Polyacrylamide nanoparticle delivery systems in photodynamic therapy

In treating many diseases, including cancer and bacterial infections, drug resistance has emerged as a major obstacle limiting the therapeutic efficacy of chemotherapeutic agents. One area which may prove to be particularly attractive is Photodynamic Therapy (PDT). Reactive oxygen species (ROS) which cause damage to tumour tissue are not generated until the drug is activated by light, minimising generalised toxicity and giving a high degree of spatial control over the clinical effect. The application of nanoparticles (NPs) in the field of drug delivery is extensively studied as a potential for delivering high payloads of drugs site selectively. They can be targeted towards, and accumulate in, tumour tissue by the enhanced permeability and retention effect, if sequestration by the reticuloendothelial system (RES) is avoided. This project aimed to develop an efficient drug delivery system based on nano-sized particles. Polyacrylamide nanoparticles were chosen to deliver photosensitisers due to the chemical and biological inertness of polyacrylamide, in addition to its optical transparency. The porous three-dimensional particles produced from microemulsion polymerisation reactions are typically prepared in the nanometre range. In the study, two types of NPs loaded with photodynamic sensitisers are synthesised: photosensitiser (i.e. phthalocyanine) entrapped NPs (PCNP) and photosensitiser (i.e. phthalocyanine) entrapped NPs coated with a second photosensitiser (i.e. porphyrin) (PCNP-P) to enhance the capacity for ROS generation, and hence therapeutic potential. The mean sizes of these particles were 45±10nm and 95±10nm respectively.NP uptake by human Caucasian colon adenocarcinoma cells (HT29) was determined by flow cytometry and confocal microscopy. Cell viability assays using two chosen NPs (PC-NP and PCNP-P) corresponding to the minimum uptake time (<5 minutes) and maximum uptake time (25 hours), quantified by flow cytometry, demonstrated that these cancer cells can be damaged by activation of the photodynamic NPs both when in the external media and post internalisation. Results suggest that in order to induce photodynamic damage, the NPs need only be associated with the tumour cell closely enough to deliver singlet oxygen - their internalisation within target cells may not be necessary. Clinically, this could be of great importance as it may help to combat the ability of many cancer cells to actively expel conventional anti-cancer drugs

Photosensitiser functionalised nanofiber fabric for efficient light driven water disinfection

Sunlight-driven water disinfection system could help provide clean water to some of the world’s poorest regions where contaminated surface water is a major public health problem and bright solar irradiation is available for free. In this work, photosensitiser - 5, 10, 15, 20-tetrakis (1-methyl-4-pyridinio) porphyrin tetra p-toluene sulfonate (TMPyP) was chosen and immobilised onto chitosan nanofiber mats and chitosan membranes for photodynamic disinfection of water since preliminary studies with TMPyP in solution showed it caused a high rate of photodynamic inactivation (PDI) of model viral organisms (bacteriophages MS2 and Qβ, murine norovirus and bovine enterovirus 2). Native gel electrophoresis, SDS-PAGE and western blotting, TEM and DLS were used to analyse pre- and post-PDI samples of the model viruses. The rate of PDI in model viruses was in the order MS2 > phage Qβ > murine norovirus > bovine enterovirus 2. Our data showed that PDI caused aggregation of MS2 particles and crosslinking of MS2 coat protein. However, the aggregation and crosslinking did not correlate to the rate of PDI we observed in MS2. Using sequence specific antibodies raised against MS2 A-protein (host attachment protein), our results suggest that the rate of PDI is relative to loss of antigenicity of sites on the A-protein. The differences in the rate of PDI were compared to amino acid compositions and surface accessibility of host attachment proteins/sites of the model viruses. Possible modes of action are discussed as a means to gaining insight to the targets and mechanisms of PDI of viruses. Chitosan electrospun nanofibers and chitosan membranes were modified by pyromellitic dianhydride in order to introduce carboxyl groups and facilitate adsorption of the cationic TMPyP. The physico-chemical properties of these modified nanofibers and membranes were investigated by microscopy, absorption spectroscopy, Fourier-transform infrared spectroscopy and Midland surface blotting approaches.The chitosan nanofiber/membrane-TMPyP composite showed photodynamic inactivation of MS2 and E. coli BL21

Synthesis, aggregation and photophysical studies of A2B2 phthalocyanines: Novel archetypes for anticancer and antimicrobial photodynamic therapy

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Orgánica. Fecha de lectura: 14-02-2020Esta tesis tiene embargado el acceso al texto completo hasta el 14-08-2021En esta tesis doctoral se han desarrollado nuevos fotosensibilizadores para su aplicación en terapia fotodinámica. Este tratamiento aplica luz del espectro visible, sobre una molécula fotosensible capaz de activar el oxígeno molecular presente en el entorno celular, produciendo especies activadas citotóxicas de oxígeno, como oxígeno singlete, capaces de inducir muerte celular. Concretamente se han preparado fotosensibilizadores de segunda generación de tipo porfirinoide basados en ftalocianinas de Zn(II) (Zn(II)Pcs). En concreto, en este trabajo hemos elegido como objetivo la preparación de Zn(II)Pcs formadas por dos tipos de isoindoles con diferente sustitución, denominadas comúnmente como A2B2, que pueden presentar dos variantes estructurales, es decir, ABAB y AABB. Estas geometrías altamente orientadas, permiten combinar sustituyentes hidrófobos e hidrófilos para dar lugar a anfífilos con la capacidad de interaccionar con medios lipófilos celulares así como facilitar el transporte en medios acuosos. La Parte 1 de esta tesis doctoral está orientada a la preparación de Zn(II)Pcs ABAB. La síntesis en todos los casos está dirigida por el uso de un ftalonitrilo que presenta grupos voluminosos que impiden la formación de la especie AABB. Estos grupos voluminosos, además, poseen unidades fluoradas que dotan a la estructura final de unas características electrónicas peculiares. Esta particular sustitución, no solo evita fenómenos de agregación, unos de los principales factores limitantes del uso de macrociclos porfirinoides en terapia fotodinámica, sino que además potencia la capacidad de las ftalocianinas para producir especies reactivas de oxígeno, como oxígeno singlete. Hemos sido capaces de combinar este patrón de sustitución con sustituyentes hidrófilos formando anfífilos. La introducción de sustituyentes con cadenas de tipo polietilenglicol destruye eficientemente cultivos celulares in vitro de cáncer de piel y de cuello de útero, y se ha demostrado cómo el patrón de sustitución ABAB es más efectivo que otros dentro de la misma familia (A3B y A4). Por otra parte, la incorporación de grupos funcionales cargados positivamente convierte estos compuestos en antimicrobianos frente S. aureus y E. coli. En la Parte 2 de esta tesis doctoral se ha trabajado con Zn(II)Pcs de tipo AABB, las cuales se han preparado utilizando un conector binaftol que evita la formación de especies ABAB. En este caso el conectol binaftol presenta quiralidad por restricción conformacional, lo que ha permitido obtener Zn(II)Pcs quirales que pueden ser estudiadas y caracterizadas por dicroísmo circular. Este tipo de compuestos pueden presentar cierta capacidad de auto-ensamblaje dependiendo del medio elegido, y que está influida por el conector quiral. De nuevo la incorporación de sustituyentes hidrófilos permite la formación de especies anfifílicas, de tal forma que la organización inducida en medio acuoso ha dado lugar a la formación de nanoestructuras de tipo micelar o vesicular, mientras que en medios más apolares como puede ser el interior celular, se encuentran molecularmente disueltas. Concretamente se han preparado derivados AABB funcionalizados con cargas positivas, efectivos en la inactivación de bacterias Gram positivas y Gram negativas. Además, también se ha decorado el conector binaltol con cadenas de polietilenglicol y se ha hecho un estudio en profundidad de su capacidad de organización en medios polares como el agua

Susceptibility of mucositis-associated gram-negative bacteria to photodynamic action.

Opportunistic pathogens such as enteric Gram-negative bacilli in the oral cavity of cancer patients undergoing radio/chemotherapy may contribute to the mucosal lesions (mucositis) caused by these cytotoxic therapies and also constitute a source of infection. What is more, resistance of organisms to conventional antibacterial regimes is a growing cause of concern. Photodynamic action (PDA), elimination of target cells by use of a photosensitising compound in combination with light, may be a potential alternative therapeutic regime for topical infections. This study evaluated the effectiveness of PDA on a number of potential pathogens associated with mucositis. The PDA of toluidine blue O (TBO) with light from a HeNe gas laser on the viability of a range of Gram-negative bacilli considered to play a role in mucositis was assessed. The susceptibility of other opportunistic pathogens together with the indigenous oral flora was also studied. Gram-negative bacilli were shown to be killed by PDA the effectiveness of which was dependent on both the TBO concentration and light dose. All the organisms tested were susceptible to PDA, indicating its broad spectrum effect. Studies of the effects of physiological factors on the efficacy of PDA showed that bacteria could be killed under all the physiological conditions tested. However, PDA in saliva and serum was not as effective as it was in saline, and greater kills were obtained at increased pHs. Mechanistic studies demonstrated that TBO was an effective photosensitiser when outside the bacteria and indicated that the outer membrane may be the site where initial photo-damage takes place through the production of singlet oxygen and, to a lesser extent, hydroxyl radicals. PDA was also shown to impair some virulence factors (lipopolysaccharide and proteolytic enzyme activity) of bacteria. An animal study was conducted to elucidate any possible adverse effects on host tissues and this demonstrated that at all doses tested, PDA had no adverse effect on the buccal mucosa of rats. Furthermore, fluorescence microscopy studies demonstrated a high level of TBO in the epithelium but no detectable levels in the underlying connective tissue. Although it was shown that killing by PDA of both laboratory and clinical strains of organisms is possible, further animal studies are needed to establish the effectiveness of this novel technique for the treatment of topical, localised infections

Molecular level biological effects of silver and titania nanoparticles on zebrafish embryos

Nanotechnology raises issues concerning the toxic impact of nanoparticles (NPs) in organisms and the environment. Nanoparticles have been defined as materials with dimensions that are equal to or less than 100 nm. It is important to develop early warning tools of NP-induced biological effects to be able to monitor and manage for any possible impacts. In the current study, two types of NPs have been selected based on their wide use: silver-NPs (AgNPs) and titanium dioxide-NPs (TiO2NPs). Early zebrafish (Danio rerio) embryos exposed in vitro to 4-nm and 10-nm AgNPs, and to silver ions alone, and TiO2NPs have been used to measure the expression level of selected target genes. A global transcriptomic approach employing Suppression Subtractive Hybridization (SSH) was used in parallel to identify novel genes that may be involved in the fish embryo response as a result of exposure to NPs. TiO2NPs coated with different layers of anionic (PSS) and cationic (PAH) polyelectrolytes were also used to measure viability, morphology, and the expression level of selected target genes. The results indicate that pathways expressed in response to NP exposure differ among both AgNPs and TiO2NPs, either due to the size, concentration, exposure time, exposure conditions, surface chemistry and surface charge of coatings of the NPs. The responses indicate that D. rerio embryos respond to NPs with not only an oxidative stress response, but with transcripts associated with fertility and metabolic functions such as membrane transport and mitochondrial metabolism. This information may be used to inform early warning biomarker development for environmental monitoring applications in future

Laser sterilization of microorganisms

The effect of light from a variety of laser sources was investigated in a microbicidal capacity under a number of different conditions. Initially, Escherichia coli, Serratia marcescens, Staphylococcus aureus, Deinococcus radiodurans, Micrococcus luteus, Bacillus subtilis, B. stearothermophilus, Candida albicans and Saccharomyces cerevisiae were exposed to near infrared light, from a high-powered, 1064 nm, Neodymium:Yttrium Aluminium Garnet (Nd:YAG) laser, as lawned cultures on agar plates. The effect of the different Nd:YAG laser operation parameters (pulse repetition frequency, pulse energy, pulse width and beam diameter) were also investigated on lawned cultures of E. coli. A system of quantifying the effect of the laser light on microorganisms lawned on agar plates, which utilised the non-uniform energy distribution of the laser beam, was developed and the different organisms and laser parameters were compared. Nd:YAG laser light killed all microorganisms mentioned above, producing clear areas within the microbial lawns. For each microbial species, a dose/response curve was obtained of area of clearing versus energy density (Jcm-2). The energy density that produced an inactivated/sterilized area (IA) equal to 50 percent of the beam area was designated the IA50-value and was plotted. Average IA50-values were all within a three fold range and varied from 1768 Jcm-2 for Serratia marcescens to 4489 Jcm-2 for vegetative cells of Bacillus stearothermophilus. The differences in sensitivity could not be attributed to cell shape, size, pigmentation, or Gram reaction. The PRF, pulse energy and beam diameter all affected the sterilization capability of Nd:YAG laser on a lawned culture of E. coli. The average IA50-value at a PRF of 10 Hz was 2800 Jcm-2 which was decreased to 2200 Jcm-2 as the PRF was increased to 30 Hz. Similarly, an increase in the pulse energy from 10 J to 30 J enhanced the sterilization efficiency of the Nd:YAG laser on E. coli lawned on agar surfaces with a reduction in the average IA50-value from 2700 Jcm-2 to 1850 Jcm-2 respectively. Variation in the beam diameter was shown to have a large effect on the threshold energy density (the lowest ED required to sterilize an area in the bacterial lawn). Laser light at a beam diameter of 2.75 cm had a threshold energy density of 1400 Jcm-2 which was reduced to 800 Jcm-2, with a beam diameter of 1.25 cm. There was not such a notable effect on the IA50-values with only 200 Jcm-2 separating both of these beam diameters. The pulse width of Nd:YAG laser light did not affect the bactericidal ability of laser light. At the lowest energy densities where sterilization was achieved for the majority of organisms (around 2000 Jcm-2), no effect was observed on the nutrient agar surface, but as the energy density was increased, a depression in the agar surface was formed, followed by localised melting of the agar. Also around the periphery of the cleared areas microcolonies were frequently observed which indicated sub-lethal damage to the microbial cells. Preliminary investigations indicate that these colonies contain auxotrophic organisms

Synthesis and Characterization of Novel Nanomaterials Based on Nanocrystalline Cellulose

openModern technologies have contributed to develop our society in all different fields. The trend in current research pulls towards the development of sustainable materials, to address concerns about environmental impact. Cellulose, one of the most ubiquitous and abundant bio-resources originating from higher plants, tunicate, bateria and algae, has become attractive from the viewpoint of sustainable development. Nanocrystalline cellulose (NCC), a rod-shaped nanoscale material with exceptional strength and physicochemical properties, can be prepared from this inexpensive renewable biomass. Besides its potential use as a reinforcing agent for industrial biocomposites, pristine NCC exhibits low toxicity and poses no serious environmental concerns, providing impetus for its use in bioapplications. The chemistry of NCC is dominated by the abundance of hydroxyl groups on its surface, which can be readily converted into other functional groups or used directly to bind compounds in a non-covalent way. Both approaches are exploited in this Thesis to obtain new NCC based hybrid nanomaterials with applications ranging from energy to catalysis to drug delivery. In all cases, the synthesis and full characterization are presented. Concerning non-covalent chemistry, NCC was proposed as exfoliating agent of 2D materials in water to obtain sustainable, cost-effective energy storage devices. Regarding covalent functionalization, two different pathways were applied to modify NCC surface. The first one consisted on its oxidation to introduce carboxylic groups followed by the attachment of amino-terminated fullerene derivatives for phodynamic therapy (PDT) applications. In the second approach, nucleophilic substitution was employed to obtain an azide functionalized NCC derivative and PAMAM type dendrons were introduced afterwards via Click Chemistry. These hybrids were used to stabilize gold nanoparticles for catalysis in aqueous media.SCUOLA DI DOTTORATO DI RICERCA IN SCIENZE E TECNOLOGIE CHIMICHE E FARMACEUTICHEHADAD CAROLINEembargoed_20170420HERREROS LOPEZ, ANA MARIAHERREROS LOPEZ, ANA MARI

Photodynamic Therapy

This book is dedicated to a topic related to the effects of photodynamic therapy organized by Biomedicines in 2022 (https://www.mdpi.com/topics/photodynamic_therapy). In medicine, the use of photodynamic therapy for the treatment of oncological and non-oncological diseases has been widely documented and well codified. In dermatology, the use varies from oncological to the treatment of chronic wounds, as well as in cosmetology for photo-rejuvenation. The 19 manuscripts published in this book cover all aspects of this therapy, including the discovery of new natural and synthetic photosensitizers, biomaterials and nanotechnology, in vitro and in vivo studies, and clinical trials

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

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