removed fluxing nitrogen, until a deep yellowish-brown film was obtained. P1-Ir40: Iridium(Ir)(III) loading, LIr (mol%) = 40, Mw (kDa) = 211.7, PDI = 1.03. P1-Ir23 was obtained following the same procedure reported above, employing 50 mg of polymer precursor P1 and 9.3 mg (8.8 µmol) of C1. P1-Ir23: LIr (mol%) = 23, Mw (kDa) = 174.5, PDI = 1.13. P1-Ir10 was obtained following the same procedure reported above, employing 50 mg of polymer precursor P1 and 4 mg (3.8 µmol) of C1. LIr (mol%) = 10, Mw (kDa) = 178.6, PDI = 1.10. 1H NMR (400 MHz, CDCl3) (P1-Ir40): δ (ppm) = 8.55-8.50 (m, 1 H), 7.847.75 (m, 1 H), 7.50 (m, 2H), 7.20 (m, 1 H), 6.77 (m, 1 H), 6.63 (m, 1 H), 6.19 (m, 1 H), 4.73 (m, 1 H), 4.31 (m., CH3COCH2CO), 4.13 (m., CH2CO2CH2CH2), 4.05 (m., CH2CO2C), 3.62-3.51 (m., OCH2CH2O), 3.34 (COCH3), 2.27 (s., CH3COCH2), 2.091.78 (m., CH2CCH3), 0.99-0.85 (m., CH2CCH3). 13C NMR (100 MHz, CDCl3): δ (ppm) = 30.79, 45.46, 45.76, 50.30, 59.69, 64.60, 69.14, 71.25, 72.60. 124 NaOAc respectively. AA-9 was carried out following the general procedure, using 1 mL of binary solution water/DMA (DMA 10%) as solvent. α-Arylation of N-Arylamines in Water. Non-supported catalyst procedure (AA8). To a 4 mL oven dried vial, equipped of a magnetic stir bar, was added 0.36 mg (0.58 µmol) of C2, 3.7 mg (29 µmol) of 1,4-dicyanobenzene and 203 mg (2.47 mmol) of sodium acetate. The vial was then sealed with a rubber septum. After three consecutive vacuum-evacuation/Argon backfill cycles, 1 mL of extensively degassed deionized water and 12.5 µL (87 µmol) of N-phenyl pyrrolidine were added under Argon positive pressure. The resulting mixture was left stirring at r.t. and under LED illumination (λmax = 450 nm) for 12 hours. After this time, the mixture was extracted three times with 6 mL of ethyl acetate each. The collected organic fractions were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The extracted crude was then diluted with 6.5 mL of CDCl3 for quantitative NMR analysis. No conversion to the desired product was observed. α-Arylation of N-Arylamines in Water. Procedure in organic solvents (AA-1, AA-2, AA-3). An oven-dried 15 mL vial equipped with a rubber septum and a magnetic stir bar was charged with 0.5% mol of photocatalyst (3.08 mg of C2 for AA-1 and AA3, 50 µL of 20 mgmL-1 P1-Ir40 stock solution in methylene chloride for AA-2), 128.1 mg (1 mmol) of 1,4-dicyanobenzene and 164,1 mg (2 mmol) of NaOAc. The Schlenk was then purged with 3 consecutive vacuum evacuation/argon backfill. Then, 4 mL of degassed solvent (DMA for AA-1, AA-2, MeOH for AA-3) was added under positive nitrogen pressure followed by 434 µL (3 mmol) of N-phenylpirrolidine. The reaction mixture was furtherly degassed via three cycles of vacuum evacuation/argon backfill. After degassing, the vial was sealed with parafilm and left under LED illumination (λmax = 450 nm) for 12 hours. After this time, the reaction was then diluted with ethyl acetate and added to a separatory funnel containing 25 mL of a saturated Na2CO3 aqueous solution. The layers were separated, and the aqueous layer was extracted with EtOAc. The collected organic fractions were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The extracted crude was then 125 purified via silica gel column chromatography to afford the title compound 7 (nhexane/ethyl acetate 10:1 to 2:1). 126 3.4.7. “In water” β-Hydroxysulfonylation of α-Methyl Styrene General Procedure 200 µL of a stock solution of polymeric carrier P1-Ir23 in methylene chloride ([polymer] = 20 mg/mL) and 9 mg (35 µmol) of tosyl chloride were put in an oven dried 4 mL vial, previously equipped with a magnetic stir bar. After complete removal of the volatiles, the vial was sealed with a rubber septum and the resulting polymeric film was degassed via three consecutive vacuum-pump/Argon-backfill cycles. 1 mL of extensively degassed deionized water and 3.4 mg (29 µmol) of α-methyl styrene were then added under Argon positive pressure. The resulting solution was left stirring at r.t. and under LED light (λmax = 450 nm) irradiation for 12 hours. After this time, the crude was extracted three times with 6 mL of ethyl acetate each. The collected organic fractions were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. After solvent removal, the crude product was either purified by column chromatography on silica gel (n-hexane/ethyl acetate 4:1) to afford the title compound 10a or added of a known amount of 1,4-dicyanobenzene and diluted with 0.6 mL of CDCl3 for quantitative 1HNMR analysis. Isolated yield (weight %) = 58 %. Conversion NMR (mol%) = 67 %. 1H NMR (400 MHz, CDCl3): δ (ppm) = 7.49 (d, 2H), 7.29 – 7.27 (m, 2H), 7.18 (td, 5H), 4.63 (br.s., 1H), 3.71 – 3.57 (m, 2H), 2.39 (s, 3H), 1.71 (s, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) = 21.73, 29.85, 60.54, 62.31, 121.88, 126.38, 128.08, 128.51, 128.84, 129.65, 136.76, 142.43. HS-1 was carried out according to the general procedure, using 100 µL of P1Ir40 stock solution in methylene chloride ([polymer] = 20 mg/mL). HS-3 was carried 127 out according to the general procedure, using 400 µL of P1-Ir10 stock solution in methylene chloride ([polymer] = 20 mg/mL). HS-4 was carried out following the general procedure using 8.2 mg (35 µmol) of paratoluensulfonyl bromide in substitution of the tosyl chloride. HS-5 was carried out following the general procedure using 7.4 mg (35 µmol) of 2-naphtalenesulfonyl fluoride in substitution of tosyl chloride. β-Hydroxysulfonylation of Aromatic Alkenes. Non-supported catalyst procedure (HS-6). To a 4 mL oven dried vial, equipped of a magnetic stir bar, was added 0.36 mg (0.58 µmol) of C2, 9 mg (35 µmol) of tosyl chloride were put in an oven dried 4 mL vial, previously equipped with a magnetic stir bar. After complete removal of the volatiles, the vial was sealed with a rubber septum and the resulting polymeric film was degassed via three consecutive vacuum-pump/Argon-backfill cycles. 1 mL of extensively degassed deionized water and 3.4 mg (29 µmol) of α-methyl styrene were then added under Argon positive pressure. The resulting solution was left stirring at r.t. and under LED light (λmax = 450 nm) irradiation for 12 hours. After this time, the crude was extracted three times with 6 mL of ethyl acetate each. The collected organic fractions were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. After solvent removal, the crude product was added of a known amount of 1,4-dicyanobenzene and diluted with 0.6 mL of CDCl3 for quantitative 1HNMR analysis. HS-7 was carried out following the general procedure and without adding any photocatalyst. No conversion to the desired product was observed. 128 3.5. Conclusions In summary, artificial photosynthases (APS) arise by endowing enzymemimetic single-chain nanoparticles, SCNPs, with broad visible-light photocatalytic activity for challenging “in water” organic reactions. We introduce a first generation of APS resulting from the decoration of an amphiphilic high-molecular-weight copolymer, poly- (OEGMA300-r-AEMA), with iridium(III) cyclometalated complex pendants followed by its own self-assembly in water. APS enabled efficient visiblelight photocatalysis of a variety of organic transformations in an aqueous solution at room temperature and under LED illumination (λmax = 450 nm). This work broadens the possibilities for performing challenging “in water” organic transformations via APS-mediated visible-light photocatalysis. 129 3.6. References (1) Emmanuel, M. A.; Bender, S. G.; Bilodeau, C.; Carceller, J. M.; DeHovitz, J. S.; Fu H.; Liu, Y.; Nicholls B. T.; Ouyang, Y.; Page, C. G.; Qiao, T.; Raps, F. C.; Sorigué, D. 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Milestones in the development of phototherapy and photodynamic therapy over the past century.32-35 The experiment reported by Meyer-Betz is worth mentioning, being far more than a scientific curiosity, he applied on his own skin 200 mg of hematoporphyrin and documented the extended inflammation response upon exposure to sunlight (in Figure 4.1, the picture of Friedrich Meyer-Betz captured after having a walk) is the first document describing the effects of a porphyrin system in humans: porphyrins are now the more extensively studied photosensitizers for PDT applications. These seminal and fundamental observations paved the way to what is now a consolidated field of research and, in a non-irrelevant number of cases, an approved medical treatment for a variety of malignant solid tumors. Phototherapy is the use of light in the treatment of a disease, while photodynamic therapy (PDT), which can be considered as a specific photochemotherapy, involves a combination of the administration of a photosensitizer and the use of light to treat a certain pathological condition. More specifically, PDT can be defined as: a therapeutic modality, which involves two individually non-toxic component (a photosensitizer and light) that are combined to induce cellular and tissue effect in an oxygen-dependent manner.34 PDT involved the administration, usually either oral, or topic, or intravenous, of a photosensitizer (PS) molecule, which is 141 biodistributed according to the biology of the organism under study and can, or not, selectively accumulate in organs of interest for the treatment. After the PS being absorbed, the application of light induces the promotion of PS ground state to its excited states and, as for every photocatalytic process, longer-living excited states (usually the triplets) induces oxidative stress within the cellule in which the PS have been internalized. This latter can be the resultant of the direct reaction of PS excited states with a substrate important for cell survival (type-I reactions), generating reactive radicals which can produce oxygenated products upon reaction with cellular oxygen. Alternatively, PS triplet state can directly react with cellular oxygen, forming singlet oxygen and reactive oxygen species (ROS), which are then capable to induce high oxidative stress in the cell, finally degenerating into cell apoptosis (type-II reactions). In Figure 4.2, the substantial different mechanistic pathways with which PDT can actuate in the cellular medium are illustrated. The proportion of these two types of reaction taking place relies on the properties of the specific PS used, in all cases, PDT is an oxygen-dependent process whose fundamental principles rely on photocatalysis in the biological, still aqueous, environment. Figure 4.2. Pictorial representation of the basic principles of PDT.34, 36, 37 4.1.2. Traditional Photosensitizers for PDT Ideal photosensitizers for PDT should possess a good lipophilic balance, to simultaneously ensure efficient biodistribution and accumulation in the tumoral tissues, 142 as well as high light absorption coefficients, preferably at as-long-as-possible wavelengths, high ISC efficiencies, negligible dark cytotoxicity and low photobleaching. At the date, different PS have been deeply investigated and widely used for therapeutic applications, and can be classified in three subgroups, or generations. Porphyrins like hematoporphyrin, whose chemical structure is shown in Figure 4.3, are first-generation photosensitizers and are traditionally natural occurring substance with high quantum yields to produce ROS.38 Figure 4.3. Structures of commonly employed molecular architectures for first and second-generation photosensitizer for PDT with their respective wavelength of activation. a) hematoporphyrin, b) protoporphyrin IX, c) chlorin e6, d) iodine-substituted BODIPY, e) Lutexaphyrin,39 f) modified aza-BODIPY, g) Ru[(dmb)2(IP-3T)]Cl2 (TLD1433).40 Hematoporphyrins and their analogues have been extensively reported as effective PDT agents since the very first reports on the field, however, with their 143 extended polycyclic aromatic structure low solubility in water usually is of central concern.40 Second-generation PS are commonly substances designed to overcome these major issues, as for chlorin e6 and protoporphyrin IX and Lu-texaphyrin (shown in Figure 4.3), the basic structure of first-generation photosensitizers were modified to improve critical features like solubility in water, wavelength of absorbance and enhance ROS generation quantum efficiency. Completely novel molecular structures as variously functionalized 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPYs) as well as water-soluble cyclometalated transition-metal complexes (Figure 4.3) can be classified as second-generation catalyst. In particular, BODIPY appeared to be an interesting molecular structure for their low photobleaching degrees, higher stability in biological environment, low dark-toxicity and high light absorption coefficients, however, due to the high luminescence they usually present low singlet oxygen generation quantum yields. Regarding cyclometalated transition-metal complexes, they commonly present ROS efficiency of comparable entity than that of phthalocyanine and porphyritic systems and, helped by the rich chemistry of their electronic excited states, usually manifest high phototoxcitities.40,41 For these reasons, they have been of particular interest as systems that can enable PDT in hypoxic condition and at low oxygen tension tissues, as in the case of TLD1433, the ruthenium(II) polypyridyl complex Ru[(dmb)2(IP-3T)]Cl2 (Figure 4.3) being the first organometallic to reach human clinical trials, which exploits long-living triplet states for either singlet-oxygen sensitization and to initiate intracellular cytotoxic radical pathways.40 4.1.3. Nanostructures in PDT As a general aspect, the induction of effective and selective destruction of damaged and cancerous cells while forbearing the surrounding healthy tissues is of pivotal importance for PDT. Despite the promising results and progress registered over the last century, PDT is currently an approved clinical solution only for very specific cases of tumors, which generally must be superficial and flat, or accessible with endoscopes to ensure good illumination of the affected region. PDT is not applicable to highly diffused, metastatic tumors, as is not possible to illuminate the entire body of 144 a patient with the technology we currently dispose of. The same can be said for solid, deep-seated tumors, in which light penetration, and so PSs activation, is of major constraint.42 Selectivity for cancer tissue is usually achieved by taking advantage of the abnormal physiology of this latter, which commonly present either higher vascularization, poor lymphatic drainage or decreased pH, all features which can be exploited for designing smarter PSs capable of selective accumulation within the tumor. In this sense, the use of nanoparticles for PDT efficiency improvement is a particularly promising approach for several reasons. An important benefit arising from the use of a nanostructure for PSs delivery for PDT is given by the possibility of inducing enhanced permeability and retention (EPR) effect. Altered vascularization of tumoral tissue normally results in abnormal internalization of circulating species in the blood systems of the affected organisms and, when the size of the nanostructures employed for the drug delivery is appropriately selected, this abnormal physiology can lead to selective accumulation of the bigger objects within the tumor (retention).43 This way, the use of nano-sized objects has been exploited to move a step forward a more selective PDT, permitting a more site-specific accumulation of the photoactive species, furtherly improving the cellular uptake, biodistribution, pharmacokinetics and minimizing the unwanted side effects arising from scarcely selective accumulation.44 Other benefits of using nanoparticles in PDT include, large surface-to-volume ratios, which always corresponds to enhanced efficacy of the delivered species in the target cells, the premature delivery of the PSs is also prevented, with this being especially true when a non-biodegradable, inert nanoparticle is used as a passive carrier and, not less importantly, nanoparticles can be prepared in a variety of fashions, topologies, dimensions and with the possibility of almost inexhaustible ways of chemical functionalization for improving biodistribution and PSs performance.42-45 Concerning inorganic-based systems, gold nanoparticles (AuNPs) have demonstrated to be employable as effective passive PSs nanocarriers for PDT-applications. Apart from being usually stable, hard systems which present optimal biocompatibility, with this explaining its wide use in nanomedicine more in general, AuNPs can be easily functionalized either covalently or non-covalently via the easily accessible metallic gold-thiolate chemistry. In Figure 4.4 are shown two examples of the different 145 approaches which can be followed for incorporating a PS into AuNPs, which can be either covalent, as reported by Russel et al. in the functionalization of PEGylated AuNPs with zinc(II)-phthalocyanine containing thiolate moieties,24 or non-covalently, exploiting strong ionic interactions between opposite charged NPs and PS, as reported by Pérez-García et al. in the preparation of positively charged, water-soluble AuNPs loaded with anionic porphyrins.46 Figure 4.4. Illustration of two exemplificative strategies towards PDT-active AuNPs. On top, covalent functionalization of the surface of metallic gold nanoparticles with hydrophilic PEG-based pendants and thiolate-bearing zinc(III)phthalocyanine.47 On the bottom, the water-soluble photoactive AuNPs system assembled via coulombic forces between pyridinium species and anionic porphyrins.46 Analogously, nanostructured silica also offers a plethora of possibilities in terms of its application in nanomedicine. Its derivative nanostructures are well-known for their biocompatibility and the possibility of subsequent functionalization, as well for the usually high colloidal stability of the final product. Silica-based nanoparticles 146 have already been applied for the fabrication of PDT-active materials, as PSs can be incorporated into mesoporous silica nanoparticles, or used for chemical modification of silica nano-objects and, notably, silica can be used to coat pre-existing nanoparticles, thanks to the possibility of layer-by-layer deposition.48,49 Amongst the different opportunities provided by inorganic-based materials, iron oxide nanoparticles (IONPs) are worth mentioning, due to the possibility offered by the application of local magnetic fields, either static for directing to the desired site of action or alternating for inducing magnetic hyperthermia, IONPs gained special attention in nanomedicine in recent times50,51 and, for what concerns PDT, they have already been demonstrated to expand the possibilities of multi-directed approaches (coupling PDT with magnetic induced hyperthermia) when included with PSs in liposomes and cell-mimicking structures.52 Nanoparticles can often guarantee augmented PSs solubilization in the complex, aqueous medium of the biological systems. E.g., the use of polymeric micelles to carry strongly hydrophobic PSs by means of their non-covalent encapsulation within the amphiphilic nanoparticles and / or covalent anchoring into the hydrophobic core have been demonstrated in several applications, including Pluronic, PEG-based lipids, pH-responsive Poly(N-isopropyl acrylamide) (PNIPAM)-based micelles and poly-ion complex micelles, to cite a few.53 As a general behavior, the micellar core is responsible for the drug-carrying capability of these polymeric nanovectors, since a variety of hydrophobic drugs can be incorporated into the core by noncovalent interactions. For this reason, PS are usually incorporated into polymeric micelles through hydrophobic interactions with the segment that forms the micellar core, by means of physical entrapment. PS can also be carried through covalent bonds between functional groups of the PS and pendent group of hydrophobic segments of the polymeric scaffold used for the supramolecular assembly formation. It is worth mentioning that the stability of the physical trapping of the PS depends on the magnitude of the hydrophobic interactions between the PS and the hydrophobic part of the copolymer.54 Amongst all the possibilities offered by the construction of nano-objects of controlled structure and shape, single-chain polymeric nanoparticles (polymeric 147 SCNPs) provide an interesting scaffold for medicinal chemistry. Thanks to their defined shape, tunable morphology resembling the one of enzymes and naturally occurring proteins, as well as the ease of functionalization, good water-solubility, and ultra-small dimensions (2-20 nm), SCNPs already have been proved to be efficient nanocarriers for drug-delivery applications and imaging. Due to the by-definition unimolecular character, SCNPs are usually more dilution-resistant than micelles and liposomes, whose major constraint is indeed coalescence in the bloodstream often leading to PS aggregation, which is usually detrimental to the pharmacokinetics and PSs efficacy over time. Also due to their ultra-reduced dimensions, SCNPs are usually highly compatible with the intracellular space.55,56 In addition, the collapse/folding of individual polymeric chains allows the formation of individual pockets in which the photoactive units can be isolated from themselves, which is generally associated with the enhancement of their optical properties, hence resulting in augmentation of ROS generation in the biological media. In a seminal, recent work reported by Meijer et al. the use of a novel class of porphyrin-containing, water-soluble SCNPs is proposed as innovative scaffold for aqueous photosensitizations.57 The authors developed the photoactive single-chain nanocarrier by means of a post-polymerization functionalization of an ultra-high molecular weight polymeric precursor Poly(pentafluoro styrene), while let reacting specific amount of the porphyrin-based photosensitizer and Jeffamine, conferring water-solubility to the final product. The readily prepared photoactive polymer resulted to self-assemble in aqueous environment into the collapsed/folded unimolecular structure were then evaluated for their feasibility in aqueous photosensitization by means of a spectrophotometric investigation, both in the UV-Vis range for the determination porphyrin aggregationextent and infra-red (IR) emission spectroscopy for singlet-oxygen generation ability estimation.57 In a later work, Liu et al. developed analogous SCPNs-based systems bearing photocatalytic porphyrins, which were successfully introduced into the lysosomal region of HeLa cells via endocytosis.58 Interestingly, the authors reported no significant changes in the emission spectra of the porphyrins upon cell internalization, indicating minimal aggregation or modification of the functional unit. The porphyrin based SCPN induced significant cell death after irradiating the cells with λ exc max = 403 148 nm light, which agrees with their ability to generate cytotoxic 1O2 singlet oxygen. The remarkable singlet-oxygen capability of the PS-loaded SCNP, in comparison to the free-porphyrin system, could not only be due to an augmented stabilization in water of the PS, but also to the fact that SCNPs possess a compact but rather open and flexible structure, allowing to the reactants (triplet molecular oxygen) and the products (ROS) to diffuse easily in and out the nanoparticles. To summarize, with a photoactive, porphyrin-containing SCNPs being evaluated for PDT-effect in a proper biological system, although these pioneering works demonstrate that the inclusion of a hydrophobic, highly aggregating PDT-suitable photosensitizer within the architecture of a water-soluble SCNP constitute a valid strategy for ROS-generating dyes for therapeutic applications, still a lot of effort have to be put for revealing the hidden potential of photocatalytic SCNPs to be exploited as efficient, last generation PDTnanodrugs. 4.2. Objectives When planning an encapsulation strategy, supramolecular approaches constitute a valid option towards the formulation of more performative PSs. The types of aggregates arising from different noncovalent interactions can confer to materials intriguing photochemical properties, very different from the single constituents taken alone.59 In Nature, photocatalytic processes are enabled in water by aminoacidic singlechain nanoparticles, which we call proteins, whose precise folding allows the stabilization and efficient functioning of photosensitizing molecules in the chemically complex, aqueous, biological media. As it happens for the most abundant photocatalyzed reaction on Earth, the chlorophyll-based photosynthesis, the careful assemble of proteins and pigments (often hydrophobic molecules) is required for the catalytic process to take place. E.g., higher plants photosystem I (PSI) is composed, amongst others, by the unique assembly of four different light-harvesting proteins (LHCI), which welcome and enclose in their folded structure a total of 165 chlorophylls and 5 additional electron-carrying cofactors as phylloquinones and Fe-S clusters, as reported in the structure shown in Figure 4.5.60,61 149 Figure 4.5. Crystal structures o part of the constitutive units of the PSI from Pisum Sativum. Specifically, the contact region of Lhca1 (green) and Lhca4 (grey) is shown. The magenta solid line follows schematically the backbone of the proteinic single-chain unit Lhca1. Enclosed chlorophylls are depicted in blue if bound to LHCA monomers, in red when present a linker function.60 Due to the extended electron delocalization in the tetrapyrrolic structure, it is worth noting that phthalocyanines are capable of specifically interact through π-π interactions with the aromatic ring systems of proteins, to the point that have been reported to be able of modulation of unwanted amyloid aggregation of proteins.62 Organic chromophores like anthracene, perylene etc. are, in fact, knowingly prone to aggregation and, although their properties in the condensed state are difficult to predict and control, several strategies to tune their assembly are often desirable when thinking on advanced application of their optoelectronic functionalities. The use of metal organic frameworks,63,64 co-assembly with biomacromolecules,65 and host-guest encapsulation,66 are some of the possible approaches toward the design of tunableaggregation of phthalocyanine PSs-based materials. The present work aimed at the rational design and preparation of improved amphiphilic single-chain polymer nanoparticles (SCNP) for imaging and photodynamic therapy (PDT) in zebrafish embryo xenografts. As described in the previous chapters, SCNPs are ultra-small polymeric nanoparticles with sizes similar to