Of impurities, drugs and metabolites - ToF-SIMS application studies for use in the pharmaceutical sciences

This thesis aims to explore the use of Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) for applications in the pharmaceutical sciences through three separate studies with different pharmaceutical applications. The first study investigated the use of ToF-SIMS for the surface characterisation of pharmaceutical crystals using 4-nitrophenol and 4-acetamidobenzoic acid impurity incorporations in acetaminophen (paracetamol) crystals. A range of impurity deposition scenarios were examined to study the impurity intensity and distribution patterns as well as impurity-induced changes to the crystal surfaces. In the second study, the characteristics of human coronary artery endothelial cells (HCAECs) before and after application of an anti-thrombotic drug were analysed using ToF-SIMS. A sample preparation method was developed to analyse the cells as well as identify and image the drug on the cell surface. Subsequently, untreated and treated cells were prepared using the trialled procedure to investigate the effects of the drug on the cells. The final study explored the use of ToF-SIMS for bacterial analysis, in particular agar-based bacteria and the tracking of bacterial metabolites. In this scenario, various sample preparation and drying methods were trialled and the most successful method applied to attempt the identification and tracking of tetracycline, a known streptomyces metabolite and antibiotic, in the bacterial growth medium. In addition to the ToF-SIMS experimental studies, a software tool for the selection of spectral pre-processing methods for NIR and other spectral applications was developed and assessed. The software toolbox enables a design-of-experiment-centred approach to selecting viable pre-processing methods to correct spectral data prior to further usage in applications such as regression modelling. Two data use-cases are presented that stem from the chemical and pharmaceutical sciences and demonstrate the applicability of the tool.This thesis aims to explore the use of Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) for applications in the pharmaceutical sciences through three separate studies with different pharmaceutical applications. The first study investigated the use of ToF-SIMS for the surface characterisation of pharmaceutical crystals using 4-nitrophenol and 4-acetamidobenzoic acid impurity incorporations in acetaminophen (paracetamol) crystals. A range of impurity deposition scenarios were examined to study the impurity intensity and distribution patterns as well as impurity-induced changes to the crystal surfaces. In the second study, the characteristics of human coronary artery endothelial cells (HCAECs) before and after application of an anti-thrombotic drug were analysed using ToF-SIMS. A sample preparation method was developed to analyse the cells as well as identify and image the drug on the cell surface. Subsequently, untreated and treated cells were prepared using the trialled procedure to investigate the effects of the drug on the cells. The final study explored the use of ToF-SIMS for bacterial analysis, in particular agar-based bacteria and the tracking of bacterial metabolites. In this scenario, various sample preparation and drying methods were trialled and the most successful method applied to attempt the identification and tracking of tetracycline, a known streptomyces metabolite and antibiotic, in the bacterial growth medium. In addition to the ToF-SIMS experimental studies, a software tool for the selection of spectral pre-processing methods for NIR and other spectral applications was developed and assessed. The software toolbox enables a design-of-experiment-centred approach to selecting viable pre-processing methods to correct spectral data prior to further usage in applications such as regression modelling. Two data use-cases are presented that stem from the chemical and pharmaceutical sciences and demonstrate the applicability of the tool

Impact of paracetamol impurities on face properties : investigating the surface of single crystals using TOF-SIMS

Understanding the mechanism of interaction between pharmaceutical molecules (APIs) and impurities on crystal surfaces is a key concept in understanding purification and for the design of pharmaceutical crystallization processes. Several techniques may be used to study crystal surface properties, such as scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), which provide detailed imaging and elemental surface characterization. Time of flight secondary ion mass spectrometry (TOF-SIMS) is valuable in determining molecular identity and distribution. By combining TOF-SIMS, SEM, and optical (OM) and Raman microscopies, we can evaluate the usefulness of TOF-SIMS as a surface characterization technique for pharmaceutical crystals. 4-Nitrophenol has been selected as an impurity that can be incorporated during crystallization of acetaminophen (paracetamol). This study explores the distribution of impurity and its concentration on the different crystal faces of samples obtained by crystallization over a range of impurity loadings and supersaturation conditions. Raman maps of paracetamol single crystal faces were analyzed using the characteristic Raman peak intensity of 4-nitrophenol to identify regions where it accumulated; Raman maps of three single crystals produced in the presence of 4-nitrophenol using different crystallization procedures highlight how it can be difficult to detect very low concentrations of similar chemical species. In contrast, the 4-nitrophenol monoisotopic mass obtained via TOF-SIMS was shown to be detectable in all the three single crystals produced. This indicates that TOF-SIMS can be a valuable technique for single crystal impurity distribution mapping even when the impurity concentration is very low

Non-leaching, highly biocompatible nanocellulose surfaces that efficiently resist fouling by bacteria in an artificial dermis model

Peer reviewe

Non-leaching, Highly Biocompatible Nanocellulose Surfaces That Efficiently Resist Fouling by Bacteria in an Artificial Dermis Model

Bacterial biofilm infections incur massive costs on healthcare systems worldwide. Particularly worrisome are the infections associated with pressure ulcers and prosthetic, plastic, and reconstructive surgeries, where staphylococci are the major biofilm-forming pathogens. Non-leaching antimicrobial surfaces offer great promise for the design of bioactive coatings to be used in medical devices. However, the vast majority are cationic, which brings about undesirable toxicity. To circumvent this issue, we have developed antimicrobial nanocellulose films by direct functionalization of the surface with dehydroabietic acid derivatives. Our conceptually unique design generates non-leaching anionic surfaces that reduce the number of viable staphylococci in suspension, including drug-resistant Staphylococcus aureus, by an impressive 4-5 log units, upon contact. Moreover, the films clearly prevent bacterial colonization of the surface in a model mimicking the physiological environment in chronic wounds. Their activity is not hampered by high protein content, and they nurture fibroblast growth at the surface without causing significant hemolysis. In this work, we have generated nanocellulose films with indisputable antimicrobial activity demonstrated using state-of-the-art models that best depict an "in vivo scenario". Our approach is to use fully renewable polymers and find suitable alternatives to silver and cationic antimicrobials. © 2020 American Chemical Society

ToF-SIMS : methods & applications

Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a powerful tool for investigating the elemental and molecular variation across surfaces and through sub-surface layers

Antimicrobial Photodynamic Treatment with Mother Juices and Their Single Compounds as Photosensitizers

The potent antimicrobial effects of antimicrobial photodynamic therapy (aPDT) with visible light plus water-filtered infrared-A irradiation and natural compounds as photosensitizers (PSs) have recently been demonstrated. The aim of this study was to obtain information on the antimicrobial effects of aPDT with mother juices against typical cariogenic oral Streptococcus pathogens in their planktonic form and determine its eradication potential on total human salivary bacteria from volunteers. Mother juices of pomegranate, bilberry, and chokeberry at different concentrations were used as PSs. The unweighted (absolute) irradiance was 200 mW cm−2, applied five minutes. Planktonic cultures of Streptococcus mutans and Streptococcus sobrinus and total mixed bacteria from pooled saliva of volunteers were treated with aPDT. Up to more than 5 log10 of S. mutans and S. sobrinus were killed by aPDT with 0.4% and 0.8% pomegranate juice, 3% and 50% chokeberry juice, and 12.5% bilberry juice (both strains). Concentrations of at least 25% (pomegranate) and >50% (chokeberry and bilberry) eradicated the mixed bacteria in saliva samples. This pilot study has shown that pomegranate mother juice is superior to the berry juices as a multicomponent PS for killing pathogenic oral bacteria with aPDT

The impact of paracetamol impurities on face-specific properties : investigating the surface of single crystals using TOF-SIMS

Understanding the mechanism of interaction between pharmaceutical molecules (API’s) and impurities on a crystal surfaces is key concept in understanding purification and designing pharmaceutical crystallization processes. Different techniques may be used to study crystal surface properties, such as scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) that provide detailed imaging or elemental surface characterization. However, for studying molecular mechanisms of interaction time of flight secondary ion mass spectrometry (TOF-SIMS) is valuable in determining molecular identification and distribution. A primary ion pulse is accelerated and focused on the sample surface under ultra-high vacuum to emit secondary particles from the crystal surface. The emitted particles comprise; molecules, fragments of molecules and atoms. Analyzing these secondary particles provides information about the molecular and elemental species present on the surface. By combining TOF-SIMS, SEM and OM analysis we can determine the usefulness of TOF-SIMS as a surface characterization technique for pharmaceutical crystals. The added value of this surface characterization technique is to understand the interactions between different molecular species during purification. 4-nitrophenol has been selected as an impurity which can be incorporated during crystallization of acetaminophen (paracetamol). Our study explores the distribution of impurity concentration on the different crystal faces of samples obtained by cooling crystallization over a range of impurity loadings and supersaturation conditions. HPLC, Raman spectroscopy and single crystal XRD are used to verify the overall impurity presence and concentration and to confirm the identity of the crystal faces investigated

The interaction potential of of herbal medicinal products: a luminescence-based screening platform for assessing effects on cytochrome P450 and its use with Devil\u27s Claw (Harpagophyti Radix) preparations

Objectives  Potential interactions between herbal medicinal products and the cytochrome (CYP) P450 system are an important safety concern. We set out to develop a screening panel for assessing such interactions and use it to evaluate the interaction potential of devil\u27s claw. Methods  The panel consisted of luminescence-based inhibition assays for CYP1A2, 2C9, 2C19, 2D6 and 3A4, and a reporter gene (luciferase) assay for pregnane X receptor (PXR) activation and CYP3A4 induction. Caftaric acid and chlorogenic acid, two compounds with strong fluorescence quenching properties, were used to demonstrate the assay\u27s resistance to interference. We tested 10 commercial devil\u27s claw preparations as well as harpagoside and harpagide, two important constituents of devil\u27s claw. Key findings  Five preparations were found to weakly inhibit CYP3A4 (IC50 124.2–327.6 µg/ml) and five were found to weakly activate PXR (EC50 10.21–169.3 µg/ml). Harpagoside and harpagide did not inhibit CYP3A4. In agreement with published data, bergamottin, a natural product known to interact with CYP3A4, was shown to inhibit CYP3A4 with an IC50 of 13.63 µm and activate PXR with an EC50 of 6.7 µm. Conclusions  Devil\u27s claw preparations are unlikely to have a clinically relevant effect on CYP function. The assay panel proved effective in screening devil\u27s claw preparations, demonstrating its suitability for use with plant extracts. It showed superior sensitivity and resistance to interference