13 research outputs found

    Porphyrin–Schiff base conjugates bearing basic amino groups as antimicrobial phototherapeutic agents

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    New porphyrin–Schiff base conjugates bearing one (6) and two (7) basic amino groups were synthesized by condensation between tetrapyrrolic macrocycle-containing amine functions and 4-(3-(N,N-dimethylamino)propoxy)benzaldehyde. This approach allowed us to easily obtain porphyrins substituted by positive charge precursor groups in aqueous media. These compounds showed the typical Soret and four Q absorption bands with red fluorescence emission (ΦF ~ 0.12) in N,N-dimethylformamide. Porphyrins 6 and 7 photosensitized the generation of O2 (1∆g) (Φ∆ ~ 0.44) and the photo-oxidation of L-tryptophan. The decomposition of this amino acid was mainly mediated by a type II photoprocess. Moreover, the addition of KI strongly quenched the photodynamic action through a reaction with O2 (1∆g) to produce iodine. The photodynamic inactivation capacity induced by porphyrins 6 and 7 was evaluated in Staphylococcus aureus, Escherichia coli, and Candida albicans. Furthermore, the photoinactivation of these microorganisms was improved using potentiation with iodide anions. These porphyrins containing basic aliphatic amino groups can be protonated in biological systems, which provides an amphiphilic character to the tetrapyrrolic macrocycle. This effect allows one to increase the interaction with the cell wall, thus improving photocytotoxic activity against microorganisms.Fil: Pérez, María Eugenia. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; ArgentinaFil: Durantini, Javier Esteban. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones en Tecnologías Energéticas y Materiales Avanzados. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Tecnologías Energéticas y Materiales Avanzados; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; ArgentinaFil: Reynoso, Eugenia. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; ArgentinaFil: Alvarez, María Gabriela. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; ArgentinaFil: Milanesio, María Elisa. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; ArgentinaFil: Durantini, Edgardo Néstor. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentin

    The meta-substituted isomer of TMPyP enables more effective photodynamic bacterial inactivation than para-TMPyP in vitro

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    Porphyrinoid-based photodynamic inactivation (PDI) provides a promising approach to treating multidrug-resistant infections. However, available agents for PDI still have optimization potential with regard to effectiveness, toxicology, chemical stability, and solubility. The currently available photosensitizer TMPyP is provided with a para substitution pattern (para-TMPyP) of the pyridinium groups and has been demonstrated to be effective for PDI of multidrug-resistant bacteria. To further improve its properties, we synthetized a structural variant of TMPyP with an isomeric substitution pattern in a meta configuration (meta-TMPyP), confirmed the correct structure by crystallographic analysis and performed a characterization with NMR-, UV/Vis-, and IR spectroscopy, photostability, and singlet oxygen generation assay. Meta-TMPyP had a hypochromic shift in absorbance (4 nm) with a 55% higher extinction coefficient and slightly improved photostability (+6.9%) compared to para-TMPyP. Despite these superior molecular properties, singlet oxygen generation was increased by only 5.4%. In contrast, PDI, based on meta-TMPyP, reduced the density of extended spectrum β-lactamase-producing and fluoroquinolone-resistant Escherichia coli by several orders of magnitude, whereby a sterilizing effect was observed after 48 min of illumination, while para-TMPyP was less effective (p < 0.01). These findings demonstrate that structural modification with meta substitution increases antibacterial properties of TMPyP in PDI

    From the Making to the Tuning to the Use of Chlorins for Biomedical Applications

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    Chlorins are porphyrins missing a double bond. These pigments are optimal platforms for the development of novel dyes that display drug-like attributes such as photodynamic therapy (PDT) agents. More recently, it was demonstrated that chlorins can serve both as a PDT agent and as a modality for fluorescence or PET imaging. Thus, multifunctional chlorins eliminate the differences that may occur in specificity, uptake, and distribution between separate compounds or constructs for imaging and therapy. The overall goal of this dissertation is to take advantage of the reputed intrinsic attributes of chlorins as a viable tool in biomedical applications. In this endeavor, we demonstrate that three successive click reactions yield multifunctional chlorin platforms appended with either S-linked or O-linked polyethyleneglycol chains (S-PEG or O-PEG, respectively) and a carboxylate-linker to a free base of a pyrrolidine ring form from a 1,3-dipolar cycloaddition. This strategy allows rapid conjugation to biological and biotargeting motifs. As a demonstration, the soluble chlorin platforms were conjugated to lysozyme and to an amino terminated tether on a short oligonucleotide. These multi-purpose chlorins possess stability to bleaching and their photophysical properties allow use as biomolecular trackers, photodynamic therapy agents, and for fluorescence diagnostic imaging. Chelation with zinc(II) further modulates the photophysical properties. It is our desire that our studies and findings contribute to the body of knowledge of chemists, clinicians, technicians and physicians in a manner that sets a precedent as a stepping stone to produce the next generation of chlorins that are beneficial for diagnostics and treatment of human disease

    Photodynamic Inactivation of Legionella pneumophila Biofilm Formation by Cationic Tetra- and Tripyridylporphyrins in Waters of Different Hardness

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    The bacterium Legionella pneumophila is still one of the probable causes of waterborne diseases, causing serious respiratory illnesses. In the aquatic systems, L. pneumophila exists inside free-living amoebae or can form biofilms. Currently developed disinfection methods are not sufficient for complete eradication of L. pneumophila biofilms in water systems of interest. Photodynamic inactivation (PDI) is a method that results in an antimicrobial effect by using a combination of light and a photosensitizer (PS). In this work, the effect of PDI in waters of natural origin and of different hardness, as a treatment against L. pneumophila biofilm, was investigated. Three cationic tripyridylporphyrins, which were previously described as efficient agents against L. pneumophila alone, were used as PSs. We studied how differences in water hardness affect the PSs’ stability, the production of singlet oxygen, and the PDI activity on L. pneumophila adhesion and biofilm formation and in biofilm destruction. Amphiphilic porphyrin showed a stronger tendency for aggregation in hard and soft water, but its production of singlet oxygen was higher in comparison to tri- and tetracationic hydrophilic porphyrins that were stable in all water samples. All three studied porphyrins were shown to be effective as PDI agents against the adhesion of the L. pneumophila to polystyrene, against biofilm formation, and in the destruction of the formed biofilm, in their micromolar concentrations. However, a higher number of dissolved ions, i.e., water hardness, generally reduced somewhat the PDI activity of all the porphyrins at all tested biofilm growth stages

    New Dyes for Cancer Theranostics

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    Porphyrinoids are robust heterocyclic dyes studied extensively for applications in medicine and as photonic materials because of their tunable photophysical properties, diverse means of modifying the periphery, and the ability to chelate most transition metals. Commercial applications include phthalocyanine dyes in optical discs, porphyrins in photodynamic therapy, and as oxygen sensors. Most applications of these dyes require exocyclic moieties to improve solubility, target disease, modulate photophysical properties, or direct self-organization into architectures with desired photonic properties. The synthesis of the porphyrinoid depends on the desired application, but the de novo synthesis often involves several steps, is time consuming, and results in low isolated yields. Thus, applications of core porphyrinoid platforms that can be rapidly and efficiently modified to evaluate new molecular architectures allows researchers to focus on the design concepts rather than the synthetic methods and opens porphyrinoid chemistry to a broader scientific community. This dissertation focuses on two widely available, commercially viable porphyrinoids as platforms: meso-perfluorophenylporphyrin, and perfluorophthalocyanine. The perfluorophenylporphyrin is readily converted to the chlorin, bacteriochlorin, and isobacteriochlorin. Derivatives of all six of these core platforms can be efficiently and controllably made via mild nucleophilic aromatic substitution (NAS) reactions using primary S, N, and O nucleophiles bearing a wide variety of functional groups. The remaining fluoro groups enhance the photo and oxidative stability of the dyes and can serve as spectroscopic signatures to characterize the compounds or in imaging applications using 19F NMR. Using simple NAS click-type chemistry, we designed a physical organic chemistry experiment for second-year college students. Students performed NAS reactions on 5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)porphyrin (TPPF20) using three different nucleophiles. Substitution occurs preferentially at the 4-position (para) because it is thermodynamically favored, and the 2- and 6- (ortho) positions are kinetically disfavored because of steric interactions with the porphyrin ring. The activation energy depends heavily on the nucleophile. An open-source software (ImageJ from NIH) was used to quantify relative intensities of spots on a TLC plate obtained from different times and varying temperatures. These data were used to generate Arrhenius plots allowing students to determine relative activation energies for three different primary nucleophiles. The experiment was developed by five undergraduates and evaluated by 40 organic chemistry II students and eight students in a physical chemistry laboratory. Students gained a deeper understanding of the relationships between the NAS mechanism, Arrhenius plots, and activation energy. The experiment is published in the Journal of Chemical Education. Using the same NAS click-type chemistry on perfluorophthalocyanines (Pc), we developed facile synthetic methods for generating Pc derivatives with near IR absorption. The photophysics of these molecules can be fine-tuned via simple click-type substitution chemistry resulting in a decrease in the HOMO-LUMO gap for each substitution. This leads to a red-shifted absorbance that should be ideal for various biomedical and materials applications. A commercially available, cost effective surfactant, isodecyloxypropyl-1,3-diaminopropane (tomamine®), is appended onto zinc 1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25-hexadecafluorophthalocyanine (ZnF16Pc). This substitution red-shifts the UV-vis absorbance and helps in solubility. Varying the equivalents of tomamine, we were able to isolate mono, di, tri and tetra-substituted products with UV-visible absorbance, lowest energy Q bands, at ca. 748, 765, 786 and 805 nm respectively. An unexpected discovery showed that addition of a 7-membered ring on the outside of ZnF16Pc induces steric interactions that cause the otherwise planar Pc macrocycle to twist and distort. This distortion results in an unexpectedly large shift in the UV-visible spectral peaks. The resulting peak is also much broader because of flexibility in the macrocycle. These compounds are highly soluble in organic and aqueous solvents and showed high photoacoustic and photothermal conversions for photoacoustic imaging and photothermal therapy of cancers. To increase the targeting efficiency of dyes, the use of glycosylated compounds is actively pursued as a therapeutic strategy for cancer due to the overexpression of various types of sugar receptors and transporters on most cancer cells. Conjugation of saccharides to photosensitizers such as porphyrins provides a promising strategy to improve the selectivity and cell uptake of the photosensitizers, enhancing the overall photosensitizing efficacy. Most porphyrin-carbohydrate conjugates are linked via the carbon-1 position of the carbohydrate because this is the most synthetically accessible approach. However, carbon-1 galactose derivatives diminish binding since the hydroxyl group in the carbon-1 position of the sugar is a hydrogen bond acceptor in the galectin-1 sugar binding site. We therefore synthesized two isomeric porphyrin-galactose conjugates using click chemistry: one linked via the carbon-1 of the galactose, and one linked via carbon-3. Free base and zinc analogues of both conjugates were synthesized. We assessed the uptake and photodynamic therapeutic (PDT) activity of the two conjugates in both monolayer and spheroidal cell cultures of four different cell lines. For both the monolayer and spheroid models we observe that the uptake of both conjugates is proportional to the protein levels of galectin-1, as measured by fluorescence spectroscopy. Compared to the carbon-1 conjugate, the uptake of the carbon-3 conjugate was greater in cell lines containing high expression of galectin-1. After photodynamic activation, MTT and lactate dehydrogenase assays demonstrated that the conjugates induce phototoxicity in both monolayers and spheroids of cancer cells. Finally, because of the general need for other metal derivatives of the PcF16 platform other than Cu, Ni, and Zn, we embarked on the synthesis of the free base macrocycle based on both literature reports and our experiences with phthalocyanine synthesis. We found that most of the literature methods have serious drawbacks that arise mostly from the unwanted substitution of the F on the macrocycle (via either reactions with the tetrafluorphthalonitrile or of the product). Thus, we have developed a procedure that minimizes the substitution side reactions and shown that the starting materials are reasonably unreactive to substitution reactions

    Espectrometria de massa de compostos catiónicos e dos seus aductos não covalentes com oligonucleotídeos

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    Doutoramento em QuímicaO objectivo do presente trabalho é a aplicação da espectrometria de massa e espectrometria de massa tandem, ambas com ionização por electrospray (ESIMS e ESI-MS/MS respectivamente) nos modos positivo e negativo ao estudo de compostos catiónicos e das suas interacções com oligonucleotídeos. Usando ESI-MS e ESI-MS/MS no modo positivo, estudaram-se clorinas e isobacterioclorinas catiónicas com anéis de pirrolidina fundidos ao macrociclo. Estes compostos sofrem reacções de cicloreversão em fase gasosa que ocorrem por eliminação de espécies neutras ou carregadas. No caso de algumas espécies catiónicas, observaram-se processos de redução envolvendo um electrão e perdas de radicais metilo, que ocorrem, provavelmente, através da formação de um radical pirrolidínio hipervalente. Foram também estudadas porfirinas catiónicas isómericas (como bases livres ou metaladas) substituídas nas posições β-pirrólicas por grupos vinilmetilpiridil, utilizando ESI-MS e ESI-MS/MS no modo de ionização positivo. Uma das bases livres apresentou, em fase gasosa, um comportamento singular, que pode estar relacionado com a sua diferente distribuição de densidade electrónica. ESI-MS e ESI-MS/MS foram igualmente usadas para estudar um conjunto de complexos de ruténio(II) com um ligando comum, um tioéter de coroa e diferentes ligandos (diiminas)com potencialidade de intercalação ao ADN. Foram observadas perdas por fragmentação da coroa e para alguns compostos, perdas das diiminas intactas. A labilidade do ião halogénio coordenado e a possível mudança de hapticidade de k2 a k3 do ligando tripodal trispirazolilmetano nos complexos de ruténio(II) com outro tioéter de coroa foram também investigados por ESI-MS. As técnicas de ESI-MS e ESI-MS/MS foram aplicadas ao estudo de vários oligonucleotídeos e dos seus aductos com cinco complexos de ruténio nos modos positivo e negativo. Foram detectados aductos dos complexos com cadeias duplas nos modos positivo e negativo, sendo a razão das abundâncias das cadeias duplas em relação às das cadeias simples maior no modo positivo do que no modo negativo. Experiências de competição nos modos positivo e negativo foram igualmente realizadas. Um dos complexos estudados mostrou consideravelmente maior afinidade pelas cadeias duplas do que os outros quatro, nos dois modos, positivo e negativo. Como os dados obtidos apontam para mais de um tipo de ligação nos aductos de complexos de ruténio com oligonucleotídeos de cadeia dupla, é provável que a afinidade superior referida seja devida à predominância de hemi-intercalação.The aim of the present work is the application of electrospray mass spectrometry (ESI-MS) and electrospray tandem mass spectrometry (ESIMS/ MS) in the positive and negative ion mode to the study of cationic compounds and of their interactions with oligonucleotides. Using ESI-MS and ESI-MS/MS in the positive ion mode we have observed that cationic pyrrolidine-fused chlorins and isobacteriochlorins undergo cycloreversion reactions in the gas phase, through loss of either neutral or charged moieties. In the case of some cationic species, one-electron reduction with methyl loss was observed, occurring probably through hypervalent pyrrolidinium radical formation. Isomeric β-pyrrolic vinylmethylpyridyl substituted cationic porphyrins (freebases or metallated) have been also studied by ESI-MS and ESI-MS/MS in the positive ion mode. The free-base isomer has a unique gas-phase behaviour that may be related to its different electronic density distribution. ESI-MS and ESI-MS/MS were also used to study a series of ruthenium(II) complexes with a common ligand. Thiacrown fragmentation and intact diimine loss are the two major types of gas-phase reactions observed. The lability of a halogen ion ligand and a possible hapticity change from k2 to k3 of the tripodal ligand trispyrazolylmethane in ruthenium(II) complexes with another thioether was investigated by ESI-MS. ESI-MS and ESI-MS/MS were used in the positive and negative ion modes to study several oligonucleotides and their adducts with five ruthenium complexes. Adducts of the complexes with double strands were detected both in the positive and in the negative ion mode. In the positive ion mode, the ratio of the abundances of double stranded to single stranded species was higher than in the negative ion mode. Competition experiments both in the positive and negative ion mode were also undertaken. One of the complexes studied displayed considerably higher affinity for the double strands than the other four, both in the positive and negative ion mode. As more than one binding mode may occur in the doublestrand- complex adducts, it is probable that this higher affinity is due to predominance of hemi-intercalation.El objetivo del presente trabajo es la aplicación de la espectrometría de masas y espectrometría de masas tandem, ambas con ionización por electrospray (ESI-MS y ESI-MS/MS respectivamente) en los modos positivo y negativo, al estudio de compuestos catiónicos y de sus interacciones con oligonucleótidos. Utilizando ESI-MS y ESI-MS/MS en el modo positivo, fueron estudiadas clorinas e isobacterioclorinas catiónicas con anillos de pirrolidina unidos al macrociclo. Estos compuestos sufren reacciones de cicloreversión en fase gaseosa a través de la eliminación de especies neutras o cargadas. En el caso de algunas especies catiónicas, se observaron procesos de reducción incluyendo un electrón y pérdidas de radicales metilo, que probablemente ocurren mediante formación de un radical pirrolidinio hipervalente. Fueron también estudiadas porfirinas catiónicas isoméricas (como bases libres o metaladas) substituidas en las posiciones β-pirrólicas por grupos vinilmetilpiridil, utilizando ESI-MS y ESI-MS/MS en el modo de ionización positivo. Una de las bases presentó en fase gaseosa un comportamiento singular, que puede estar relacionado con su diferente distribución de densidad electrónica. Se utilizaron así mismo ESI-MS y ESI-MS/MS para estudiar un conjunto de complejos de rutenio(II) con un ligando común, un tioéter de corona y diferentes ligandos (diiminas) potencialmente intercaladores al ADN. Se observaron pérdidas por fragmentación de la corona y para algunos compuestos, pérdidas de diiminas intactas. La labilidad del ión halógeno coordinado y el posible cambio de hapticidad de k2 a k3 del ligando tripodal trispirazolilmetano en los complejos de rutenio (II) con otro tioéter de corona fueron también investigados por ESI-MS. Las técnicas de ESI-MS y ESI-MS/MS fueron aplicadas al estudio de varios oligonucleótidos y de sus aductos con cinco complejos de rutenio en los modos positivo y negativo, siendo detectados aductos de los complejos con cadenas dobles en los modos positivo y negativo, con una razón mayor de abundancias de cadenas dobles en relación con las simples en el modo positivo que en el modo negativo. Se llevaron a cabo experimentos de competición en los modos positivo y negativo. Uno de los complejos estudiados mostró una considerable mayor afinidad por las cadenas dobles de ADN que los otros cuatro en ambos modos, positivo y negativo. Como los datos obtenidos apuntan a la existencia de más de un tipo de enlace de los aductos con complejos de rutenio con oligonucleótidos de cadena doble, es probable que la superior afinidad sea debida a la predominancia de hemiintercalación

    Formulações à base de solventes eutécticos profundos para o desenvolvimento de sistemas de administração de fármacos

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    The pharmaceutical industry is characterized by constant changes and demands, driven by the need to develop new drugs, or drug formulations of existing drugs, that are more efficient and safer. In this context, strategies such as drug reformulation are frequently applied to overcome many of the drawbacks associated with the existing pharmaceuticals, given the lower cost of this approach. Approaches to enhance drug solubility, stability, and permeation are crucial to improve their therapeutic efficacy. Therefore, understanding the problems associated with the existing active pharmaceutical ingredients, developing more effective strategies for their solubilization and administration, as well as to select proper solvents or excipients, are main aspects to improve therapeutic efficacy. Additionally, the possibility to incorporate these components, namely the solvents, in the drug delivery system, to tune their properties is appealing. Recently, alternative solvents, such as deep eutectic solvents (DES), have been explored in the pharmaceutical field, showing high solvation ability and high drug permeation across biological membranes, as well as drug stabilization. Furthermore, the versatility displayed by these solvents enables their incorporation into different drug delivery systems, being even possible the use of hydrophilic biopolymers. This partnership results in positive effects in the properties of biopolymer-based materials, allowing to particularly tune the mechanical properties and the respective drug release profiles. In this context, the main goal of this thesis is focused on the development of DES formulations of existing drugs and their incorporation in delivery systems, envisioning the improvement of their efficacy. The works developed involved the rational design of these formulations and the evaluation of their impact on system’s performance. Given the rising concerns associated with antimicrobial agents, including antibiotics, this was the class mainly studied. The first study focused on the development of DES aqueous solutions of cholinium chloride:urea:malonic acid, proline:urea:malonic acid and citric acid:xylitol, to remarkably improve the solubility, stability, and therapeutic efficacy of the antibiotic ciprofloxacin. The developed formulations enhanced the drug solubility up to 430-fold, in comparison to water, and the susceptibility of Gram-negative and Gram-positive bacteria to ciprofloxacin by 2- to 4-fold, respectively, while being non-toxic to human cells at the studied concentrations. The ability to improve the therapeutic efficacy of the antibiotic while avoiding the development of antimicrobial tolerance was demonstrated. The following study aimed to use betaine-based DES aqueous solutions, such as betaine:glycerol and betaine:xylitol, in the development of ocular drug delivery systems, namely thermo-responsive microemulsions that increase their viscosity upon contact with the ocular environment. These systems allowed a sustained-release and a higher permeation of the antibiotic chloramphenicol through the cornea. Finally, a higher antimicrobial activity and faster action in case of infection caused by multi-resistant bacteria was demonstrated using these microemulsions in comparison to a commercialized formulation. The versatility of DES formulations was also explored in the development of biopolymer-based drug delivery systems. In this sense, pullulan-based adhesive films were developed for application in antimicrobial photodynamic therapy (aPDT). For this purpose, betaine-based DES (betaine:levulinic acid) were applied to improve the solubility and photostability of the natural photosensitizer, curcumin. The incorporation of the DES formulations in the films, permitted to tune pullulan’s properties, obtaining systems with higher extensibility than the pristine materials. These films also present capability to absorb skin moisture and transit into a hydrogel with and higher adhesiveness than commercial hydrogels. The use of these systems in combination with an aPDT approach, allowed to eradicate common drug-resistant strains below the detection limit in ex vivo skin samples while being non-toxic to skin cells. The partnership between DES and biopolymers was also investigated for the transdermal delivery of anti-inflammatory drugs. DES aqueous solutions, based on arginine:glycerol, were used to increase the solubility of ibuprofen (up to 7917-fold, in comparison to water). These formulations were non-cytotoxic to macrophages and shown to preserve the anti-inflammatory action of the drug. Their incorporation into alginate-based hydrogels resulted in materials with higher flexibility, that presented a sustained release of the drug. Additionally, these hydrogels promoted an enhancement in the drug permeation across human skin in comparison to their counterpart containing only ibuprofen. In conclusion, the present thesis demonstrates the versatility and advantages of DES formulations in the improvement of drug delivery and therapeutic efficacy of known drugs.A indústria farmacêutica é caraterizada por mudanças e exigências constantes, originadas pela necessidade de desenvolver novos fármacos, ou formulações de fármacos existentes, que sejam mais eficazes e seguras. Neste contexto, são frequentemente utilizadas estratégias como a reformulação farmacêutica para ultrapassar muitas das desvantagens associadas aos fármacos existentes, dado o menor custo desta abordagem. As estratégias usadas para aumentar a solubilidade, estabilidade e permeação de fármacos são cruciais para melhorar a sua eficácia terapêutica. Assim, compreender os problemas associados aos princípios ativos já existentes, desenvolver estratégias mais eficazes para a sua solubilização e administração, assim como a seleção de solventes ou excipientes apropriados, são aspetos cruciais para melhorar a eficácia terapêutica. Adicionalmente, a possibilidade de incorporar estes componentes, nomeadamente os solventes, em sistemas de administração de fármacos é apelativa para modelar as suas propriedades. Recentemente, solventes alternativos como os solventes eutécticos profundos (DES) têm sido explorados na área farmacêutica, mostrando uma elevada capacidade de solvatação e de permeação de fármacos através de membranas biológicas, bem como da sua estabilização. Adicionalmente, a versatilidade destes solventes possibilita a sua incorporação em diferentes sistemas de administração de fármacos, sendo inclusive possível o uso de biopolímeros hidrofílicos. Esta combinação resulta em efeitos positivos nas propriedades dos materiais biopoliméricos, permitindo em particular o ajuste das suas propriedades mecânicas e dos perfis de libertação dos respetivos fármacos. Neste contexto, o principal objetivo da presente tese centrou-se no desenvolvimento de formulações de DES e na sua utilização em novos sistemas de administração de fármacos já existentes, visando a melhoria da sua eficácia. Os trabalhos desenvolvidos envolveram o design racional dessas formulações e a avaliação do seu impacto no desempenho do sistema. Dada as crescentes preocupações associadas aos agentes antimicrobianos, incluindo antibióticos, esta classe de fármacos foi uma das mais estudadas. O primeiro trabalho focou-se no desenvolvimento de soluções aquosas de DES, tais como cloreto de colina:ureia:ácido malónico, prolina:ureia:ácido malónico e ácido cítrico:xilitol, para melhorar a solubilidade, a estabilidade e a eficácia terapêutica do antibiótico ciprofloxacina.As formulações desenvolvidas permitiram aumentar a solubilidade do fármaco 430 vezes comparativamente à sua solubilidade em água, e a suscetibilidade de bactérias Gram-negativas e Gram-positivas à ciprofloxacina 2 a 4 vezes, respetivamente, sendo não tóxicas para células humanas nas concentrações estudadas. Foi ainda demonstrada a capacidade de melhorar a eficácia terapêutica do antibiótico sem promover o desenvolvimento de tolerância antimicrobiana ao mesmo. O trabalho seguinte teve como objetivo a utilização de soluções aquosas de DES à base de betaína, nomeadamente betaína:glicerol e betaína:xilitol, no desenvolvimento de sistemas de administração de fármacos para administração ocular, especificamente microemulsões com carácter termo-responsivo, cuja viscosidade aumenta quando em contato com o ambiente ocular. Estes sistemas permitiram obter uma libertação contínua e uma maior permeação do antibiótico cloranfenicol através da córnea. Por fim, foi demonstrada uma maior atividade antimicrobiana e uma ação mais rápida em caso de infeção por bactérias multirresistentes, usando estas microemulsões, comparativamente com uma formulação comercial. A versatilidade das formulações aquosas de DES foi também explorada no desenvolvimento de sistemas tópicos de administração de fármacos à base de biopolímeros. Neste sentido, foram desenvolvidos filmes adesivos baseados em pululano para aplicação em terapia fotodinâmica antimicrobiana (TFDa). Com este propósito, foram utilizados DES à base de betaína (betaína:ácido levulínico)) para melhorar a solubilidade e fotoestabilidade da curcumina, um fotossensibilizador natural. A incorporação das formulações de DES nos filmes de pululano permitiu ajustar as suas propriedades, tendo-se obtido sistemas com maior extensibilidade do que os correspondentes originais. Estes filmes possuem ainda capacidade de absorver a humidade da pele e passar para a forma de hidrogel com maior adesividade do que os hidrogéis comerciais. O uso destes sistemas em combinação com uma abordagem de TFDa permitiu erradicar estirpes resistentes a antibióticos comuns, abaixo do limite de deteção do método, em amostras de pele ex vivo, sendo estes sistemas não tóxicos para as células da pele. A combinação de DES e biopolímeros foi também investigada para a administração transdérmica de fármacos anti-inflamatórios. Foram utilizadas soluções aquosas de DES à base de arginina:glicerol para aumentar a solubilidade do ibuprofeno (até 7917 vezes em comparação com a solubilidade em água). Estas formulações não apresentaram citotoxicidade para macrófagos e preservaram a ação anti-inflamatória do fármaco. A sua incorporação em hidrogéis de alginato resultou em materiais com maior flexibilidade, e que apresentam uma libertação sustentada do fármaco. Adicionalmente, estes hidrogéis promoveram um aumento da permeação do fármaco na pele humana em comparação com o sistema homólogo contento apenas ibuprofeno. Em suma, a presente tese demonstra a versatilidade e as vantagens das formulações aquosas de DES na melhoria da administração e na eficácia terapêutica de diferentes fármacos existentes.Programa Doutoral em Bioquímic

    Non-covalent interactions in organotin(IV) derivatives of 5,7-ditertbutyl- and 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine as recognition motifs in crystalline self- assembly and their in vitro antistaphylococcal activity

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    Non-covalent interactions are known to play a key role in biological compounds due to their stabilization of the tertiary and quaternary structure of proteins [1]. Ligands similar to purine rings, such as triazolo pyrimidine ones, are very versatile in their interactions with metals and can act as model systems for natural bio-inorganic compounds [2]. A considerable series (twelve novel compounds are reported) of 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp) and 5,7-diphenyl- 1,2,4-triazolo[1,5-a]pyrimidine (dptp) were synthesized and investigated by FT-IR and 119Sn M\uf6ssbauer in the solid state and by 1H and 13C NMR spectroscopy, in solution [3]. The X-ray crystal and molecular structures of Et2SnCl2(dbtp)2 and Ph2SnCl2(EtOH)2(dptp)2 were described, in this latter pyrimidine molecules are not directly bound to the metal center but strictly H-bonded, through N(3), to the -OH group of the ethanol moieties. The network of hydrogen bonding and aromatic interactions involving pyrimidine and phenyl rings in both complexes drives their self-assembly. Noncovalent interactions involving aromatic rings are key processes in both chemical and biological recognition, contributing to overall complex stability and forming recognition motifs. It is noteworthy that in Ph2SnCl2(EtOH)2(dptp)2 \u3c0\u2013\u3c0 stacking interactions between pairs of antiparallel triazolopyrimidine rings mimick basepair interactions physiologically occurring in DNA (Fig.1). M\uf6ssbauer spectra suggest for Et2SnCl2(dbtp)2 a distorted octahedral structure, with C-Sn-C bond angles lower than 180\ub0. The estimated angle for Et2SnCl2(dbtp)2 is virtually identical to that determined by X-ray diffraction. Ph2SnCl2(EtOH)2(dptp)2 is characterized by an essentially linear C-Sn-C fragment according to the X-ray all-trans structure. The compounds were screened for their in vitro antibacterial activity on a group of reference staphylococcal strains susceptible or resistant to methicillin and against two reference Gramnegative pathogens [4] . We tested the biological activity of all the specimen against a group of staphylococcal reference strains (S. aureus ATCC 25923, S. aureus ATCC 29213, methicillin resistant S. aureus 43866 and S. epidermidis RP62A) along with Gram-negative pathogens (P. aeruginosa ATCC9027 and E. coli ATCC25922). Ph2SnCl2(EtOH)2(dptp)2 showed good antibacterial activity with a MIC value of 5 \u3bcg mL-1 against S. aureus ATCC29213 and also resulted active against methicillin resistant S. epidermidis RP62A

    Supramolecular Luminescent Sensors

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    There is great need for stand-alone luminescence-based chemosensors that exemplify selectivity, sensitivity, and applicability and that overcome the challenges that arise from complex, real-world media. Discussed herein are recent developments toward these goals in the field of supramolecular luminescent chemosensors, including macrocycles, polymers, and nanomaterials. Specific focus is placed on the development of new macrocycle hosts since 2010, coupled with considerations of the underlying principles of supramolecular chemistry as well as analytes of interest and common luminophores. State-of-the-art developments in the fields of polymer and nanomaterial sensors are also examined, and some remaining unsolved challenges in the area of chemosensors are discussed

    Cationic Pyrrolidine/Pyrroline-Substituted Porphyrins as Efficient Photosensitizers against E. coli

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    New porphyrin&ndash;pyrrolidine/pyrroline conjugates were prepared by revisiting 1,3-dipolar cycloaddition reactions between a porphyrinic azomethine ylide and a series of dipolarophiles. Cationic conjugates obtained by alkylation of the pyrrolidine/pyrroline cycloadducts showed ability to generate singlet oxygen and to produce iodine in presence of KI when irradiated with visible light. Some of the cationic derivatives showed photobactericidal properties towards a Gram-negative bioluminescent E. coli. In all cases, these features were significantly improved using KI as coadjutant, allowing, under the tested conditions, the photoinactivation of the bacterium until the detection limit of the method with a drastic reduction of the required photosensitizer concentration and irradiation time. The obtained results showed a high correlation between the ability of the cationic porphyrin derivative to produce singlet oxygen and iodine and its E. coli photoinactivation profile
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