Fighting infections with light:targeted antimicrobial photodynamic therapy

Bacterial infections represent a major concern for today’s healthcare system, especially in nosocomial settings where frail and immunocompromised patients are threatened by increasingly drug-resistant pathogens. Since antimicrobial resistance leads to high morbidity and mortality, there is a pressing need to develop alternative antimicrobial therapies to which bacteria can neither adapt nor acquire resistance. Bacteria-targeted antimicrobial photodynamic therapy (aPDT) can potentially meet this challenge. Targeted aPDT relies on the combination of a targeting agent that is chemically coupled to a photo-activatable drug that is referred to as photosensitizer. Upon activation by light, the bacteria-targeted photosensitizer will generate reactive oxygen species that simultaneously destroy multiple essential components of the targeted bacterial cells. The present PhD research was aimed at identifying effective targeting molecules for aPDT of infections caused by methicillin-resistant Staphylococcus aureus (MRSA). These targeting molecules included S. aureus-specific monoclonal antibodies, or molecules with a much broader target spectrum, such as antibiotics or the bacteria-binding domain from a bacteriophage. The different molecules were conjugated to a near-infrared photosensitizer, which is already clinically applied in anti-cancer PDT. This resulted in a portfolio of novel conjugates for targeted aPDT, which were shown to be highly effective in killing MRSA. Notably, these conjugates were even effective when the bacteria were hiding within human cells, or in hard-to-eradicate multicellular communities known as biofilms. Altogether, the results show that targeted aPDT holds great promise for clinical application in the treatment of bacterial infections, even if the bacteria are highly resistant to antibiotics

Galacto-ftalocianinas de silício para o tratamento do cancro da bexiga

Mestrado em Bioquímica - Bioquímica ClínicaA terapia fotodinâmica (PhotodynamicTherapy, PDT) é uma metodologia emergente no tratamento de diversas doenças oncológicas e tem por base o uso de oxigénio molecular, luz e um fotossensibilizador (FS) para seletivamente destruir as células tumorais. Em oncologia, a PDT leva à indução de espécies reativas de oxigénio (reactive oxygen species, ROS) no tecido tumoral, no qual ocorreu previamente o uptake preferencial e/ou a retenção de um FS. As ftalocianinas têm-se vindo a revelar FSs promissores na PDT devido às suas propriedades foto-físicas. Contudo, estes compostos para além de pouco solúveis em água, têm problemas de agregação e de especificidade para os tecidos tumorais. Assim, o trabalho apresentado nesta tese teve como objetivo principal conjugar co-axialmente ftalocianinas de silício (silicon phthalocyanines, SiPcs) com duas moléculas de galactose (SiPcGal2) e com duas unidades dendríticas de galactose (SiPcGal4) para que estes FSs fossem reconhecidos por galectinas (e.g. galectina-1) sobrexpressas em células tumorais. Contudo, os compostos desejados finais não foram obtidos, uma vez que a remoção dos grupos isopropilideno, protetores dos grupos hidroxilo das unidades de galactose, não foi conseguida. Assim, foram avaliadas as propriedades foto-físicas e foto-químicas das SiPcs com as galactoses protegidas, comparando com a SiPc dihidróxido (SiPc(OH)2), de forma a estudar a influência da conjugação co-axial de biomoléculas no core destes tipo de FSs. Infelizmente, a solubilidade das SiPcs em solventes aquosos não foi conseguida, contudo o seu espectro de absorção UV-visível evidenciou elevada absorção a altos comprimentos de onda (650-700 nm), janela espectrofotométrica onde ocorre uma penetração mais profunda da luz nos tecidos. Para além disso, estes FSs demonstraram-se excelentes marcadores fluorescentes, estáveis após irradiação e bons geradores de 1O2. Foram ainda realizados estudos in vitro com o objetivo de validar o seu potencial fotodinâmico no tratamento do cancro da bexiga, sendo que a SiPcGal4 e a SiPcGal2 agregaram nas células, tendo assim um baixo uptake, baixa toxicidade após foto-ativação e baixa produção de ROS. No geral, as SiPcs demonstraram um grande potencial como futuros FSs para a PDT, dado as suas excelentes propriedades foto-físicas, o que nos incentiva na descoberta de novas técnicas que diminuam a sua agregação nas células, como a utilização de bio-formulações estáveis e a desproteção das moléculas de galactose, que também irá aumentar a sua especificidade para células tumorais.Photodynamic Therapy (PDT) relies on the combination of a photosensitizer (PS), light and molecular oxygen (O2) to generate reactive oxygen species (ROS), which can trigger cell death pathways. In oncology, the PS needs to be preferentially accumulated in cancer cells and a good generator of ROS (especially singlet oxygen, 1O2). Phthalocyanines (Pcs) are promising PSs in PDT due to their photochemical and photophysical properties. However, Pcs present solubility and aggregation problems, as well as low selectivity to the cancer tissue. Therefore, it will be conjugated a silicon phthalocyanine (SiPc) with two galactose molecules (SiPcGal2) and another with two galacto-dendritic units (SiPcGal4), both in axial positions. The aim of that conjugation is to promote the binding of the PS with galactose-binding proteins such as galectins (e.g. galectin-1) which are found to be overexpressed in cancer cells. Nevertheless, the desired compound were not obtained, once the hydrolysis of the isopropylidene galactose-protective groups didn’t work. Thereby, the photophysical and photochemical properties of those two SiPcs with the galactose-protective groups were studied in comparison with the SiPc dihydroxide (SiPc(OH)2), in order to study the SiPc core properties as well as the influence of an axial conjugation of biomolecules. The PSs solubility was compromised in an aqueous solution, however their absorption UV-Visible spectra showed high absorption peaks at a high wavelengths range (650–700 nm), which is the ideal therapeutical window where there is a higher penetration of light into the tissues. Furthermore, these SiPcs demonstrated to be good fluorescence labels, photostable and good 1O2 generators. In vitro studies were performed with the aim of validating them as photodynamic therapeutic agents against bladder cancer cells, however SiPcGal4 and SiPcGal2 aggregated on cells, having a low uptake, phototoxicity and ROS production. Overall, SiPcs have demonstrated a great potential as future PSs for PDT, thanks to their excellent photophysical properties, which prompt us in the discovery of different approaches that diminished their aggregation on cells, such as the incorporation of PSs into bio-stable formulations and the deprotection of the galactose molecules, which will also increase their specificity to tumoral cells

A Facile and Reproducible Synthesis of Near-Infrared Fluorescent Conjugates with Small Targeting Molecules for Microbial Infection Imaging

Optical imaging of microbial infections, based on the detection of targeted fluorescent probes, offers high sensitivity and resolution with a relatively simple and portable setup. As the absorbance of near-infrared (NIR) light by human tissues is minimal, using respective tracers, such as IRdye800CW, enables imaging deeper target sites in the body. Herein, we present a general strategy for the conjugation of IRdye800CW and IRdye700DX to small molecules (vancomycin and amphotericin B) to provide conjugates targeted toward bacterial and fungal infections for optical imaging and photodynamic therapy. In particular, we present how the use of coupling agents (such as HBTU or HATU) leads to high yields (over 50%) in the reactions of amines and IRDye-NHS esters and how precipitation can be used as a convenient purification strategy to remove excess of the targeting molecule after the reaction. The high selectivity of the synthesized model compound Vanco-800CW has been proven in vitro, and the development of analogous agents opens up new possibilities for diagnostic and theranostic purposes. In times of increasing microbial resistance, this research gives us access to a platform of new fluorescent tracers for the imaging of infections, enabling early diagnosis and respective treatment

Comparison of two fluorescent probes in preclinical non-invasive imaging and image-guided debridement surgery of Staphylococcal biofilm implant infections

Abstract Implant-associated infections are challenging to diagnose and treat. Fluorescent probes have been heralded as a technologic advancement that can improve our ability to non-invasively identify infecting organisms, as well as guide the inexact procedure of surgical debridement. This study’s purpose was to compare two fluorescent probes for their ability to localize Staphylococcus aureus biofilm infections on spinal implants utilizing noninvasive optical imaging, then assessing the broader applicability of the more successful probe in other infection animal models. This was followed by real-time, fluorescence image-guided surgery to facilitate debridement of infected tissue. The two probe candidates, a labelled antibiotic that targets peptidoglycan (Vanco-800CW), and the other, a labelled antibody targeting the immunodominant Staphylococcal antigen A (1D9-680), were injected into mice with spine implant infections. Mice were then imaged noninvasively with near infrared fluorescent imaging at wavelengths corresponding to the two probe candidates. Both probes localized to the infection, with the 1D9-680 probe showing greater fidelity over time. The 1D9-680 probe was then tested in mouse models of shoulder implant and allograft infection, demonstrating its broader applicability. Finally, an image-guided surgery system which superimposes fluorescent signals over analog, real-time, tissue images was employed to facilitate debridement of fluorescent-labelled bacteria

A Facile and Reproducible Synthesis of Near-Infrared Fluorescent Conjugates with Small Targeting Molecules for Microbial Infection Imaging

Optical imaging of microbial infections, based on the detection of targeted fluorescent probes, offers high sensitivity and resolution with a relatively simple and portable setup. As the absorbance of near-infrared (NIR) light by human tissues is minimal, using respective tracers, such as IRdye800CW, enables imaging deeper target sites in the body. Herein, we present a general strategy for the conjugation of IRdye800CW and IRdye700DX to small molecules (vancomycin and amphotericin B) to provide conjugates targeted toward bacterial and fungal infections for optical imaging and photodynamic therapy. In particular, we present how the use of coupling agents (such as HBTU or HATU) leads to high yields (over 50%) in the reactions of amines and IRDye-NHS esters and how precipitation can be used as a convenient purification strategy to remove excess of the targeting molecule after the reaction. The high selectivity of the synthesized model compound Vanco-800CW has been proven in vitro, and the development of analogous agents opens up new possibilities for diagnostic and theranostic purposes. In times of increasing microbial resistance, this research gives us access to a platform of new fluorescent tracers for the imaging of infections, enabling early diagnosis and respective treatment