Mid-IR Imaging and Multivariate Analysis of Dynamic Processes in Pharmaceutically Relevant Microparticles

Sustained release microparticles used for parenteral drug delivery must be well characterized in terms of their size range, morphology and function. It is widely understood that the chemistry and morphology of microparticles have a degree of interdependence which strongly affects drug release behaviour from microparticles. This thesis investigates, for the first time, the use of mid-IR imaging along with the development and optimisation of relevant multivariate image analysis methods for studying the real-time degradation of pharmaceutically relevant biodegradable polymer microparticles and the real-time release of protein based drugs from such microparticle systems. The application of attenuated total reflection - Fourier transform infrared spectroscopic(ATR-FTIR) imaging and analysis to monitor the degradation of a single microparticle is optimised and the developed methodology is detailed. A series of time resolved images of a PLGA microparticle undergoing hydrolysis at 70 °C are obtained using ATR-FTIR imaging for the first time. A novel partially supervised non-linear curve fitting (NLCF) tool is developed and the output from the NLCF is evaluated by direct quantitative comparison with a traditional peak height (PH) data analysis approach and multivariate curve resolution alternating least squares (MCR-ALS) analysis for the same images, in order to develop an image analysis strategy. The NLCF method is shown to facilitate the calculation of hydrolysis rate constants for both the glycolic (kG)and lactic (kL) segments of the PLGA copolymer. This results in improved spatial resolution on time-resolved microparticle images, so providing better insight into the dimensions of hydration layers and particle dimension changes during hydrolysis when compared to images derived from both PH measurements and MCR-ALS. The MCRALS routine is shown to be faster than NLCF and its images are found to provide sufficient contrast to be used for qualitative comparison. The optimised mid-IR-ATR procedures are then applied to investigate several factors influencing the hydrolytic degradation of a family of PLGA microparticles. Degradation rate constants for glycolic and lactic units are shown to increase (whilist maintaining a ~1.3 ratio between each other) with increasing initial glycolic content of the copolymer,temperature or γ-radiation exposure. Differential scanning calorimetry (DSC) and gel permeation chromatography (GPC) results indicate a chain scission based degradation in PLGA upon γ exposure. The distribution of lactic acid is probed with IR during the hydrolysis of a PLA microparticle for the first time, showing a diffusional pathway from the degrading microparticle outwards into surrounding water. Utilising the chemical selectivity of the infrared methodology, ATR-FTIR imaging is applied for the first time to monitor the redistribution and release of human growth hormone (hGH) from a range of CriticalMixTM processed PLGA/PLA microparticles during a set of dissolution experiments at 37 °C in D2O. Increasing the γ dose is shown to have a profound influence on the release mechanism, with higher γ doses leading to a dramatic increase in the initial burst release followed by retardation in the sustained release and a lower total level of hGH release over the dissolution experiment. These changes are shown to be the result of: (i) protein aggregation as a function of applied γ-dose as studied by size exclusion chromatography; (ii) decrease in overall porosity as studied by SEM; (iii) decrease in Mw of all of the component polymers post γ irradiation indicating a chain scission mechanism as studied by GPC and DSC; and (iv) the increase in the number of oxygenated components in the Poloxamer 407 excipient, thereby increasing the strength of interaction between the microparticle and the entrapped hGH. These findings suggest that any γ sterilisation dose should be less than 25 kGy and that other sterilisation methods may need to be considered, due to the stability of the studied formulations

Next Generation Sequencing Based Multiplex Long-Range PCR for Routine Genotyping of Autoinflammatory Disorders

Background: During the last decade, remarkable progress with massive sequencing has been made in the identification of disease-associated genes for AIDs using next-generation sequencing technologies (NGS). An international group of experts described the ideal genetic screening method which should give information about SNVs, InDels, Copy Number Variations (CNVs), GC rich regions. We aimed to develop and validate a molecular diagnostic method in conjunction with the NGS platform as an inexpensive, extended and uniform coverage and fast screening tool which consists of nine genes known to be associated with various AIDs. Methods: For the validation of basic and expanded panels, long-range multiplex models were setup on healthy samples without any known variations for MEFV, MVK, TNFRSF1A, NLRP3, PSTPIP1, IL1RN, NOD2, NLRP12 and LPIN2 genes. Patients with AIDs who had already known causative variants in these genes were sequenced for analytical validation. As a last step, multiplex models were validated on patients with pre-diagnosis of AIDs. All sequencing steps were performed on the Illumina NGS platform. Validity steps included the selection of related candidate genes, primer design, development of screening methods, validation and verification of the product. The GDPE (Gentera) bioinformatics pipeline was followed. Results: Although there was no nonsynonymous variation in 21 healthy samples, 107 synonymous variant alleles and some intronic and UTR variants were detected. In 10 patients who underwent analytical validation, besides the 11 known nonsynonymous variant alleles, 11 additional nonsynonymous variant alleles and a total of 81 synonymous variants were found. In the clinical validation phase, 46 patients sequenced with multiplex panels, genetic and clinical findings were combined for diagnosis. Conclusion: In this study, we describe the development and validation of an NGS-based multiplex array enabling the “long-amplicon” approach for targeted sequencing of nine genes associated with common AIDs. This screening tool is less expensive and more comprehensive compared to other methods and more informative than traditional sequencing. The proposed panel offers advantages to WES or hybridization probe equivalents in terms of CNV analysis, high sensitivity and uniformity, GC-rich region sequencing, InDel detection and intron covering

Investigation of factors influencing the hydrolytic degradation of single PLGA microparticles

Abstract Poly lactide-co-glycolide (PLGA) is an important polymer matrix used to provide sustained release across a range of active pharmaceutical ingredients (APIs) and works by hydrolytic degradation within the body, thereby releasing entrapped drug. Processing and sterilisation can impact on the morphology and chemistry of PLGA therefore influencing the hydrolysis rate and in turn the release rate of any entrapped API. This paper has looked at the effect of supercritical carbon dioxide (scCO2) processing, gamma irradiation, comonomer ratio and temperature on the hydrolysis of individual PLGA microparticles, using a combination of Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) imaging, Scanning Electron Microscopy (SEM), Differential Scanning Calorimetery (DSC) and Gel Permeation chromatography (GPC) to facilitate a better understanding of the physiochemical factors affecting the hydrolysis rate. This work has shown that scCO2 processing influences hydrolysis rates by increasing the porosity of the PLGA microparticles, increasing the lactide comonomer ratio decreases hydrolysis rates by reducing the hydrophilicity of the PLGA microparticles and increasing the gamma irradiation dose systematically increases the rate of hydrolysis due to reducing the overall molecular weight of the polymer matrix via a chain scission mechanism. Moreover this work shows that ATR-FTIR imaging facilitates the determination of a range of physicochemical parameters during the hydrolysis of a single PLGA microparticle including water ingress, water/polymer interface dimensions, degradation product distribution and hydrolysis rates for both lactide and glycolide copolymer units from the same experimen

Multi-omics Prediction from High-content Cellular Imaging with Deep Learning

High-content cellular imaging, transcriptomics, and proteomics data provide rich and complementary views on the molecular layers of biology that influence cellular states and function. However, the biological determinants through which changes in multi-omics measurements influence cellular morphology have not yet been systematically explored, and the degree to which cell imaging could potentially enable the prediction of multi-omics directly from cell imaging data is therefore currently unclear. Here, we address the question of whether it is possible to predict bulk multi-omics measurements directly from cell images using Image2Omics -- a deep learning approach that predicts multi-omics in a cell population directly from high-content images stained with multiplexed fluorescent dyes. We perform an experimental evaluation in gene-edited macrophages derived from human induced pluripotent stem cell (hiPSC) under multiple stimulation conditions and demonstrate that Image2Omics achieves significantly better performance in predicting transcriptomics and proteomics measurements directly from cell images than predictors based on the mean observed training set abundance. We observed significant predictability of abundances for 5903 (22.43%; 95% CI: 8.77%, 38.88%) and 5819 (22.11%; 95% CI: 10.40%, 38.08%) transcripts out of 26137 in M1 and M2-stimulated macrophages respectively and for 1933 (38.77%; 95% CI: 36.94%, 39.85%) and 2055 (41.22%; 95% CI: 39.31%, 42.42%) proteins out of 4986 in M1 and M2-stimulated macrophages respectively. Our results show that some transcript and protein abundances are predictable from cell imaging and that cell imaging may potentially, in some settings and depending on the mechanisms of interest and desired performance threshold, even be a scalable and resource-efficient substitute for multi-omics measurements

Studying the release of hGH from gamma-irradiated PLGA microparticles using ATR-FTIR imaging

Attenuated total reflection-Fourier transform infrared (ATR-FTIR) imaging has been applied for the first time to monitor the redistribution and release of hGH from a range of PLGA/PLA microparticles during a set of dissolution experiments at 37 °C in D2O. The effect of gamma-irradiation, a common sterilisation method, on hGH release kinetics from such systems has been demonstrated. Increasing the gamma dose was shown to have a profound influence on the nature of the release mechanism, with higher gamma doses leading to a dramatic increase in the initial burst release followed by a retardation in the sustained release and a lower total level of hGH release over the dissolution experiment. These changes were shown to be the result of a combination of factors; firstly, via scanning electron microscopy (SEM), gamma-irradiation was shown to strongly influence the morphology of the PLGA/PLA microparticles; reducing their overall porosity and reducing the available surface area, whilst forcing some of the entrapped hGH to the microparticle surface. Secondly, from FTIR measurements, gamma-irradiation was shown to increase the number of oxygenated components in the Poloxamer 407 excipient, by a process of chain scission, thereby increasing the strength of interaction between the microparticle and the entrapped hGH

Human rhinovirus promotes STING trafficking to replication organelles to promote viral replication

Human rhinovirus (HRV), like coronavirus (HCoV), are positive-strand RNA viruses that cause both upper and lower respiratory tract illness, with their replication facilitated by concentrating RNA-synthesizing machinery in intracellular compartments made of modified host membranes, referred to as replication organelles (ROs). Here we report a non-canonical, essential function for stimulator of interferon genes (STING) during HRV infections. While the canonical function of STING is to detect cytosolic DNA and activate inflammatory responses, HRV infection triggers the release of STIM1-bound STING in the ER by lowering Ca2+, thereby allowing STING to interact with phosphatidylinositol 4-phosphate (PI4P) and traffic to ROs to facilitates viral replication and transmission via autophagy. Our results thus hint a critical function of STING in HRV viral replication and transmission, with possible implications for other RO-mediated RNA viruses

The application of non-linear curve fitting routines to the analysis of mid-infrared images obtained from single polymeric microparticles

For the first time, we report a series of time resolved images of a single PLGA microparticle undergoing hydrolysis at 70 °C that have been obtained using attenuated total reflectance-Fourier transform infrared spectroscopic (ATR-FTIR) imaging. A novel partially supervised non-linear curve fitting (NLCF) tool was developed to identify and fit peaks to the infrared spectrum obtained from each pixel within the 64 × 64 array. The output from the NLCF was evaluated by comparison with a traditional peak height (PH) data analysis approach and multivariate curve resolution alternating least squares (MCR-ALS) analysis for the same images, in order to understand the limitations and advantages of the NLCF methodology. The NLCF method was shown to facilitate consistent spatial resolution enhancement as defined using the step-edge approach on dry microparticle images when compared to images derived from both PH measurements and MCR-ALS. The NLCF method was shown to improve both the S/N and sharpness of images obtained during an evolving experiment, providing a better insight into the magnitude of hydration layers and particle dimension changes during hydrolysis. The NLCF approach facilitated the calculation of hydrolysis rate constants for both the glycolic (kG) and lactic (kL) acid segments of the PLGA copolymer. This represents a real advantage over MCR-ALS which could not distinguish between the two segments due to colinearity within the data. The NLCF approach made it possible to calculate the hydrolysis rate constants from a single pixel, unlike the peak height data analysis approach which suffered from poor S/N at each pixel. These findings show the potential value of applying NLCF to the study of real-time chemical processes at the micron scale, assisting in the understanding of the mechanisms of chemical processes that occur within microparticles and enhancing the value of the mid-IR ATR analysis

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Background: Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. // Methods: We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung's disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. // Findings: We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung's disease) from 264 hospitals (89 in high-income countries, 166 in middle-income countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in low-income countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. // Interpretation: Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between low-income, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Design of a touch sensation application including surface tension sample using a haptic device in engineering education

This paper illustrates the effects of force feedback haptic applications as an educational material on the attitudes of students in engineering education. For this purpose, a simulation design has been carried out on a surface tension subject from physical chemistry. The availability of haptic devices in educational environments has accelerated. Although surface tension occurs at the atomic and molecular levels, the effects can be seen and the results are noticeable in daily life. These and similar applications make it necessary for this to be used as educational material by modeling and simulating small scale events such as surface. The aim of this study is to reach the conclusion that the haptic simulations that take place outside the scale of our daily lives (micro & macro) have a positive effect on the attitudes of students. It is also one of the expected results that the measurement values related to the research subject are proportionally transferred to the haptic device, consistently. In this application, a haptic device and its supporting application programming interfaces (API) for render operations have been used. A sample consisting of 48 engineering students has been divided into survey and control groups. While the survey group was working with the haptic application, the control group worked with conventional teaching methods. The data obtained from the sample have been analysed statistically, and the results have been evaluated. The findings of the research show that the survey group had a more positive attitude towards the haptic applications than the control group.WOS:0004603547000062-s2.0-8505551440