This dissertation contains the synthesis and characterization of squaraine based new functional materials. In the first part of this thesis work, a water soluble benzothiazolium squaraine dye was synthesized with pyridium pendents, and controlled aggregation properties were achieved. After formation of partially reversible J-aggregation on a polyelectrolyte (poly(acryl acid) sodium salt) template, the nonlinear, two-photon absorption cross section per repeat unit was found to be above 30-fold enhanced compared with nonaggregate and/or low aggregates. Using a similar strategy, sulfonate anions were introduced into the squaraine structure, and the resulting compounds exhibited good water solubilities. A ‘turn on’ fluorescence was discovered when these squaraine dyes interacted with bovine serum albumin (BSA), titration studies by BSA site selective reagents show these squaraine dyes can bind to both site I and II of BSA, with a preference of site II. Introduction of these squaraine dyes to BSA nanoparticles generated near-IR protein nano fabricates, and cell images were collected. Metal sensing properties were also studied using the sulfonates containing a benzoindolium squaraine dye, and the linear response of the absorption of the squaraine dye to the concentration of Hg2+ makes it a good heavy metal-selective sensing material that can be carried out in aqueous solution. Later, a squaraine scaffold was attached to deoxyribonucleosides by Sonogashira coupling reactions, in which the reaction conditions were modified. Iodo-deoxyuridine and bromo-deoxyadenosine were used as the deoxyribonucleosides building blocks, and the resulting squaraine dye-modified deoxyribonucleosides exhibited near-IR absorption and emission properties due to the squaraine chromophore. Interestingly, these non-natural deoxyribonucleosdies showed viscosity dependent photophysical properties, which make them nice candidates for fluorescence viscosity sensors at the cellular level. After incubation with cells, these iv viscosity sensors were readily uptaken by cell, and images were obtained showing regions of high viscosity in cells.

The pursuit of more suitable drugs intended for possible biological applications are a continuously growing topic of research within the scientific community. One of these suitable qualities includes the need for hydrophilicity and or some appropriate delivery system for the drug to enter into biological systems. A system of analyzing and following these compounds would then, however, be necessary to conduct any kind of mechanistic or interaction studies for he said drug within the biological system. Just to name a few, fluorescence and X-ray computed tomography (CT) methods allow for imaging of biological systems but require the need of compounds with specific qualities. Finally, even with a means of entering and following a oaded drug, it would not be complete without a way of targeting its intended location. Herein, the first chapter reports the synthesis and characterization of a fluorene-based pyridil bis-?-diketone compound with suitable one- and two-photon fluorescent properties and its encapsulation into Pluronic F127 micelles for the possible application of tracking lysosomes. Next the synthesis and characterization of a BODIPY-based fluorophore with excellent fluorescence ability is reported. This compound was conjugated to two triphenylphosphine (TPP) groups and is shown as a potential mitochondria probe within HCT-116 cells. Finally, the synthesis and characterization of diatrizoic acid (DA) based derivatives conjugated to silica nanoparticles, as well as unconjugated, are reported as potential CT contrast agents. The derivatives were also functionalized with maleimide moieties facilitating subsequent potential bioconjugation of a targeting protein via a thiol group.

In this work, we investigated the possible applications of Nd3+ ions doped lanthanium trifluoride (LaF3) nanocrystals as infrared constrast agents in the first and second biological windows of the electromagnetic spectrum, which extend from 700 to 1400 nm. For this, we use the three emissions of Nd3+ ions centered around 900, 1060, and 1330 nm, corresponding to transitions generated from the metastable state 4F3/2. In comparison with other fluorescent nanoparticles (NPs) used as biolables agents, such as semiconductor quantum dots and multiphotonic luminescent NPs, the Nd3+ doped LaF3 NPs present several advantages such as high fluorescence quantum efficiency and high chemical and spectral stabilities. We have demonstrated that, with the emission around 1060 nm is possible to obtain high brightness images of cancer cells and high penetration images of animal models (mices). Additionally, we have demonstrated that the emission around 900 nm has an appreciable thermal sensitivity that allows the use of such NPs as optical nanothermometers. As the Nd3+ concentration is increased to values around 25 mol%, this thermal sensitivity comes with a high conversion efficiency of light-to-heat, so that the NPs work as multifunctional agents capable of generating heat and measuring, simultaneously, induced local temperature. This has allowed the development of real time controlled thermal therapies of cancerous tumors in animal models (mices). / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nesta tese avaliamos as possíveis aplicações de nanocristais de trifluoreto de lantânio (LaF3) dopados com íons de Nd3+ como agentes de contrastes infravermelho na primeira e segunda janelas biológicas do espectro eletromagnético, as quais se estendem de 700 a 1400 nm. Para isso usamos as três emissões de íons de Nd3+ centradas em torno de 900, 1060 e 1330 nm, correspondentes a transições geradas a partir do estado metaestável 4F3/2. Na comparação com outras nanopartículas (NPs) fluorescentes usadas como agentes de bio-contrastes, tais como, pontos quânticos de semicondutores e NPs multifotônicas luminescentes, as NPs de LaF3 dopadas com íons de Nd3+ apresentam diversas vantagens, tais como, alta eficiência quântica de fluorescência e altas estabilidades química e espectral. Nós demonstramos com a emissão em torno de 1060 nm que é possível obter imagens de alto brilho de células cancerígenas e imagens de alta penetração de modelos animais (ratos). Adicionalmente, demonstramos que a emissão em torno de 900 nm apresenta uma apreciável sensibilidade térmica que permite utilizar tais NPs como nanotermômetros ópticos. Quando a concentração de íons de Nd3+ é elevada para valores em torno de 25 mol%, esta sensibilidade térmica vem acompanhada de uma alta eficiência de conversão luz-calor, fazendo as NPs se comportarem como agentes multifuncionais capazes de gerar calor e medir, de forma simultânea, a temperatura local induzida. Isto tem permitido o desenvolvimento de terapias térmicas, controladas em tempo real, de tumores cancerígenos em modelos animais (ratos).

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5.4pt;mso-para-margin-top:0cm;mso-para-margin-right:0cm;mso-para-margin-bottom:8.0pt;mso-para-margin-left:0cm;line-height:107%;mso-pagination:widow-orphan;font-size:11.0pt;font-family:"Calibri","sans-serif";mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-ansi-language:EN-US;mso-fareast-language:EN-US;}</style><![endif]--></p><p>Luminescentni nanokristali (nanofosfori) na bazi fluorapatita (FAP-a) dopirani elementima retkih zemalja idealni su kontrastni agenti za bio-medicinske primene, kao &scaron;to su detekcije, snimanja, praćenja i terapije ćelija kancera. Kancer je jedna od najče&scaron;ćih bolesti modernog doba čiji uspeh lečenja zavisi od rane dijagnostike i neinvazivnog tretmana. Luminescentne nanočestice mogu uneti inovativnu paradigmu u lečenje kancera kombinovanjem biosnimanja, dijagnostike i tretmana. Za studije fluorescentnih biosnimanja nanokristali fluorapatita dopirani retkim zemljama kao kontrastni agenti pružaju značajne prednosti u vidu velikih kontrasta i dugotrajnosti luminescencije, i &scaron;to je jo&scaron; važnije visoke biokompatibilnosti, netoksičnosti i bioaktivnosti. Glavni ciljevi ove doktorske disertacije su sinteza novih luminescentnih multifotonskih bionanomaterijala na bazi fluorapatita dopiranih jonima prazeodimijuma (Pr³⁺), njihova karakterizacija i evaluacija&nbsp; primene za fluorescentna biosnimanja kancera. Sintezom nanoprahova u umerenim uslovima metodom ko-precipitacije, a potom su&scaron;enjem na 110 ^oC i kalcinacijom na temperaturama od 700 i 1000 ^oC očekuje se pronalaženje najboljih uslova za dobijanje novih nanofosfora koji bi na&scaron;li i različite bio-medicinske primene u oblasti fluorescentnih biosnimanja. Proučavane su tri vrste PrFAP nanokristala, sa 0,1%, 0,5% i 1% atomskih procenta Pr³⁺, zajedno sa nedopiranim FAP kontrolnim uzorkom. Nivoi energije aktivator jona Pr³⁺ sadrže metastabilna multipletna stanja koja nude mogućnosti efikasnih emisionih linija u vi&scaron;e boja u FAP nanokristalima, kao i u infracrvenoj i ultravioletnoj oblasti spektra. Metodom ko-precipitacije na sobnoj temperaturi (25 ^oC), a potom su&scaron;enjem na 110 ^oC, sintetisani su monofazni heksagonalni nanokristali PrFAPs nepravilnog sfernog oblika. Termičkom analizom sintetisanih uzoraka, na&nbsp;osnovu detektovanih temperaturnih opsega procesa dekarbonacije i dehidroksilacije, utvrđene su temperature kalcinacije od 700 i 1000 oC. Termička analiza i karakterizacija uzoraka su pokazale da Pr³⁺ joni dovode do stabilizacije FAP strukture na vi&scaron;im temperaturama, &scaron;to je pripisano unosu lantanoidnih jona sa specifičnim magnetnim osobinama u sistem i stvaranju jačih privlačnih sila sa O^2-anjonima. Nanokristali su&scaron;eni na 100 ^oC i kalcinisani na 1000 ^oC, zbog prisustva defekata kristalne re&scaron;etke koji zadržavaju emisiju Pr³⁺ jona, nisu pokazali luminescentne karakteristike od značaja za primene u medicinskim fluorescentnim biosnimanjima. Kalcinacijom uzoraka na 700 ^oC izrađen je novi tip aktiviranih fluorapatitnih nanokristala dopira / <p>Luminescent nanocrystals (nanophosphorus) based on fluorapatite (FAP) doped with rare earth elements are ideal contrast agents for biomedical applications such as cancer cell detection, imaging, tracking and therapy. Cancer is one of the most common diseases of the modern times whose success of the cure depends on early diagnosis and non-invasive treatment. Luminescent nanoparticles can bring an innovative paradigm into the treatment of cancer by combining bioimaging, diagnostics and treatment. Rare earth doped fluorapatite nanocrystals as contrast agents for studies of fluorescence bioimaging, offer significant advantages in terms of high contrasts and long-term luminescence, and more importantly high biocompatibility, non-toxicity and bioactivity. The main objectives of this doctoral dissertation are the synthesis of novel luminescent multiphoton bionanomaterials based on fluorapatites doped with praseodymium ions (Pr³⁺), their characterization and evaluation of their application for cancer fluorescence bioimaging. Synthesis of nanopowders under moderate conditions by the co-precipitation method, followed by dried at 110 &deg;C and calcination at 700 and 1000 &deg;C, is expected to find the best conditions for obtaining new nanophosphors that would find different bio-<br />medical applications in the field of fluorescence bioimaging. Three types of PrFAP nanocrystals were studied, with 0,1%, 0,5%, and 1% atomic percentages of Pr³⁺, together with an undoped FAP control sample. Energy levels of the Pr³⁺ ion activator contain metastable multiplet states that offer the possibility of efficient multi-color emission lines in FAP nanocrystals as well as in the infrared and ultraviolet regions of the spectrum. Single-phase hexagonal nanocrystals PrFAPs of irregular spherical shape were synthesized by the method of co-precipitation at room temperature (25 ^oC) and then drying at 110 ^oC. Thermal analysis of the synthesized samples, based on the detected temperature ranges of the decarbonation and dehydroxylation processes, determined calcination temperatures of 700 and 1000 ^oC. Thermal analysis with characterization showed that Pr³⁺ ions lead to stabilization of the FAP structure at higher temperatures,&nbsp;which was attributed to the entry of lanthanoid ions with specific magnetic properties into the system and the creation of stronger attractive forces with O^2- anions. Nanocrystals dried at 100 ^oC and calcined at 1000 ^oC, due to the presence of crystal lattice defects that quench the emission of Pr³⁺ ions, did not show luminescent characteristics of significance for applications in medical fluorescence imaging. Calcination of the samples at 700 ^oC produced a new type of activated praseodymium doped fluorapatite nanocrystals (PrFAPa) with excitation-emission profiles in the visible part of the spectrum. Physicochemical characterization confirmed spherical crystals of hexagonal structure up to a nanometer size of about 20 nm. Quantum-chemical calculations predicted that Pr³⁺ ions would be embedded in the crystal lattice of FAP nanocrystals at the Ca2 position (6h), which was followed by deformations of the F^- ion position. The assumed substitution mechanism is one Pr3+ ion for one Ca²⁺, with partial substitution of F^&ndash;anions with O^2&ndash; and OH^&ndash; and creation of vacancies due to achieving system neutrality. The results of in vitro biocompatibility and hemocompatibility showed that PrFAP nanocrystals were not toxic to living cells. In addition, the internalization of PrFAPa nanocrystals by skin (A431) and lung (A549) cancer cells was studied using fluorescence-based confocal microscopy and wide-field microscopy. The nanocrystals show characteristic green emission at 545 nm (³P₀&rarr;³H₅ transition of Pr³⁺ ion) and orange emission at 600 nm (¹D₂&rarr;³H₄), which we use to discriminate from cell autofluorescence. Studies of the images obtained by confocal microscopy in the blue, green, and red channels revealed that nanocrystals could recognize the cell surface and adhere to it, but they did not confirm the entry of nanocrystals into the cells. The wide-field microscopy detected emission transitions in green and orange color, and confirmed that the luminescent signal was coming from inside the cells. Using resonant excitation of PrFAP nanocrystals at 488 nm and emission of 600 nm, confocal microscopy extracted the fluorescence signal from inside the cancer cells. Orthogonal projections across 3D confocal stacks show that the nanocrystals are able to enter the cells positioning themselves within the cytoplasm. Overall, the obtained PrFAPa nanocrystals are biocompatible and of the tested types, the 0,5% Pr³⁺ doped nanocrystals show the highest promise as a tracking nanoparticle probe for bioimaging applications.</p>

In this work, hydrophilic fluorene-based derivatives that contain ethylene oxide substituents, have been synthesized and characterized for potential use as new fluorophores for bioimaging applications and for fluorescence sensing of heavy metals. Symmetrical and unsymmetrical fluorene derivatives based on structural types of acceptor-pi-acceptor, acceptor-pi-donor, and donor-pi-donor were characterized by TGA, UV-vis absorption, fluorescence emission, lifetime, anisotropy, and two-photon absorption (2PA) cross section. They were found to possess high thermal stability, high photostability, high fluorescence quantum yields, and generally large two-photon absorption cross sections, making them quite suitable for new probes in single-photon absorption and two-photon absorption fluorescence microscopy imaging. Novel hydrophilic fluorene derivatives were synthesized from fluorene in multiple steps employing the metal-catalyzed Heck coupling reaction, the Stille reaction, the Sonogashira reaction, the Ullmann condensation reaction, and "click" chemistry. To increase the hydrophilicity of the new compounds, ethylene oxide substituents were utilized for to impart water solubility. An alternative alkylation methodology using ethyleneoxy tosylates was introduced for the synthesis of ethylene oxide-containing fluorene derivatives. Several of these hydrophilic derivatives were incubated into various cell lines as new probes for both conventional and two-photon absorption fluorescence bioimaging. These compounds were biocompatible, exhibiting low cytotoxicity as determined by cell viability studies, and displayed colocalization for selected cellular organelles. In addition, hydrophilic bis(1,2,3-triazolyl)fluorene derivatives were found to exhibit sensitive fluorescence responses in the presence of certain heavy metal, and were selective for sensing zinc and mercury over other a number of other metal ions relevant to living cells or other biological environments. The UV-vis absorption and fluorescence emission spectra of the complexes exhibited a blue-shifted absorption and emission for selective metal chelation upon binding to zinc and mercury(II) ions, resulting in an approximately two-fold enhanced fluorescence response. Fluorescence titration studies revealed that the complexes of 1:2 and 1:3 ligand to metal formed with binding constant values of 108 and 1014 for zinc and mercury ions, respectively. Finally, preliminary experiments were performed to explore the possibility of employing select hydrophilic fluorene-based derivatives in the synthesis of hydrophilic fluorescent gold nanoparticles. Although results are very preliminary, the aim is to use such materials for other biomedical applications, such as surface enhanced scattering resonance and noninvasive photothermal therapy to diagnose and to treat cancers. Thus, this research had led to the discovery of alternative methodologies for synthesis of hydrophilic fluorene derivatives by alkylation with alkyl tosylates and synthesis of hydrophilic fluorescent molecule capped gold nanoparticles. Furthermore, several novel hydrophilic fluorene-based derivatives were synthesized and characterized for their linear and nonlinear photophysical properties, and are now available for further examination of their bioimaging and sensing applications.