Design of Photosensitizing Agents for Targeted Antimicrobial Photodynamic Therapy

Photodynamic inactivation of microorganisms has gained substantial attention due to its unique mode of action, in which pathogens are unable to generate resistance, and due to the fact that it can be applied in a minimally invasive manner. In photodynamic therapy (PDT), a non-toxic photosensitizer (PS) is activated by a specific wavelength of light and generates highly cytotoxic reactive oxygen species (ROS) such as superoxide (O2-, type-I mechanism) or singlet oxygen (1O2*, type-II mechanism). Although it offers many advantages over conventional treatment methods, ROS-mediated microbial killing is often faced with the issues of accessibility, poor selectivity and off-target damage. Thus, several strategies have been employed to develop target-specific antimicrobial PDT (aPDT). This includes conjugation of known PS building-blocks to either non-specific cationic moieties or target-specific antibiotics and antimicrobial peptides, or combining them with targeting nanomaterials. In this review, we summarise these general strategies and related challenges, and highlight recent developments in targeted aPDT

Evaluation of metallophthalocyanine functionalized photocatalytic asymmetric polymer membranes for pollution control and antimicrobial activity

The conceptualisation of photosensitizing water treatment polymer membranes using phthalocyanine based photosensitizers is reported in this thesis. The key to successful preparation of stable photoactive polymer membranes was established as the covalent anchorage of the photosensitizer to a polymer, which was proven by singlet oxygen generation by the membranes without photosensitizer deterioration. Despite this limitation, the covalent linkage-incapable unsubstituted zinc (II) phthalocyanine (complex 2) was applied as a nanoconjugate of graphene quantum dots (2π(GQDs)). 2π(GQDs) was formed through π-π stacking, and was then covalently anchored, as a proof of concept. This concept was also applied to 2-(4-carboxyphenoxy) phthalocyaninato zinc (II) (complex 3) which is capable of covalent linkage but proved to deteriorate the efficiency of singlet oxygen formation with comparison to the covalent conjugates. Singlet oxygen generation by functionalized polymer membranes rendered them photocatalytic in the degradation of organic pollutants and microorganisms in water. Organic pollutant degradation capability was exemplified by 2π(GQDs) and a porphyrin-phthalocyanine heterodyad (complex 10) functionalized membranes (2π(GQDs)-memb and 10-memb respectively), where a MPc loading of approximately 0.139 μmol MPc/g of membrane was able to achieve a 4-chlorophenol degradation rate of 3.77 × 10−6 mol L−1 min−1 in a second order reaction with an initial 4-chlorophenol concentration of 3.24 × 10−4 mol L−1 for 2π(GQDs)-memb as an example. Antibacterial studies against S.aureus using a quaternized MPc and conjugates of silver triangular nanoprisms with zinc (II) and indium (III) MPcs showed note-worthy improvements in photodynamic antimicrobial chemotherapy (PACT) activity in comparison to the unquaternized MPc precursor, and the free zinc and indium MPcs respectively. Functionalization of polymer membranes with these higher activity photosensitizers translated to the formation of potentially superior biological fouling resistant membranes. The use of porphyrin-phthalocyanine polynuclei arrays (complex 10) in polymer membrane functionalization resulted in the use of a wider wavelength range (white light). The findings from this work as a whole, thus presents the potential applicability of phthalocyanine functionalized polymer membranes in water treatment technology

Like a bolt from the blue : phthalocyanines in biomedical optics

The purpose of this review is to compile preclinical and clinical results on phthalocyanines (Pcs) as photosensitizers (PS) for Photodynamic Therapy (PDT) and contrast agents for fluorescence imaging. Indeed, Pcs are excellent candidates in these fields due to their strong absorbance in the NIR region and high chemical and photo-stability. In particular, this is mostly relevant for their in vivo activation in deeper tissular regions. However, most Pcs present two major limitations, i.e., a strong tendency to aggregate and a low water-solubility. In order to overcome these issues, both chemical tuning and pharmaceutical formulation combined with tumor targeting strategies were applied. These aspects will be developed in this review for the most extensively studied Pcs during the last 25 years, i.e., aluminium-, zinc- and silicon-based Pcs

Important Advances in Antibacterial Nanoparticle-Mediated Photodynamic Therapy

Earlier applications of photodynamic therapy (PDT) were accomplished by direct or intravenous injection of the photosensitizer, followed by preferential accumulation in cancerous tissues after systemic circulation. Nowadays, nanoparticles are used as carriers and delivery systems, which also facilitate combinations of PDT with other non-invasive technologies. PDT has expanded to disease types other than cancers. Nanoparticle-mediated target specific PDT can reduce the emergence of resistance, and has introduced chemotherapy combinations with PDT, and potential repurposing of chemotherapy drugs that are being used less because of resistance. The novel discoveries of inorganic and organic dye nanoconjugate photosensitizers discussed in this chapter have enhancement PDT efficacy. This review describes the type I and II mechanisms of PDT, some of the first- and second-generation photosensitizers in the market, and the roles played by nanomaterials across the PDT clinical translation value chain. It discusses nanoparticles as delivery systems for photosensitizers, smart stimulus-responsive, and disease-targeting nanoparticles, focusing on folate, glycan-based, pH, and external stimulus-responsive targeting. Well-known in anticancer applications, folate targeting is now debuting in antibacterial applications. Other targeting technologies are discussed. Nanoparticles applications as agents for combining PDT with other therapies are discussed. The World Health Organization has identified PDT as a promising new technology

A study of the photophysicochemical and antimicrobial properties of two zinc phthalocyanine–silver nanoparticle conjugates

This work reports conjugation of two mono carboxy substituted zinc phthalocyanines with glutathione capped silver nanoparticles (GSH-AgNPs). The photophysicochemical behaviour of the novel phthalocyanines–silver nanoparticle conjugates was investigated together with simple mixtures of the silver nanoparticles with low symmetry phthalocyanines (i.e. with no direct chemical bond formation). It was observed that upon conjugation of the phthalocyanines to the silver nanoparticles, a blue shifting of the Q band was induced. The triplet lifetimes and quantum yields improved upon conjugation as compared to the phthalocyanines alone. Fluorescence lifetimes and quantum yields decreased for conjugates compared to the phthalocyanines alone due to quenching caused by the silver nanoparticles. The low symmetry phthalocyanines and their conjugates showed antimicrobial activity against Escherichia coli in the presence and absence of light

Low symmetry metallophthalocyanines and their nanoparticle conjugates for photodynamic antimicrobial chemotherapy

This thesis reports on the syntheses of novel low symmetrically substituted Zn, Sn, Ge and Ti MPc complexes containing a single carboxylic or cysteinyl group available for attachments to MNPs. The complexes were extensively characterized by various techniques to ensure their purity. Various metallic nanoparticles consisting of silver (AgNPs), gold (AuNPs) as well as quantum dots (QDs) were successfully prepared and conjugated to the low symmetry phthalocyanine complexes. The conjugates were successfully characterized using many techniques. The Q-band maxima of the MPcs were observed at completely different wavelength regions depending on the nature of the substituents and the central metal used. Blue shifting of the Q band in the absorption spectra was observed for the complexes in the presence of AuNPs, while aggregation was observed in the presence of quantum dots. The complexes were successfully electrospun into polymer fibers for the antimicrobial inhibition of bacteria. The photophysical and photochemical properties of these complexes were extensively investigated. Higher triplet and singlet oxygen quantum yields were achieved for the Ge Pc complexes, with all the complexes giving reasonable singlet oxygen quantum yields. An enhancement in triplet and singlet oxygen quantum yields was observed for all the complexes in the presence of metal nanoparticles. However, the singlet oxygen quantum yields decreased for all the complexes when incorporated into electrospun fibers. The antimicrobial behaviour of the complexes was investigated against Bacillus Subtilis and Staphylococcus Aureus in solution and in the fiber matrix. High antimicrobial inhibitions were observed for the Ge complexes followed by the ZnPc derivatives. All the low symmetry ZnPc derivatives were conjugated to AgNPs and their antimicrobial behaviour was compared to their symmetrical counterparts. The best antimicrobial inhibition behaviour was observed for the low symmetry Pcs when compared to their symmetrical counterparts. In the absence and in the presence of AgNPs, axially ligated SiPc also showed better antimicrobial activity when it was compared to the unsubstituted ZnPc complex

Photophysicochemical and photodynamic antimicrobial chemotherapeutic studies of novel phthalocyanines conjugated to silver nanoparticles

This work reports on the synthesis, characterization and the physicochemical properties of novel unsymmetrically substituted zinc phthalocyanines: namely tris{11,19, 27-(1,2- diethylaminoethylthiol)-2-(captopril) phthalocyanine Zn ((ZnMCapPc (1.5)), hexakis{8,11,16,19,42,27-(octylthio)-1-(4-phenoxycarboxy) phthalocyanine} Zn (ZnMPCPc(1.7)) and Tris {11, 19, 27-(1,2-diethylaminoethylthiol)-1,2(caffeic acid) phthalocyanine} Zn ((ZnMCafPc (1.3)). Symmetrically substituted counterparts (tetrakis(diethylamino)zinc phthalocyaninato (3.8), octakis(octylthio)zinc phthalocyaninato (3.9) and tetrakis (carboxyphenoxy)zinc phthalocyaninato (3.10) complexes) were also synthesized for comparison of the photophysicochemical properties and to investigate the effect of the substituents on the low symmetry Pcs. The complexes were successfully characterized by IR, NMR, mass spectral and elemental analyses. All the complexes showed the ability to produce singlet oxygen, while the highest triplet quantum yields were obtained for 1.7, 1.5 and 3.9 (0.80, 0.65 and 0.62 respectively and the lowest were obtained for 1.3 and 3.10 (0.57 and 0.47 respectively). High triplet lifetimes (109-286 μs) were also obtained for all complexes, with 1.7 being the highest (286 μs) which also corresponds to its triplet and singlet quantum yields (0.80 and 0.77 respectively). The photosensitizing properties of low symmetry derivatives, ZnMCapPc and ZnMCafPc were investigated by conjugating glutathione (GSH) capped silver nanoparticles (AgNP). The formation of the amide bond was confirmed by IR and UV-Vis spectroscopies. The photophysicochemical behaviour of the novel phthalocyanine-GSH-AgNP conjugates and the simple mixture of the Ag NPs with low the symmetry phthalocyanines were investigated. It was observed that upon conjugation of the phthalocyanines to the GSH-AgNPs, a blue shift in the Q band was induced. The triplet lifetimes and quantum yields improved upon conjugation as compared to the phthalocyanines (Pc) alone. Complex 1.5 triplet lifetimes increased from 109 to 148 and triplet quantum yield from 0.65 to 0.86 upon conjugation. Fluorescence lifetimes and quantum yields decreased for the conjugates compared to the phthalocyanines alone, due to the quenching caused by the Ag NPs. The antimicrobial activity of the zinc phthalocyanines (complexes 1.3 and 1.5) and their conjugates against Escherichia coli was investigated. Only 1.3 and 1.5 complexes were investigated because of the availability of the sample. In general phthalocyanines showed increase in antibacterial activity with the increase in phthalocyanines concentration in the presence and absence of light. The Pc complexes and their Ag NP conjugates showed an increase in antibacterial activity, due to the synergistic effect afforded by Ag NP and Pcs. Improved antibacterial properties were obtained upon irradiation. 1.5-AgNPs had the highest antibacterial activity compared to 1.3-AgNPs conjugate; these results are in agreement with the photophysical behaviour. This work demonstrates improved photophysicochemical properties of low sym

Photophysicochemical and photodynamic antimicrobial chemotherapeutic studies of novel phthalocyanines conjugated to silver nanoparticles

This work reports on the synthesis, characterization and the physicochemical properties of novel unsymmetrically substituted zinc phthalocyanines: namely tris{11,19, 27-(1,2- diethylaminoethylthiol)-2-(captopril) phthalocyanine Zn ((ZnMCapPc (1.5)), hexakis{8,11,16,19,42,27-(octylthio)-1-(4-phenoxycarboxy) phthalocyanine} Zn (ZnMPCPc(1.7)) and Tris {11, 19, 27-(1,2-diethylaminoethylthiol)-1,2(caffeic acid) phthalocyanine} Zn ((ZnMCafPc (1.3)). Symmetrically substituted counterparts (tetrakis(diethylamino)zinc phthalocyaninato (3.8), octakis(octylthio)zinc phthalocyaninato (3.9) and tetrakis (carboxyphenoxy)zinc phthalocyaninato (3.10) complexes) were also synthesized for comparison of the photophysicochemical properties and to investigate the effect of the substituents on the low symmetry Pcs. The complexes were successfully characterized by IR, NMR, mass spectral and elemental analyses. All the complexes showed the ability to produce singlet oxygen, while the highest triplet quantum yields were obtained for 1.7, 1.5 and 3.9 (0.80, 0.65 and 0.62 respectively and the lowest were obtained for 1.3 and 3.10 (0.57 and 0.47 respectively). High triplet lifetimes (109-286 μs) were also obtained for all complexes, with 1.7 being the highest (286 μs) which also corresponds to its triplet and singlet quantum yields (0.80 and 0.77 respectively). The photosensitizing properties of low symmetry derivatives, ZnMCapPc and ZnMCafPc were investigated by conjugating glutathione (GSH) capped silver nanoparticles (AgNP). The formation of the amide bond was confirmed by IR and UV-Vis spectroscopies. The photophysicochemical behaviour of the novel phthalocyanine-GSH-AgNP conjugates and the simple mixture of the Ag NPs with low the symmetry phthalocyanines were investigated. It was observed that upon conjugation of the phthalocyanines to the GSH-AgNPs, a blue shift in the Q band was induced. The triplet lifetimes and quantum yields improved upon conjugation as compared to the phthalocyanines (Pc) alone. Complex 1.5 triplet lifetimes increased from 109 to 148 and triplet quantum yield from 0.65 to 0.86 upon conjugation. Fluorescence lifetimes and quantum yields decreased for the conjugates compared to the phthalocyanines alone, due to the quenching caused by the Ag NPs. The antimicrobial activity of the zinc phthalocyanines (complexes 1.3 and 1.5) and their conjugates against Escherichia coli was investigated. Only 1.3 and 1.5 complexes were investigated because of the availability of the sample. In general phthalocyanines showed increase in antibacterial activity with the increase in phthalocyanines concentration in the presence and absence of light. The Pc complexes and their Ag NP conjugates showed an increase in antibacterial activity, due to the synergistic effect afforded by Ag NP and Pcs. Improved antibacterial properties were obtained upon irradiation. 1.5-AgNPs had the highest antibacterial activity compared to 1.3-AgNPs conjugate; these results are in agreement with the photophysical behaviour. This work demonstrates improved photophysicochemical properties of low sym

Phototheranostic agents for breast cancer treatment

La llum juga un paper crucial en teràpies modernes, particularment en el camp de la teràpia fotodinàmica. La teràpia fotodinàmica es una alternativa de tractament en el camp de l’oncologia, la qual aprofita la interacció de la llum amb agents fotosensibilitzadors per produir mort cel·lular. Tot i així, la seva aplicació clínica s’ha vist limitada per la falta de selectivitat i eficàcia en la destrucció de cèl·lules canceroses, així com el desafiament d’entregar de manera eficient els fotosensibilitzadors en la zona tumoral. En vista de la problemàtica presentada, el present treball de tesi estima abordar dues propostes, una basada en l’ús d’anticossos i l’altra en l’ús de nanoestructures. La primera proposta oferix selectivitat al fotosensibilitzador mitjançant l’ús d’anticossos monoclonals. S’han desenvolupat fotoimmunoconjugats amb l’anticòs trastuzumab i el fotosensibilitzador IRDye®700DX (IR700). S’ha avaluat les seves propietats fotofísiques i fotoquímiques, així com l’activitat fototòxica. La conjugació proporciona selectivitat contra cèl·lules tumorals que sobre expressen el receptor HER2 (HER2-positives). A la vegada, s’ha realitzat la conjugació amb un biosimilar del trastuzumab, Herzuma®, demostrant activitat fotodinàmica indistinguible i selecció cel·lular. A més, la conjugació de trastuzumab amb IR700 ofereix acció fotodinàmica contra cèl·lules HER2-positives resistents a trastuzumab. També, s’ha desenvolupat un fotoquimioimmunoconjugat, basat en la unió de trastuzumab amb IR700 i doxorrubicina (Dox), amb alliberació de l’agent quimioterapèutic provocada per l’exposició a la llum. S’ha avaluat l’efecte fototòxic cel·lular, així com la seva fotofísica. La unió de Dox al conjugat provoca una reducció en la capacitat de tractament. El fotoquimioimmunoconjugat, presenta selectivitat per cèl·lules que sobre expressen el receptor HER2. Finalment, s’ha desenvolupat un altre fotoimmunoconjugat basat en l’ús d’un derivat d’aza-BODIPY unit a l’anticòs trastuzumab. S’ha avaluat el fotosensibilitzador de manera fotofísica i fotoquímica, així com l’efecte fotodinàmic. Tot i no demostrar gran producció d’oxigen singlet, ha sigut efectiu en erradicar cèl·lules tumorals. Al unir-lo amb l’anticòs ha demostrat preferència fotodinàmica contra cèl·lules que sobre expressen el receptor HER2. La segona proposta aborda l’estudi de fotosensibilitzadors autoassemblatge formant nanoestructures entregant de manera mes eficient al teixit diana. S’han utilitzat ftalocianines de zinc(II) (ZnPc) amfifíliques amb capacitat d’autoassemblar-se en aigua. S’han avaluat les seves propietats fotofísiques i fotoquímiques, així com la fototoxicitat cel·lular. Els derivats de nanopartícules de ZnPc han demostrat la capacitat de desassemblar-se amb l’interacció cel·lular, donant especies fotoquímicament actives. Han provat ser efectius pel tractament fotodinàmic. El resultats obtinguts demostren noves vies en el disseny de teràpia fotodinàmica i exalten les necessitats d’abordar les complexitats associades en l’entrega i interacció de fotosensibilitzadors per millorar els resultats i poder arribar a clínica.La luz desempeña un papel crucial en terapias modernas, particularmente en el campo de la terapia fotodinámica. La terapia fotodinámica es una alternativa de tratamiento en el campo de la oncología, que aprovecha la interacción de la luz con agentes fotosensibilizadores para producir la muerte celular. Sin embargo, su aplicación clínica se ha visto limitada por la falta de selectividad y eficacia en la destrucción de células cancerosas, así como el desafío de entregar de forma eficiente el fotosensibilizador en la zona tumoral. Ante la problemática presentada, el presente trabajo de tesis estima abordar dos propuestas, una basada en el uso de anticuerpos y la otra en el uso de nanoestructuras. La primera propuesta ofrece selectividad al fotosensibilizador mediante el uso de anticuerpos monoclonales. Se han desarrollado fotoinmunoconjugados con el anticuerpo monoclonal trastuzumab y el fotosensibilizador IRDye®700DX. Se han evaluado sus propiedades fotofísicas y fotoquímicas, así como la actividad fototóxica. La conjugación proporciona selectividad contra células tumorales que sobre expresan el receptor (HER2-positivas). A su vez, se ha realizado la conjugación con un biosimilar del trastuzumab, Herzuma®, demostrando la misma actividad fotodinámica y selección celular. Además, la conjugación de trastuzumab con IR700 ofrece acción fotodinámica contra células HER2-positivas resistentes a trastuzumab. También, se ha desarrollado un fotoquimioinmunoconjugado, basado en la unión de trastuzumab con IR700 y doxorrubicina (Dox), con liberación del agente quimioterapéutico provocada por la exposición a la luz. Se ha evaluado el efecto fototóxico celular, así como la fotofísica. La unión de Dox en el conjugado provoca una reducción en la capacidad de tratamiento. El fotoquimioinmunoconjugado, presenta selectividad por células que sobre expresan el receptor. Por último, se ha desarrollado otro fotoimmunoconjugado basado en el uso de un derivado de aza-BODIPY unido al anticuerpo trastuzumab. Se ha evaluado el fotosensibilizador de manera fotofísica i fotoquímica, así como el efecto fotodinámico. A pesar de no demostrar gran producción de oxígeno singlete, ha sido efectivo al erradicar células tumorales. Al unirlo con el anticuerpo demostró preferencia fotodinámica contra células que sobre expresan el receptor HER2. La segunda propuesta aborda el estudio de fotosensibilizadores autoensamblables formando nanoestructuras para la mejora de entregar de forma eficiente al tejido diana. Se han utilizado ftalocianinas de zinc(II) ZnPc anfifílicas capaces de autoensamblarse en agua formando nanopartículas. Se han evaluado sus propiedades fotofísicas y fotoquímicas, así como la fototoxicidad celular. Los derivados de nanopartículas de ZnPc han demostrado la capacidad de desensamblarse con la interacción celular, dando especies fotoquímicamente activas. Han probado ser efectivos para el tratamiento fotodinámico. Los resultados obtenidos demuestran nuevas vías en el diseño de terapia fotodinámica y exaltan las necesidades de abordar las complejidades asociadas en la entrega e interacción de fotosensibilizadores para mejorar los resultados y poder llegar a clínica.Light plays a crucial role in modern therapies, particularly in photodynamic therapy, an alternative treatment in oncology. It uses the interaction of light with photosensitizing agents to produce cell death. However, its clinical application has been limited due to the lack of selectivity and effectiveness in destroying cancer cells, as well as the challenge of efficiently delivering photosensitisers to the tumour area. This thesis aims to address two proposals: one based on the use of antibodies and the other on the use of nanostructures. The first proposal aims to provide selectivity to the photosensitiser through monoclonal antibodies. A rational design of photoimmunoconjugates has been developed with the antibody trastuzumab and the photosensitiser IRDye®700DX (IR700). Its photo-physical and photochemical properties, as well as phototoxic activity, have been evaluated. The conjugation provides selectivity against tumour cells that overexpress the HER2 receptor (HER2-positive). At the same time, the conjugation with a trastuzumab biosimilar, Herzuma®, has been carried out, demonstrating indistinguishable photodynamic activity and cell selection. In addition, the conjugation of trastuzumab to IR700 provides photodynamic action against trastuzumab-resistant HER2-positive cells. Also, a photochemoimmunoconjugate has been developed, based on the orthogonal union of trastuzumab with IR700 and doxorubicin (Dox), with the release of the chemotherapeutic agent caused by exposure to light. The cellular phototoxic effect has been evaluated, as well as its photophysics. The binding of Dox to the conjugate causes a reduction in the treatment capacity. The photochemoimmunoconjugate has selectivity for cells that overexpress the HER2 receptor. Finally, another photoimmunoconjugate has been developed based on the use of an aza-BODIPY derivative attached to the trastuzumab antibody. The photosensitiser has been evaluated photophysically and photochemically, as well as the photodynamic effect. Despite not demonstrating a large production of singlet oxygen, it has been effective in eradicating tumour cells. By combining it with the antibody, it has shown photodynamic preference against cells that overexpress the HER2 receptor. The second proposal focuses on the study of self-assembling photosensitisers forming nanostructures for more efficient delivery to the target tissue. Amphiphilic zinc(II) phthalocyanine (ZnPc) photsensitisers capable of self-assembling in water forming nanoparticles have been used. Their photophysical and photochemical capacities, as well as cellular phototoxicity, have been evaluated. Derivatives of ZnPc nanoparticles have demonstrated the ability to disassemble with the interaction with cells, generating photochemically active species. They have demonstrated capacity in photodynamic treatment. The results obtained demonstrate new ways in the design of photodynamic therapy and highlight the need to address the complexities associated with the delivery and interaction of photosensitisers to improve results and reach the clinic

Porphyrin Derivative Nanoformulations for Therapy and Antiparasitic Agents.

Porphyrins and analogous macrocycles exhibit interesting photochemical, catalytic, and luminescence properties demonstrating high potential in the treatment of several diseases. Among them can be highlighted the possibility of application in photodynamic therapy and antimicrobial/antiparasitic PDT, for example, of malaria parasite. However, the low efficiency generally associated with their low solubility in water and bioavailability have precluded biomedical applications. Nanotechnology can provide efficient strategies to enhance bioavailability and incorporate targeted delivery properties to conventional pharmaceuticals, enhancing the effectiveness and reducing the toxicity, thus improving the adhesion to the treatment. In this way, those limitations can be overcome by using two main strategies: (1) Incorporation of hydrophilic substituents into the macrocycle ring while controlling the interaction with biological systems and (2) by including them in nanocarriers and delivery nanosystems. This review will focus on antiparasitic drugs based on porphyrin derivatives developed according to these two strategies, considering their vast and increasing applications befitting the multiple roles of these compounds in nature