Dott.ssa Željka Krpetić Scuola di Dottorato in Scienze Chimiche XXI Ciclo Università degli Studi di Milano Tutors: Prof Francesca Porta Dr Giorgio Scarì Coordinator: Prof. F. Cozzi Introduction The aim of my PhD research project was the study of metal nanoparticles and their applications in biological systems. Biological studies have been accomplished in collaboration with Dr Giorgio Scarì from the Department of Biology of Milan University. During the first two years, my research was focused on the following arguments: - Specific design and use of 15-mer peptides as stabilisers in the gold nanoparticles preparation - Bioconjugation of peptide stabilised gold nanoparticles - Preparation of gold nanoparticles stabilised with different small organic molecules for the selective cellular uptake into cancer cells In the 3rd year of my PhD studies, more sophisticated nanoparticle systems for different biological applications were studied, as listed here: - Use of Aloin A and Aloesin, two active components of Aloe vera, in the preparation of gold and silver nanoparticles and their biological applications - NMR and IR studies of simple aminoalcohol stabilised gold nanoparticles - Fluorescence spectroscopy and microscopy studies of dye stabilised gold nanoparticles and their potential use as biolabels Peptide design for the stabilisation of gold nanoparticles Gold nanoparticles can be easily functionalised with biomolecules or organic ligands, and they can be attractive tools for various applications. Stabilisation of gold nanoparticles by peptide molecules is well reported in literature [1-6]. In this PhD project, 15-mer peptides were designed and used as stabilisers for gold nanoparticles. Peptides designed, having periodical sequences, were planned to allow a parallel binding to gold surface [7]. One, H2N-GC(GGC)4-G-COOH (GC15), composed of 10 glycines and 5 cysteines, the other H2N-GK(GGK)4-G-COOH (GK15) composed of 10 glycines and 5 lysines (Figure 1). The sequences of the peptides were planned to allow the peptide to bind gold particles along its length, as observed for leucine and lysine-containing peptide bound to carboxylate-terminated thiol capped gold nanoparticles [8]. GC15 bears many potential anchor groups (SH or NH2) that can covalently bind gold particle, although the superiority of the thiol groups in covalent bonding with gold has already been stated [9]. GK15 peptide contains only primary amines that can bind gold particles in different ways depending on the pH of the sol and the pI of the peptide [10]. In particular, an electrostatic binding of GK15 can be assumed, if the NH2 groups are protonated, as observed in the case of gold-poly-lysine systems [11]. Peptides were synthesised by a standard Fmoc solid-phase procedure, purified by preparative HPLC and characterised by mass spectrometry (ESI-MS) by the professor Giovanna Speranza’s research group of Milan University. Gold nanoparticles stabilised by GC15 and GK15 were prepared via the borohydride reduction method in water at pH 3, as well as via ligand exchange method. In the borohydride reduction method, gold precursor, AuCl4-, is reduced by NaBH4, in the presence of the peptide ligand obtaining a cherry red coloured gold sol. In the ligand exchange preparation method gold nanoparticles of 15 nm diameter were obtained via the Turkevich/Frens method [12-13], subsequently protected by addition of the peptide and purified by repeated centrifugation and redispersion. By different preparation methods gold particles of different diameters were obtained. Particles were purified by dialysis or centrifugation, depending on the particles size. The particles were characterised by UV-visible, ATR-FTIR, 1H NMR spectroscopies, while the particles dimensional and morphological characterisation was performed by TEM. NMR spectrosopy has revealed to be very useful tool for the characterisation in aqueous media after the lyophilisation and redispersion of the particles. This is an important result since very few nanoparticle systems can be stored in dry state and then redispersed in water [14] and studied by the NMR. These peptides containing several regularly spaced amine (lysine) or thiol (cysteine) functions have been introduced as very strongly binding “multidentate” ligands to stabilise gold nanoparticles [15]. Spectroscopic investigations suggest an electrostatic multiple interactions of protonated NH2 groups of GK15 with anions present on negative gold surface (AuCl4-, AuCl2-), observing breaking and formation of H-bondings. While for GC15 peptide, the coordination to gold particles was observed via the thiol functionality, as expected. A multidentate peptide for stabilisation and facile bioconjugation of gold nanoparticles There is an increasing interest in the preparation of nanoparticles that are stable in aqueous media and can be readily functionalised with bio-molecules by established bioconjugation procedures. A number of different approaches to conjugating metal nanoparticles to biomolecules have also been reported. These include click chemistry [16], biotin-avidin coupling [17-19], ligand exchange [20, 21] and a range of standard bioconjugation procedures [22, 23]. After the GC15 and GK15 peptides showed novel and successful characteristics in the gold particle stabilisation, a new peptide of this family was specifically designed for the stabilisation and subsequent bioconjugation of gold nanoparticles. This ligand (H2N-GCGGCGGKGGCGGCG-COOH) (Figure 2) can bind to the nanoparticle via the thiol groups of four cysteine moieties and contains a central lysine that provides an amine function to which biomolecular functionality can be readily attached. This protection method is very robust and can be used either for the one-step synthesis of relatively small (2-4 nm) particles or for the stabilisation of pre-prepared, larger (10-20 nm) colloids. Water-soluble GCK15 peptide was purchased from Aldrich (purity>95%). The resulting GCK15 coated gold particles have been characterised by TEM, UV-vis, ATR-FTIR and 1H NMR spectroscopy. Gold nanoparticles of 2.4 nm diameters were prepared in a one-step reaction by borohydride reduction of AuCl4- in the presence of the stabilising GCK15 peptide. A clear brown solution was obtained indicating the formation of gold particles in the size range below 3 nm. This conclusion was confirmed by the absence of a plasmon absorption band in the UV-vis spectrum. Gold nanoparticles of 15 nm diameters were obtained via the Turkevich/Frens method [12, 13] subsequently protected by addition of our peptide and purified by repeated centrifugation and redispersion. The UV-vis spectrum shows a plasmon absorption band at 520 nm typical for gold particles of this size range, and red gold colloidal solution. The particles are extremely stable and can be centrifuged and redispersed in pure water many times without detectable loss of material, whereas the as prepared citrate-stabilised particles cannot be redispersed in pure water after the first centrifugation. The analysis of the ligand shell in the case of 15 nm GCK15 stabilised gold particles obtained via the ligand exchange method is more difficult due to the very small proportion of peptide present in the total amount of material, which is predominantly gold. However, using high resolution magic angle spinning (HR-MAS) well resolved 1H NMR spectra of 15 nm Au@GCK15 nanoparticles were obtained. The absence of citrate peaks (quartet centred at 2.5-2.7 ppm) suggests complete ligand exchange by exposure to the peptide. The sharp doublet centred at 2.90 ppm in the spectrum of the free peptide ligand is due to the 8 cysteine β-methylene groups vicinal to the thiol groups and disappears completely upon binding to the particles. This indicates that all cysteine thiol groups are involved in the surface binding process. As an example of facile bioconjugation, a biotin moiety has been introduced via a standard coupling procedure. Biotinylation of peptide-stabilised gold nanoparticles was achieved using the standard sulfo-NHS-biotin labelling agent. Binding of the biotinylated particles to streptavidin-modified agarose beads has been demonstrated leading to an intense red colouration of the beads as evidence for successful biotinylation. Particles that have not been biotinylated do not attach to the beads. In adittion, dot blot experiments also clearly indicate efficient biotinylation of the particles. The attachment of biomolecular functionality of choice, e.g. biotin, is possible due to the presence of a central lysine residue that is not involved in the binding of the ligand to the surface of the particles. Biological application of peptide stabilised gold nanoparticles. A study of the cellular uptake mechanism. Current studies in this research area have been focused on coating biorecognition molecules on the surface of NPs to mediate cellular accumulation in different cell compartments. In bionanotechnology it is very important to have stabilisers, which could be easily functionalised with other biologically important ligands. Understanding and controlling the interactions between nanoscale objects and living cells is of great importance for arising diagnostic and therapeutic applications of nanoparticles and for nanotoxicology studies [24]. In this PhD thesis, the intracellular uptake of differently sized spherical water-soluble peptide-coated gold nanoparticles into HeLa cells has been investigated [25]. HeLa cells are human epithelial cells from a fatal cervical carcinoma transformed by human papillomavirus 18 (HPV18), classic example of an immortalized cell line widely used in medical research. For this study, gold particles stabilised with GC15, GK15 and GCK15 peptides were successfully uptaken into HeLa cells, as well as biotinylated GCK15 peptide stabilised gold particles. A comparison has been made between gold particles prepared by two different preparation methods: borohydride direct reduction and the ligand exchange preparation method. It was found that the particles prepared by using the citrate displacement method enters HeLa cells in different fashion as compared with the particles prepared by borohydride reduction method. Intracellular uptake of gold particles was investigated using TEM microscopy. Samples for TEM observations were prepared by incubation of gold particles with HeLa cells at 37°C and 5% CO2 flow for 1h and the samples were then processed by a number of necessary steps (fixation, post fixation, staining, dexydration, embedding in epoxy resin, polymerisation, ultra thin cutting and mounting on TEM grids) in order to obtain 70 nm thick sections cutted with the ultra microtrome suitable for the TEM observations. Accumulation of gold particles into membrane-bound compartments inside cells, known as endosomes, is generally observed. It is shown that smaller particles (<4nm) entered cells in agglomerated form, this phenomenon were also described elsewhere [26, 27]. However, all the particles were found in endosomes, whether early or late endosomes. No particles were found in HeLa cells nuclei. In a similar fashion, biotinylated GCK15 stabilised gold particles were also found in HeLa cells endosomes. Microscopy observations have demonstrated that the mechanism of the cellular uptake of gold particles into HeLa cells is mediated via the receptor-mediated endocytosys, as evidenced by TEM micrographs of ultra thin cellular sections. It was possible to observe almost all the steps of this mechanism: 1. Specific adsorption of gold nanoparticles on the cell membrane 2. Specific recognition of gold nanoparticles by receptors present in the cell’s membrane 3. Invagination of the cell membrane with formation of a membrane-bound compartments known as endosomes 4. Observation of the endosomes formed carrying gold nanoparticles present in cell’s cytoplasm If the uptake mechanism of gold particles is endocytosis it is expected their exit via the exocytosis. This phenomenon would restrain their leftover time in cells, and consequently the toxicity for the organism. Selective cellular uptake of gold nanoparticles into cancer cells Current clinical X-ray contrast agents impose serious limitations on medical imaging: short imaging times, the need for catheterisation in many cases, occasional renal toxicity, and poor contrast in large patients [28]. Gold nanoparticles may overcome these limitations, as demonstrated by Hainfeld and co workers. Gold has higher absorption than iodine agents, usually used for these purposes, with less bone and tissue interference achieving better contrast with lower X-ray dose. Moreover, nanoparticles clear the blood more slowly than iodine agents, permitting longer imaging times. In this study, gold nanoparticles of 1.9 nm in diameter were injected intravenously into mice and images recorded over time with a standard mammography unit. Retention in liver and spleen resulted very low with elimination by the kidneys. These concepts were extended by using different gold nanoparticles to deliver a very large quantity of gold to tumours via intravenous injection. Combination with X-rays resulted in eradication of most tumours [29]. This PhD work was stimulated by the Hainfield’s study [30-33] where a synergistic effect was observed between gold nanoparticles and the X-ray treatment resulting in tumour reduction or eradication. The survival after one year of the combined therapy was of 70%. The success of this technique is related to the high ability of gold to accumulate within tumours and absorb X-rays. Instead of the intravenous injection in a tumour tissue, different cancer cells with a range of small sized gold nanoparticles were incubated. We have studied with confocal microscopy the intracellular uptake of small sized gold nanoparticles stabilised by different organic biocompatible ligands (5-aminovaleric acid, adipic acid, L-DOPA, glucose, glycolic acid, dopamine) and their use as nanogold bioconiugates with different cancer cells (K562-leucemia myelogenous cronica caucasica humana, PC12-pheochromocytoma). Selective entrance of these particles into cancer cells was found [34]. Negative control has been performed on human epithelial cells where no entrance of gold particles was found even after 8 h of incubation. A preliminary toxicity experiment in vivo has been performed on sane CD1 mice type. Aminovaleric acid coated gold nanoparticles were chosen as model particles and injected intraperitoneally in two mice. Survival after 2 years post injection was verified, as an exceptional result. This preliminary result leads us to conclude very low or total absence of toxicity effects on living tissue and inner organs. Aloin A and Aloesin stabilised gold and silver nanoparticles and their biological applications. The inner gel of Aloe vera (Aloe barbadensis Miller) leaf is widely used in various medical, cosmetic and nutraceutical applications [35]. Many beneficial effects and biological activities of this plant as anti-viral, anti-bacterical, laxative, anti-inflammation and immunostimulation have been attributed to the polysaccharides present in the leaf pulp. Different chemical compounds, responsible for its healing properties, have been isolated so far from this specie as alkaloids, anthraquinones, anthrones, chromones, flavonoids, coumarins and pyrones, and their chemistry was thoroughly studied and reported by Dagne and coworkers [36] and professor Speranza and professor Manitto research groups [37-40]. In the anticancer drugs research, the studies on Aloe vera components have been videly undertaken. It was found by Pecere and co-workers that a hydroxyanthraquinone, naturally present in Aloe vera leaves, has a specific in vitro and in vivo antineuroectodermal tumour activity [41]. Nanoparticles synthesis using biological entities is already reported in literature, including bacteria, yeast, funghi and plants [42, 43] as clean, non-toxic and environmetally acceptable routes. Many studies on the plant use in nanobiotechnology have appeared in literature in a size-controlled formation of gold nanoparticles. Different plants are involved in the both intra and extracellular formation of silver and gold nanoparticles, reporting the use of oat (Avena sativa) [44], lemongrass extract (Cymbopogon flexuosus) [45-47], leguminous shrub Sesbania drummondii [48], Brassica juncea [49], neem leaf broth (Azadirachta indica) [50], pine (Pinus desiflora), persimmon (Diopyros kaki), ginkgo (Ginko biloba), magnolia (Magnolia kobus) and platanus (Platanus orientalis) [51]. In the listed examples, nanoparticles formation is a consequence of the Au(III) to Au(0) reduction inside plant cells or tissues. On the other hand, use of various leaf extracts utilised both as reducing agents and stabilisers in the nanoparticles preparation has been reported, as for example Emblica Officinalis fruit extract [52], Aloe vera leaf extract [53] and Cinnamon camphora leaf extract [54]. Using Aloe vera leaf extract, the formation of gold nanotriangles has been achieved as a result of the slow reduction of aqueous tetrachloroaurate anions, AuCl4-, along with the shape-directing effects of carbonyl compounds present as constituents in the plant extract. With the aim to prepare novel water soluble and biocompatible nanoparticles for biological applications, in this PhD project two active components of Aloe vera, Aloin A and Aloesin have been utilised, as stabilisers for gold and silver nanoparticles. By using different reducing agents (sodium borohydride, citric and ascorbic acid) and varying the reaction conditions (temperature and reaction time) we were able to prepare extremely stabile, water soluble Aloin A and Aloesin stabilised gold particles sized from 4 to 50 nm diameter range and approximately 5 nm sized silver nanoparticles. Prior to characterisation, particles were purified by dialysis or centrifugation, depending on the particle’s size. Silver particles were characterised by UV-visible spectroscopy and the morphology and size of both silver and gold particles were investigated by TEM. Gold particles were characterised using UV-visible, ATR-FTIR and 1H NMR spectroscopies, which highlighted the interaction between gold and Aloin A and Aloesin ligand molecules. Although NMR studies of the particles ligand shell might be an issue, due to the very small content of the organic material present on the particles surface, HR-MAS 1H NMR technique has been used. Due to the very small amount of the sample needed for the analysis, this technique resulted very advantageous and promising in the studies of the particle ligand shell, appearing more functional and effective than usual NMR analysis in solution. By ligand exchange preparation method, involving citrate coated 15 nm gold particles, it was possible to exchange the citrate ligand with Aloin A and Aloesin molecules, stabilising the particles also in this way. The amount of Aloin A and Aloesin was finely tuned as well as pH of the colloidal solution allowing particle’s agglomeration studies. Agglomeration of the particles was followed by TEM microscopy. More agglomerated particles were found on lower pH values (5-7) in less protected colloidal samples (<5000 ligand molecules per particle). When the pH of the colloidal solution was adjusted to higher values (8-10) and approximately 10000 ligand molecules were set up for each gold particle good stabilisation of the particles was achieved. 50 nm Aloin A and Aloesin stabilised gold nanoparticles, prepared by two different methods were applied to the vehicle study into macrophage cells. For the biological experiments by a peritoneal washing procedure, macrophage cells were extracted from a CD1 mouse, pre-emptively sacrificed by CO2 asphyxia. Macrophages were then collected in the physiological solution and seeded in the cell culture medium (MEM) at 37°C in the CO2 atmosphere at 5% at sterile conditions. Subsequently, macrophages were treated with gold colloidal solution (50 nm Au-Aloin A and 40 nm Au-Aloesin particles obtained by citric acid reduction method). For the treatment, 50 μl of gold colloidal solution was added to 1 ml of the cell culture medium containing marcophage cells. Macrophages were then incubated with gold nanoparticles for 5, 15, 30 and 60 min in the same conditions (37°C, at 5% CO2). After the incubation, the samples were prepared for the confocal and fluorescence microscopy observations by a number of necessary steps (centrifugation, adittion of DAPI, fixation) obtaining incubated cells on a microscopy glass slides. DAPI (4',6-diamidino-2-phenylindole) fluorescent stain was used in order to stain the cells’ nuclei. Confocal microscopy observations have revealed the presence of Aloin A and Aloesin stabilised gold particles in macrophage cells cytoplasm, while the fluorescence microscopy has revealed, in some cases, the presence of these particles also in macrophages nuclei. It is an important result, since there is an emergent need for carriers that can carry bioactive agents into the cell nucleus for drug as well as gene delivery. However, in general, nanoparticles mainly localise in the cytosol (or extranucleus). Therefore, nanoparticles capable of localising into the nucleus are particularly inportant for cancer therapy. Because cancer cells have many intracellular mechanisms to limit drug molecules' access to the nucleus the direct delivery of the drug into the nucleus would circumvent these drug-resistance mechanisms [55]. NMR and IR studies of simple aminoalcohol stabilised gold nanoparticles Special properties of gold nanoparticles [56] led to their use in important applications in the areas of catalysis, optoelectronics, electron microscopy, and biology. However, the nature of the gold capping ligand bond usually remains unknown, especially for ligands bearing multiple anchor groups that can bind gold particles in different ways (carboxy, amino, hydroxy, thiol etc.). Several studies using variety of characterisation techniques have been carried out [57]. Some of the capping agents investigated include amines or amino derivatives [58]. Heath and co-workers proposed the formation of a partially covalent bond between the gold surface and the primary amines, pointing out that the stability of the Au-amine nanoparticle depends mainly on kinetic effects [59]. Further experiments were performed using amino derivatives and exploiting them as stabilisers and/or reducing agents [60-70] in the particles preparation. Various amino functionalities have been studied, including amino acids and amino-containing polymers, with the amino groups being particularly interesting due to their presence in biological and environmental systems. However, there is a lack of reports on aminoalcohols, which are an attractive class of compounds for their potential oxidation to amino acids. This PhD project research was stimulated by the novelty of gold-aminoalcohol systems, the discovery of aminoalcohol binding site(s) (NH2 vs OH functionality), and the nature of the Au-NH bond. Reproducible gold hydrosols stabilised by aminoalcohols have been prepared. Aminoalcohol stabilised gold and silver particles were characterised by spectroscopic and microscopic means giving a deep insight into the gold-nitrogen interaction. NMR spectroscopy was applied to investigate gold nanoparticles in organic solvents; [71, 72] however, many experimental difficulties (organic molecule quantity in the ligand shell, particles purity, redispersion of gold colloids) had to be overcome before the NMR technique could be applied as a valid tool in the investigation of small-sized water-soluble gold nanoparticles [73]. Aminoalcohol-stabilised gold nanoparticles in aqueous solution were prepared by the borohydride reduction of HAuCl4 in the presence of aminoalcohol. Different aminoalcohols were used as stabilisers for gold particles: ethanolamine, 2-(propylamino)ethanol, 2-amino-1,3-propanediol, DL-2-amino-1-pentanol, 3-amino-1-propanol, (±)-3-amino-1,2-propanediol, 4-amino-1-butanol, 5-amino-1-pentanol. Red gold sols with 4 nm mean diameter particles were obtained. The samples were characterised by ATR-FTIR spectroscopy in solid state, and by 1H NMR, UV-vis, and TEM analyses in solution. The ATR-FTIR spectra of the solid materials suggested the gold-aminoalcohol interaction. Considering NH2 and OH stretching vibration modes in the free and coordinated ligands, storng evidence of the NH3+ groups involved in an electrostatic bond is shown in the case of 4-amino-1-butanol stabilised gold particles. To test this, 4-amino-1-butanol hydrochloride was prepared (by bubbling gaseous HCl in a 1,2-dimethoxy-ethane solution of 4-amino-1-butanol) and the IR spectrum was collected. Successful NMR measurements were obtained only after accurate purification of the particles (dialysis). 1H NMR experiments indicated that the binding site of aminoalcohols studied is the protonated amino group ligated with an ionic bond to the gold surface, as strongly supported by general chemical shift trend. A difference of ca. 0.40-0.50 ppm, from free aminoalcohols to coordinated aminoalcohols on gold particles has been observed [74]. Thus, NMR technique is proving to be a powerful tool for the characterisation of these colloidal systems, but is still remains not fully utilised, because of the inability of current preparative methods to supply enough purified nanoparticles. Fluorescence spectroscopy and microscopy studies of dye stabilised gold nanoparticles and their potential use in biological labelling The introduction of labelling agents in biological systems is required for a facile microscopy detection of biological systems. Fluorescent labels nowadays represent widely developed tools in biology and medicine [75]. Fluorescence parameters are usually used to obtain informations on living cells. In this context, modified gold nanoparticles can be used as nano reporters. Substantial progress in the ability to fabricate nanoparticles and the discovery of their novel size dependent physical and chemical features has drawn the attention of researchers in the area of biomedical imaging. The development of targeted contrast agents such as fluorescent probes has made it possible to selectively view specific biological events and processes in both living and nonviable systems with improved detection limits, imaging modalities and engineered biomarker functionality. The fabrication of luminescent-engineered nanoparticles is expected to be integral to the development of next generation therapeutic, diagnosis and imaging technologies [76]. The aim of this project was the fabrication of novel dye stabilised gold nanoparticles with favourable luminescent properties as bio-labels and the in vitro studies of their cellular uptake. Cellular uptake of luminescent gold nanoparticles into macrophages and Human Neuroblastoma IMR-32 cells was studied. Dye-stabilised gold particles have been prepared by a particular preparation method, designed in order to obtain 30 to 50 nm sized particles. For the preparation, different reducing agents (NaBH4, Ascorbic and Citric acid) and stabilisers having photoluminescence (PL) signals in different Visible spectrum region have been applied. As stabilisers for gold particles fluorescent molecules: 2’,7’-Dichlorofluorescein, 4-Methylumelliferyl phosphate, Eosin Y (2′,4′,5′,7′-Tetrabromofluorescein disodium salt), HPTS (8-Hydroxy-1,3,6-pyrenetrisulfonic acid) and Thionin acetate (3,7-Diaminophenothiazin-5-ium acetate) have been utilised (Figure 3). These novel gold colloids were characterised by UV-vis spectroscopy, TEM and HR-MAS NMR spectroscopy while the luminescent properties of these particles were studied by fluorescence spectroscopy. Absorption and emission spectra showed no peaks due to free ligands, and were substancially different. PL spectra of gold nanoparticles, obtained by excitation at the absorption wavelength value (523-547 nm range), showed values in a 596-661 nm range, depending on the ligand kind and particles size. We have also found that the type of reducing agent influences gold nanoparticles PL emission. Moreover, when excited at 488 nm, gold sols showed two PL peaks, centered at 525 and 581 nm, in accordance with Fluorescence and Confocal microscopy observations. Indeed, when FITC (Fluorescein Isothiocyanate) fluorescence filter is used (λex = 488 nm) the particles internalised in cells, showed green fluorescence signals, while Texas Red fluorescence filter is used (λex = 568 nm) the particles showed red fluorescence signals. Luminescent gold nanoparticles were tested as labelling agents in two different cellular systems (macrophages and neuroblastoma IMR-32 cells). Dye stabilised gold particles were incubated with macrophage and IMR-32 cells for 1 h (37°C, 5% CO2), and the cellular uptake of the particles was confirmed by Confocal microscopy and TEM. Besides these techniques, Fluorescence microscopy observations have showed interesting results. These novel systems, coupling gold with the dye, have an advantage of being visualised by all three microscopy techniques, (TEM, Confocal and Fluorescence microscopy) as they satisfy their detection requirements (gold electronic density, gold fluorescence and ligand fluorescence) as they exibit the long fluorescence lifetime. For the dye-stabilised particles uptaken into macrophages and IMR-32 cells, an almost general correlation between the fluorescence signal colour and the particles size has been found. In the case of particles smaller than 10 nm in size (3 nm Au@Citrate and 10 nm Au@4-aminovaleric acid were taken as examples) only green fluorescence signals are found applying FITC filter. On the contrary, if the particle diameters are 15 nm or larger, red fluorescence signals are found when Texas Red fluorescence filter is applied. However, in some cases, for particles with broader size distribution, green and red fluorescence signals were both observed. 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Relazione Finale : terzo anno di Dottorato in Scienze e Tecnologie Chimiche, 21. Ciclo / Zeljka Krpetic, Francesca Porta, Giorgio Scarì. - [s.l] : null, 2008.

Relazione Finale : terzo anno di Dottorato in Scienze e Tecnologie Chimiche, 21. Ciclo

Zeljka Krpetic;Francesca Porta;Giorgio Scarì
2008

Abstract

Dott.ssa Željka Krpetić Scuola di Dottorato in Scienze Chimiche XXI Ciclo Università degli Studi di Milano Tutors: Prof Francesca Porta Dr Giorgio Scarì Coordinator: Prof. F. Cozzi Introduction The aim of my PhD research project was the study of metal nanoparticles and their applications in biological systems. Biological studies have been accomplished in collaboration with Dr Giorgio Scarì from the Department of Biology of Milan University. During the first two years, my research was focused on the following arguments: - Specific design and use of 15-mer peptides as stabilisers in the gold nanoparticles preparation - Bioconjugation of peptide stabilised gold nanoparticles - Preparation of gold nanoparticles stabilised with different small organic molecules for the selective cellular uptake into cancer cells In the 3rd year of my PhD studies, more sophisticated nanoparticle systems for different biological applications were studied, as listed here: - Use of Aloin A and Aloesin, two active components of Aloe vera, in the preparation of gold and silver nanoparticles and their biological applications - NMR and IR studies of simple aminoalcohol stabilised gold nanoparticles - Fluorescence spectroscopy and microscopy studies of dye stabilised gold nanoparticles and their potential use as biolabels Peptide design for the stabilisation of gold nanoparticles Gold nanoparticles can be easily functionalised with biomolecules or organic ligands, and they can be attractive tools for various applications. Stabilisation of gold nanoparticles by peptide molecules is well reported in literature [1-6]. In this PhD project, 15-mer peptides were designed and used as stabilisers for gold nanoparticles. Peptides designed, having periodical sequences, were planned to allow a parallel binding to gold surface [7]. One, H2N-GC(GGC)4-G-COOH (GC15), composed of 10 glycines and 5 cysteines, the other H2N-GK(GGK)4-G-COOH (GK15) composed of 10 glycines and 5 lysines (Figure 1). The sequences of the peptides were planned to allow the peptide to bind gold particles along its length, as observed for leucine and lysine-containing peptide bound to carboxylate-terminated thiol capped gold nanoparticles [8]. GC15 bears many potential anchor groups (SH or NH2) that can covalently bind gold particle, although the superiority of the thiol groups in covalent bonding with gold has already been stated [9]. GK15 peptide contains only primary amines that can bind gold particles in different ways depending on the pH of the sol and the pI of the peptide [10]. In particular, an electrostatic binding of GK15 can be assumed, if the NH2 groups are protonated, as observed in the case of gold-poly-lysine systems [11]. Peptides were synthesised by a standard Fmoc solid-phase procedure, purified by preparative HPLC and characterised by mass spectrometry (ESI-MS) by the professor Giovanna Speranza’s research group of Milan University. Gold nanoparticles stabilised by GC15 and GK15 were prepared via the borohydride reduction method in water at pH 3, as well as via ligand exchange method. In the borohydride reduction method, gold precursor, AuCl4-, is reduced by NaBH4, in the presence of the peptide ligand obtaining a cherry red coloured gold sol. In the ligand exchange preparation method gold nanoparticles of 15 nm diameter were obtained via the Turkevich/Frens method [12-13], subsequently protected by addition of the peptide and purified by repeated centrifugation and redispersion. By different preparation methods gold particles of different diameters were obtained. Particles were purified by dialysis or centrifugation, depending on the particles size. The particles were characterised by UV-visible, ATR-FTIR, 1H NMR spectroscopies, while the particles dimensional and morphological characterisation was performed by TEM. NMR spectrosopy has revealed to be very useful tool for the characterisation in aqueous media after the lyophilisation and redispersion of the particles. This is an important result since very few nanoparticle systems can be stored in dry state and then redispersed in water [14] and studied by the NMR. These peptides containing several regularly spaced amine (lysine) or thiol (cysteine) functions have been introduced as very strongly binding “multidentate” ligands to stabilise gold nanoparticles [15]. Spectroscopic investigations suggest an electrostatic multiple interactions of protonated NH2 groups of GK15 with anions present on negative gold surface (AuCl4-, AuCl2-), observing breaking and formation of H-bondings. While for GC15 peptide, the coordination to gold particles was observed via the thiol functionality, as expected. A multidentate peptide for stabilisation and facile bioconjugation of gold nanoparticles There is an increasing interest in the preparation of nanoparticles that are stable in aqueous media and can be readily functionalised with bio-molecules by established bioconjugation procedures. A number of different approaches to conjugating metal nanoparticles to biomolecules have also been reported. These include click chemistry [16], biotin-avidin coupling [17-19], ligand exchange [20, 21] and a range of standard bioconjugation procedures [22, 23]. After the GC15 and GK15 peptides showed novel and successful characteristics in the gold particle stabilisation, a new peptide of this family was specifically designed for the stabilisation and subsequent bioconjugation of gold nanoparticles. This ligand (H2N-GCGGCGGKGGCGGCG-COOH) (Figure 2) can bind to the nanoparticle via the thiol groups of four cysteine moieties and contains a central lysine that provides an amine function to which biomolecular functionality can be readily attached. This protection method is very robust and can be used either for the one-step synthesis of relatively small (2-4 nm) particles or for the stabilisation of pre-prepared, larger (10-20 nm) colloids. Water-soluble GCK15 peptide was purchased from Aldrich (purity>95%). The resulting GCK15 coated gold particles have been characterised by TEM, UV-vis, ATR-FTIR and 1H NMR spectroscopy. Gold nanoparticles of 2.4 nm diameters were prepared in a one-step reaction by borohydride reduction of AuCl4- in the presence of the stabilising GCK15 peptide. A clear brown solution was obtained indicating the formation of gold particles in the size range below 3 nm. This conclusion was confirmed by the absence of a plasmon absorption band in the UV-vis spectrum. Gold nanoparticles of 15 nm diameters were obtained via the Turkevich/Frens method [12, 13] subsequently protected by addition of our peptide and purified by repeated centrifugation and redispersion. The UV-vis spectrum shows a plasmon absorption band at 520 nm typical for gold particles of this size range, and red gold colloidal solution. The particles are extremely stable and can be centrifuged and redispersed in pure water many times without detectable loss of material, whereas the as prepared citrate-stabilised particles cannot be redispersed in pure water after the first centrifugation. The analysis of the ligand shell in the case of 15 nm GCK15 stabilised gold particles obtained via the ligand exchange method is more difficult due to the very small proportion of peptide present in the total amount of material, which is predominantly gold. However, using high resolution magic angle spinning (HR-MAS) well resolved 1H NMR spectra of 15 nm Au@GCK15 nanoparticles were obtained. The absence of citrate peaks (quartet centred at 2.5-2.7 ppm) suggests complete ligand exchange by exposure to the peptide. The sharp doublet centred at 2.90 ppm in the spectrum of the free peptide ligand is due to the 8 cysteine β-methylene groups vicinal to the thiol groups and disappears completely upon binding to the particles. This indicates that all cysteine thiol groups are involved in the surface binding process. As an example of facile bioconjugation, a biotin moiety has been introduced via a standard coupling procedure. Biotinylation of peptide-stabilised gold nanoparticles was achieved using the standard sulfo-NHS-biotin labelling agent. Binding of the biotinylated particles to streptavidin-modified agarose beads has been demonstrated leading to an intense red colouration of the beads as evidence for successful biotinylation. Particles that have not been biotinylated do not attach to the beads. In adittion, dot blot experiments also clearly indicate efficient biotinylation of the particles. The attachment of biomolecular functionality of choice, e.g. biotin, is possible due to the presence of a central lysine residue that is not involved in the binding of the ligand to the surface of the particles. Biological application of peptide stabilised gold nanoparticles. A study of the cellular uptake mechanism. Current studies in this research area have been focused on coating biorecognition molecules on the surface of NPs to mediate cellular accumulation in different cell compartments. In bionanotechnology it is very important to have stabilisers, which could be easily functionalised with other biologically important ligands. Understanding and controlling the interactions between nanoscale objects and living cells is of great importance for arising diagnostic and therapeutic applications of nanoparticles and for nanotoxicology studies [24]. In this PhD thesis, the intracellular uptake of differently sized spherical water-soluble peptide-coated gold nanoparticles into HeLa cells has been investigated [25]. HeLa cells are human epithelial cells from a fatal cervical carcinoma transformed by human papillomavirus 18 (HPV18), classic example of an immortalized cell line widely used in medical research. For this study, gold particles stabilised with GC15, GK15 and GCK15 peptides were successfully uptaken into HeLa cells, as well as biotinylated GCK15 peptide stabilised gold particles. A comparison has been made between gold particles prepared by two different preparation methods: borohydride direct reduction and the ligand exchange preparation method. It was found that the particles prepared by using the citrate displacement method enters HeLa cells in different fashion as compared with the particles prepared by borohydride reduction method. Intracellular uptake of gold particles was investigated using TEM microscopy. Samples for TEM observations were prepared by incubation of gold particles with HeLa cells at 37°C and 5% CO2 flow for 1h and the samples were then processed by a number of necessary steps (fixation, post fixation, staining, dexydration, embedding in epoxy resin, polymerisation, ultra thin cutting and mounting on TEM grids) in order to obtain 70 nm thick sections cutted with the ultra microtrome suitable for the TEM observations. Accumulation of gold particles into membrane-bound compartments inside cells, known as endosomes, is generally observed. It is shown that smaller particles (<4nm) entered cells in agglomerated form, this phenomenon were also described elsewhere [26, 27]. However, all the particles were found in endosomes, whether early or late endosomes. No particles were found in HeLa cells nuclei. In a similar fashion, biotinylated GCK15 stabilised gold particles were also found in HeLa cells endosomes. Microscopy observations have demonstrated that the mechanism of the cellular uptake of gold particles into HeLa cells is mediated via the receptor-mediated endocytosys, as evidenced by TEM micrographs of ultra thin cellular sections. It was possible to observe almost all the steps of this mechanism: 1. Specific adsorption of gold nanoparticles on the cell membrane 2. Specific recognition of gold nanoparticles by receptors present in the cell’s membrane 3. Invagination of the cell membrane with formation of a membrane-bound compartments known as endosomes 4. Observation of the endosomes formed carrying gold nanoparticles present in cell’s cytoplasm If the uptake mechanism of gold particles is endocytosis it is expected their exit via the exocytosis. This phenomenon would restrain their leftover time in cells, and consequently the toxicity for the organism. Selective cellular uptake of gold nanoparticles into cancer cells Current clinical X-ray contrast agents impose serious limitations on medical imaging: short imaging times, the need for catheterisation in many cases, occasional renal toxicity, and poor contrast in large patients [28]. Gold nanoparticles may overcome these limitations, as demonstrated by Hainfeld and co workers. Gold has higher absorption than iodine agents, usually used for these purposes, with less bone and tissue interference achieving better contrast with lower X-ray dose. Moreover, nanoparticles clear the blood more slowly than iodine agents, permitting longer imaging times. In this study, gold nanoparticles of 1.9 nm in diameter were injected intravenously into mice and images recorded over time with a standard mammography unit. Retention in liver and spleen resulted very low with elimination by the kidneys. These concepts were extended by using different gold nanoparticles to deliver a very large quantity of gold to tumours via intravenous injection. Combination with X-rays resulted in eradication of most tumours [29]. This PhD work was stimulated by the Hainfield’s study [30-33] where a synergistic effect was observed between gold nanoparticles and the X-ray treatment resulting in tumour reduction or eradication. The survival after one year of the combined therapy was of 70%. The success of this technique is related to the high ability of gold to accumulate within tumours and absorb X-rays. Instead of the intravenous injection in a tumour tissue, different cancer cells with a range of small sized gold nanoparticles were incubated. We have studied with confocal microscopy the intracellular uptake of small sized gold nanoparticles stabilised by different organic biocompatible ligands (5-aminovaleric acid, adipic acid, L-DOPA, glucose, glycolic acid, dopamine) and their use as nanogold bioconiugates with different cancer cells (K562-leucemia myelogenous cronica caucasica humana, PC12-pheochromocytoma). Selective entrance of these particles into cancer cells was found [34]. Negative control has been performed on human epithelial cells where no entrance of gold particles was found even after 8 h of incubation. A preliminary toxicity experiment in vivo has been performed on sane CD1 mice type. Aminovaleric acid coated gold nanoparticles were chosen as model particles and injected intraperitoneally in two mice. Survival after 2 years post injection was verified, as an exceptional result. This preliminary result leads us to conclude very low or total absence of toxicity effects on living tissue and inner organs. Aloin A and Aloesin stabilised gold and silver nanoparticles and their biological applications. The inner gel of Aloe vera (Aloe barbadensis Miller) leaf is widely used in various medical, cosmetic and nutraceutical applications [35]. Many beneficial effects and biological activities of this plant as anti-viral, anti-bacterical, laxative, anti-inflammation and immunostimulation have been attributed to the polysaccharides present in the leaf pulp. Different chemical compounds, responsible for its healing properties, have been isolated so far from this specie as alkaloids, anthraquinones, anthrones, chromones, flavonoids, coumarins and pyrones, and their chemistry was thoroughly studied and reported by Dagne and coworkers [36] and professor Speranza and professor Manitto research groups [37-40]. In the anticancer drugs research, the studies on Aloe vera components have been videly undertaken. It was found by Pecere and co-workers that a hydroxyanthraquinone, naturally present in Aloe vera leaves, has a specific in vitro and in vivo antineuroectodermal tumour activity [41]. Nanoparticles synthesis using biological entities is already reported in literature, including bacteria, yeast, funghi and plants [42, 43] as clean, non-toxic and environmetally acceptable routes. Many studies on the plant use in nanobiotechnology have appeared in literature in a size-controlled formation of gold nanoparticles. Different plants are involved in the both intra and extracellular formation of silver and gold nanoparticles, reporting the use of oat (Avena sativa) [44], lemongrass extract (Cymbopogon flexuosus) [45-47], leguminous shrub Sesbania drummondii [48], Brassica juncea [49], neem leaf broth (Azadirachta indica) [50], pine (Pinus desiflora), persimmon (Diopyros kaki), ginkgo (Ginko biloba), magnolia (Magnolia kobus) and platanus (Platanus orientalis) [51]. In the listed examples, nanoparticles formation is a consequence of the Au(III) to Au(0) reduction inside plant cells or tissues. On the other hand, use of various leaf extracts utilised both as reducing agents and stabilisers in the nanoparticles preparation has been reported, as for example Emblica Officinalis fruit extract [52], Aloe vera leaf extract [53] and Cinnamon camphora leaf extract [54]. Using Aloe vera leaf extract, the formation of gold nanotriangles has been achieved as a result of the slow reduction of aqueous tetrachloroaurate anions, AuCl4-, along with the shape-directing effects of carbonyl compounds present as constituents in the plant extract. With the aim to prepare novel water soluble and biocompatible nanoparticles for biological applications, in this PhD project two active components of Aloe vera, Aloin A and Aloesin have been utilised, as stabilisers for gold and silver nanoparticles. By using different reducing agents (sodium borohydride, citric and ascorbic acid) and varying the reaction conditions (temperature and reaction time) we were able to prepare extremely stabile, water soluble Aloin A and Aloesin stabilised gold particles sized from 4 to 50 nm diameter range and approximately 5 nm sized silver nanoparticles. Prior to characterisation, particles were purified by dialysis or centrifugation, depending on the particle’s size. Silver particles were characterised by UV-visible spectroscopy and the morphology and size of both silver and gold particles were investigated by TEM. Gold particles were characterised using UV-visible, ATR-FTIR and 1H NMR spectroscopies, which highlighted the interaction between gold and Aloin A and Aloesin ligand molecules. Although NMR studies of the particles ligand shell might be an issue, due to the very small content of the organic material present on the particles surface, HR-MAS 1H NMR technique has been used. Due to the very small amount of the sample needed for the analysis, this technique resulted very advantageous and promising in the studies of the particle ligand shell, appearing more functional and effective than usual NMR analysis in solution. By ligand exchange preparation method, involving citrate coated 15 nm gold particles, it was possible to exchange the citrate ligand with Aloin A and Aloesin molecules, stabilising the particles also in this way. The amount of Aloin A and Aloesin was finely tuned as well as pH of the colloidal solution allowing particle’s agglomeration studies. Agglomeration of the particles was followed by TEM microscopy. More agglomerated particles were found on lower pH values (5-7) in less protected colloidal samples (<5000 ligand molecules per particle). When the pH of the colloidal solution was adjusted to higher values (8-10) and approximately 10000 ligand molecules were set up for each gold particle good stabilisation of the particles was achieved. 50 nm Aloin A and Aloesin stabilised gold nanoparticles, prepared by two different methods were applied to the vehicle study into macrophage cells. For the biological experiments by a peritoneal washing procedure, macrophage cells were extracted from a CD1 mouse, pre-emptively sacrificed by CO2 asphyxia. Macrophages were then collected in the physiological solution and seeded in the cell culture medium (MEM) at 37°C in the CO2 atmosphere at 5% at sterile conditions. Subsequently, macrophages were treated with gold colloidal solution (50 nm Au-Aloin A and 40 nm Au-Aloesin particles obtained by citric acid reduction method). For the treatment, 50 μl of gold colloidal solution was added to 1 ml of the cell culture medium containing marcophage cells. Macrophages were then incubated with gold nanoparticles for 5, 15, 30 and 60 min in the same conditions (37°C, at 5% CO2). After the incubation, the samples were prepared for the confocal and fluorescence microscopy observations by a number of necessary steps (centrifugation, adittion of DAPI, fixation) obtaining incubated cells on a microscopy glass slides. DAPI (4',6-diamidino-2-phenylindole) fluorescent stain was used in order to stain the cells’ nuclei. Confocal microscopy observations have revealed the presence of Aloin A and Aloesin stabilised gold particles in macrophage cells cytoplasm, while the fluorescence microscopy has revealed, in some cases, the presence of these particles also in macrophages nuclei. It is an important result, since there is an emergent need for carriers that can carry bioactive agents into the cell nucleus for drug as well as gene delivery. However, in general, nanoparticles mainly localise in the cytosol (or extranucleus). Therefore, nanoparticles capable of localising into the nucleus are particularly inportant for cancer therapy. Because cancer cells have many intracellular mechanisms to limit drug molecules' access to the nucleus the direct delivery of the drug into the nucleus would circumvent these drug-resistance mechanisms [55]. NMR and IR studies of simple aminoalcohol stabilised gold nanoparticles Special properties of gold nanoparticles [56] led to their use in important applications in the areas of catalysis, optoelectronics, electron microscopy, and biology. However, the nature of the gold capping ligand bond usually remains unknown, especially for ligands bearing multiple anchor groups that can bind gold particles in different ways (carboxy, amino, hydroxy, thiol etc.). Several studies using variety of characterisation techniques have been carried out [57]. Some of the capping agents investigated include amines or amino derivatives [58]. Heath and co-workers proposed the formation of a partially covalent bond between the gold surface and the primary amines, pointing out that the stability of the Au-amine nanoparticle depends mainly on kinetic effects [59]. Further experiments were performed using amino derivatives and exploiting them as stabilisers and/or reducing agents [60-70] in the particles preparation. Various amino functionalities have been studied, including amino acids and amino-containing polymers, with the amino groups being particularly interesting due to their presence in biological and environmental systems. However, there is a lack of reports on aminoalcohols, which are an attractive class of compounds for their potential oxidation to amino acids. This PhD project research was stimulated by the novelty of gold-aminoalcohol systems, the discovery of aminoalcohol binding site(s) (NH2 vs OH functionality), and the nature of the Au-NH bond. Reproducible gold hydrosols stabilised by aminoalcohols have been prepared. Aminoalcohol stabilised gold and silver particles were characterised by spectroscopic and microscopic means giving a deep insight into the gold-nitrogen interaction. NMR spectroscopy was applied to investigate gold nanoparticles in organic solvents; [71, 72] however, many experimental difficulties (organic molecule quantity in the ligand shell, particles purity, redispersion of gold colloids) had to be overcome before the NMR technique could be applied as a valid tool in the investigation of small-sized water-soluble gold nanoparticles [73]. Aminoalcohol-stabilised gold nanoparticles in aqueous solution were prepared by the borohydride reduction of HAuCl4 in the presence of aminoalcohol. Different aminoalcohols were used as stabilisers for gold particles: ethanolamine, 2-(propylamino)ethanol, 2-amino-1,3-propanediol, DL-2-amino-1-pentanol, 3-amino-1-propanol, (±)-3-amino-1,2-propanediol, 4-amino-1-butanol, 5-amino-1-pentanol. Red gold sols with 4 nm mean diameter particles were obtained. The samples were characterised by ATR-FTIR spectroscopy in solid state, and by 1H NMR, UV-vis, and TEM analyses in solution. The ATR-FTIR spectra of the solid materials suggested the gold-aminoalcohol interaction. Considering NH2 and OH stretching vibration modes in the free and coordinated ligands, storng evidence of the NH3+ groups involved in an electrostatic bond is shown in the case of 4-amino-1-butanol stabilised gold particles. To test this, 4-amino-1-butanol hydrochloride was prepared (by bubbling gaseous HCl in a 1,2-dimethoxy-ethane solution of 4-amino-1-butanol) and the IR spectrum was collected. Successful NMR measurements were obtained only after accurate purification of the particles (dialysis). 1H NMR experiments indicated that the binding site of aminoalcohols studied is the protonated amino group ligated with an ionic bond to the gold surface, as strongly supported by general chemical shift trend. A difference of ca. 0.40-0.50 ppm, from free aminoalcohols to coordinated aminoalcohols on gold particles has been observed [74]. Thus, NMR technique is proving to be a powerful tool for the characterisation of these colloidal systems, but is still remains not fully utilised, because of the inability of current preparative methods to supply enough purified nanoparticles. Fluorescence spectroscopy and microscopy studies of dye stabilised gold nanoparticles and their potential use in biological labelling The introduction of labelling agents in biological systems is required for a facile microscopy detection of biological systems. Fluorescent labels nowadays represent widely developed tools in biology and medicine [75]. Fluorescence parameters are usually used to obtain informations on living cells. In this context, modified gold nanoparticles can be used as nano reporters. Substantial progress in the ability to fabricate nanoparticles and the discovery of their novel size dependent physical and chemical features has drawn the attention of researchers in the area of biomedical imaging. The development of targeted contrast agents such as fluorescent probes has made it possible to selectively view specific biological events and processes in both living and nonviable systems with improved detection limits, imaging modalities and engineered biomarker functionality. The fabrication of luminescent-engineered nanoparticles is expected to be integral to the development of next generation therapeutic, diagnosis and imaging technologies [76]. The aim of this project was the fabrication of novel dye stabilised gold nanoparticles with favourable luminescent properties as bio-labels and the in vitro studies of their cellular uptake. Cellular uptake of luminescent gold nanoparticles into macrophages and Human Neuroblastoma IMR-32 cells was studied. Dye-stabilised gold particles have been prepared by a particular preparation method, designed in order to obtain 30 to 50 nm sized particles. For the preparation, different reducing agents (NaBH4, Ascorbic and Citric acid) and stabilisers having photoluminescence (PL) signals in different Visible spectrum region have been applied. As stabilisers for gold particles fluorescent molecules: 2’,7’-Dichlorofluorescein, 4-Methylumelliferyl phosphate, Eosin Y (2′,4′,5′,7′-Tetrabromofluorescein disodium salt), HPTS (8-Hydroxy-1,3,6-pyrenetrisulfonic acid) and Thionin acetate (3,7-Diaminophenothiazin-5-ium acetate) have been utilised (Figure 3). These novel gold colloids were characterised by UV-vis spectroscopy, TEM and HR-MAS NMR spectroscopy while the luminescent properties of these particles were studied by fluorescence spectroscopy. Absorption and emission spectra showed no peaks due to free ligands, and were substancially different. PL spectra of gold nanoparticles, obtained by excitation at the absorption wavelength value (523-547 nm range), showed values in a 596-661 nm range, depending on the ligand kind and particles size. We have also found that the type of reducing agent influences gold nanoparticles PL emission. Moreover, when excited at 488 nm, gold sols showed two PL peaks, centered at 525 and 581 nm, in accordance with Fluorescence and Confocal microscopy observations. Indeed, when FITC (Fluorescein Isothiocyanate) fluorescence filter is used (λex = 488 nm) the particles internalised in cells, showed green fluorescence signals, while Texas Red fluorescence filter is used (λex = 568 nm) the particles showed red fluorescence signals. Luminescent gold nanoparticles were tested as labelling agents in two different cellular systems (macrophages and neuroblastoma IMR-32 cells). Dye stabilised gold particles were incubated with macrophage and IMR-32 cells for 1 h (37°C, 5% CO2), and the cellular uptake of the particles was confirmed by Confocal microscopy and TEM. Besides these techniques, Fluorescence microscopy observations have showed interesting results. These novel systems, coupling gold with the dye, have an advantage of being visualised by all three microscopy techniques, (TEM, Confocal and Fluorescence microscopy) as they satisfy their detection requirements (gold electronic density, gold fluorescence and ligand fluorescence) as they exibit the long fluorescence lifetime. For the dye-stabilised particles uptaken into macrophages and IMR-32 cells, an almost general correlation between the fluorescence signal colour and the particles size has been found. In the case of particles smaller than 10 nm in size (3 nm Au@Citrate and 10 nm Au@4-aminovaleric acid were taken as examples) only green fluorescence signals are found applying FITC filter. On the contrary, if the particle diameters are 15 nm or larger, red fluorescence signals are found when Texas Red fluorescence filter is applied. However, in some cases, for particles with broader size distribution, green and red fluorescence signals were both observed. 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Settore BIO/06 - Anatomia Comparata e Citologia
Settore CHIM/03 - Chimica Generale e Inorganica
Working Paper
Relazione Finale : terzo anno di Dottorato in Scienze e Tecnologie Chimiche, 21. Ciclo / Zeljka Krpetic, Francesca Porta, Giorgio Scarì. - [s.l] : null, 2008.
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