Surface modification of Ti-6Al-4V powder during recycling in EBM process

Surface and Interface Analysis published by John Wiley & Sons Ltd Electron beam melting is an additive manufacturing technology in vacuum that suits Ti-6Al-4V parts, which has a high affinity with oxygen. Since the high cost of the feedstock powder, the un-melted powder is often recycled in the subsequent process. In this study, the influence of powder reuse on the surface characteristics of Ti-6Al-4V powder is examined using a variety of technology including scanning electron microscopy, X-ray photoelectron spectroscopy, and Auger electron spectroscopy. The modification of surface morphology and chemistry either generally or locally are revealed and discussed, which helps the creation of powder recycling strategy. Compared with fresh virgin powder, “worn” and rougher powder particles are observed after recycling. Meanwhile, the average oxide thickness is slightly increased, and less Al enrichment was found at the surface. Locally varied chemistry/oxide thickness on different powder particles or different location on the same particle is observed

Study protocol for locoregional precision treatment of hepatocellular carcinoma with transarterial chemoembolisation (TACTida), a clinical study:idarubicin dose selection, tissue response and survival

INTRODUCTION: Hepatocellular carcinoma (HCC) is a common cause of cancer-related death, often detected in the intermediate stage. The standard of care for intermediate-stage HCC is transarterial chemoembolisation (TACE), where idarubicin (IDA) is a promising drug. Despite the fact that TACE has been used for several decades, treatment success is unpredictable. This clinical trial has been designed believing that further improvement might be achieved by increasing the understanding of interactions between local pharmacology, tumour targeting, HCC pathophysiology, metabolomics and molecular mechanisms of drug resistance. METHODS AND ANALYSIS: The study population of this single-centre clinical trial consists of adults with intermediate-stage HCC. Each tumour site will receive TACE with two different IDA doses, 10 and 15 mg, on separate occasions. Before and after each patient's first TACE blood samples, tissue and liquid biopsies, and positron emission tomography (PET)/MRI will be performed. Blood samples will be used for pharmacokinetics (PK) and liver function evaluation. Tissue biopsies will be used for histopathology analyses, and culturing of primary organoids of tumour and non-tumour tissue to measure cell viability, drug response, multiomics and gene expression. Multiomics analyses will also be performed on liquid biopsies. PET/MRI will be used to evaluate tumour viability and liver metabolism. The two doses of IDA will be compared regarding PK, antitumour effects and safety. Imaging, molecular biology and multiomics data will be used to identify HCC phenotypes and their relation to drug uptake and metabolism, treatment response and survival. ETHICS AND DISSEMINATION: Participants give informed consent. Personal data are deidentified. A patient will be withdrawn from the study if considered medically necessary, or if it is the wish of the patient. The study has been approved by the Swedish Ethical Review Authority (Dnr. 2021-01928) and by the Medical Product Agency, Uppsala, Sweden. TRIAL REGISTRATION NUMBER: EudraCT number: 2021-001257-31

Anthracyclines : Toxicity and chemotherapy-induced mucositis

Anthracyclines belong to a class of cytostatic compounds that are commonly used to treat various types of cancers, including lymphoma, breast cancer and primary liver cancer. The aim of this thesis was to study how two anthracyclines, doxorubicin and idarubicin, induced cytotoxicity in tumor cells, as well as their off-target effect on the gastrointestinal system. In Paper I, we exposed four different cancer cell lines to various concentrations of doxorubicin in two different formulations, and measured the cell viability and how much doxorubicin was taken up by the cells. We found that the cell lines differed in their uptake and sensitivity, and that the most resistant cell line had an intracellular exposure of doxorubicin that was about 100 times lower when compared to the more sensitive cell lines. To study the off-target gastrointestinal toxicity, we dosed rats with doxorubicin and studied how chemotherapy-induced mucositis developed during seven days in Paper II. The main effect parameter was intestinal villus atrophy, which was most severe after three days. This was preceded by an increased cell death and decreased proliferation in the crypts, which occurred within the first day after doxorubicin exposure. To study how different cytostatic drugs affected chemotherapy-induced mucositis, and to what extent they caused diarrhea, rats were dosed with one of six different chemotherapies, including doxorubicin and idarubicin, in Paper III. All selected chemotherapies caused similar villus atrophy three days after dosing, but only a third, including idarubicin, caused clear diarrhea. In paper IV the objective was to treat or prevent idarubicin-induced mucositis and diarrhea, by using the anti-inflammatory drugs anakinra and/or dexamethasone. Anakinra alone stopped the diarrhea, while the combination of anakinra and dexamethasone prevented villus atrophy. These positive effects encourage further investigations into the use of anakinra and dexamethasone as supportive therapies for chemotherapy-induced mucositis and diarrhea. The overall long-term impact of the different studies in this thesis is to increase the specific anti-tumoral effect of chemotherapies, while also alleviating the adverse effects. This would improve quality-of-life and treatment outcomes of cancer patients

Formulation and characterization of liposomal spray dried powders intended for inhalation

This master thesis has evaluated the possible use of a spray dryer to produce powders suitable for use as inhalants and whether the addition of liposomes to the solution to be spray dried would improve the properties of the powder. The powders created consisted of Lysozyme as model drug, Lactose as filler and stabilizer, and different types of phospholipids to create liposomes using an ethanol injection method. It was shown that powders suitable for use as inhalants were possible to create. By adjusting the parameters of both the spray dryer and by changing the proportions of the ingredients it was possible to achieve various powders, some of which displayed strong signs of having the proper parameters for use for inhalation. The quality parameter deemed most important for the powders was the fine particle fraction, which corresponds to the fraction of powder that reaches the target area of the lungs. The fine particle fraction was tested using a cascade impactor and it was discovered that this fraction was dependent on the concentration of both lysozyme and liposomes as well as the inlet temperature and feed flow rate of the spray dryer. The detected fine particle fraction varied between just a few percent for some of the powders without liposomes up to more than 50 % for one of the powders created using liposomes. Other investigated parameters were the water activity (important for the stability of the powder), the liposome size distribution (to see how this affected the particles), the yield and the outlet temperature from the spray dryer.Creating a fat powder for inhalation By using tiny fat bubbles, is it possible to improve a protein powder intended for inhalation? This question was the basis of this thesis, and the answer seems to be in the positive. So how do you get a protein medicine, such as insulin or a vaccine, into the body? The usual answer is to inject it, as it doesn’t survive the stomach, but a recent trend is to try to breath it in instead. This would be an improvement for many reasons, as first of all no one likes needles, let alone having to use one on yourself several times a day. But for it to be breathed in, there are several problems. How does one make the protein become absorbed by the lungs and enter the blood? One answer is to make sure the medicine is in particles small enough to be breathed in to the very bottom of the lungs, where it can easily pass through the thin boundary between the air and the blood. These small particles are often called fine particles, and obviously it is better for more of the medicine to consist of these fine particles, but in reality many medicines only have half of the medicine in this size range. To create such small particles special equipment is needed. One such device is the spray dryer, which is a device for turning a liquid into a fine powder. By tweaking the settings, it is possible to create a powder with particles small enough to reach the bottom of the lungs. The powder we produced consisted of lysozyme, a protein, lactose, used mainly as something to take up space and make the powder cheaper, and different types of fat in the form of liposomes. The powers were created both with and without these liposomes to test what effect their presence had. It was tested for not only its size, but also for its water activity and the yield of the spray dryer that created the powder. Water activity is a term for the humidity of the powder, which is important as it affects how long the powder can be stored before it is destroyed. An effect of most protein medicines is that when the protein has reached the blood, it quickly has its effect and is then quickly removed. This is sometimes great when you need a quick effect, but sometimes you want something with a lasting effect instead. For these occasions, one tactic is to have the protein kept inside of a tiny fat bubbles called a liposome. By having the protein inside of the liposome it takes a longer time for it to be released into the blood and in consequence makes it last longer. This effect was not studied in this project, but instead it was studied if the liposome had any other effect on the powder. It was discovered that when you added liposomes to the powder, a lot of things happened. The powder became stickier and the yield became lower. On the other hand, the amount of fine particles became larger; in fact, it more than tripled for one of the powders with liposomes when compared with a powder created the same way but without the liposomes. All powders created with liposomes had more fine particles than any created without them, leading to the conclusion that the addition of liposomes were indeed a step forward. This discovery, that there is not only a potential increase in effective time of the medicine in the body, but also a significant effect on the amount of fine particles in the powder could be used to improve the powders created for inhalation purposes after further studies

Chemotherapeutics Combined with Luminal Irritants: Effects on Small-Intestinal Mannitol Permeability and Villus Length in Rats

Chemotherapy causes intestinal mucositis, which includes villous atrophy and altered mucosal barrier function. However, there is an uncertainty regarding how the reduced small-intestinal surface area affects the mucosal permeability of the small marker probe mannitol (MW 188), and how the mucosa responds to luminal irritants after chemotherapy. The aims in this study were to determine (i) the relationship between chemotherapy-induced villus atrophy and the intestinal permeability of mannitol and (ii) how the mucosa regulate this permeability in response to luminal ethanol and sodium dodecyl sulfate (SDS). This was investigated by treating rats with a single intraperitoneal dose of doxorubicin, irinotecan, or 5-fluorouracil. After 72 h, jejunum was single-pass perfused and mannitol permeability determined at baseline and after 15 min luminal exposure to 15% ethanol or 5 mg/mL SDS. Tissue samples for morphological analyses were sampled from the perfused segment. All three chemotherapeutics caused a similar 30% reduction in villus length. Mannitol permeability increased with irinotecan (1.3-fold) and 5-fluorouracil (2.5-fold) and was reduced with doxorubicin (0.5-fold), suggesting that it is not epithelial surface area alone that regulates intestinal permeability to mannitol. There was no additional increase in mannitol permeability induced by luminal ethanol or SDS in the chemotherapy-treated rats compared to controls, which may be related to the relatively high basal permeability of mannitol compared to other common low-permeability probes. We therefore suggest that future studies should focus on elucidating the complex interplay between chemotherapy in combination with luminal irritants on the intestinal permeability of other probes

Anakinra and dexamethasone treatment of idarubicin-induced mucositis and diarrhoea in rats

Chemotherapy-induced mucositis, characterized by diarrhoea and villous atrophy, is a severe side effect contributing to reduced quality of life and premature death in cancer patients treated with cytostatics. Despite its high incidence, there is no effective supportive therapy available. The main objective of this study was to determine if the anti-inflammatory drugs anakinra and/or dexamethasone—which have different mechanisms-of-action—might be used to effectively treat idarubicin-induced mucositis in rats. Mucositis was induced through a single injection with 2 mg/kg idarubicin (with saline as control), followed by daily treatments of anakinra (100 mg/kg/day), dexamethasone (10 mg/kg/day) or both for 3 days. After 72 h, jejunal tissue was collected for morphological, apoptotic and proliferative analyses, and colonic faecal water content and body weight change were determined. The diarrhoea that was induced by idarubicin (from 63.5% to 78.6% water content in faeces) was completely reversed by anakinra alone, and the jejunal villus height reduction by 36% was prevented by a combination of anakinra and dexamethasone. Dexamethasone reduced apoptosis in the jejunal crypts, both alone and in combination with anakinra. These positive effects encouraged further investigations into the use of anakinra and dexamethasone as supportive therapies for chemotherapy-induced intestinal mucositis and diarrhoea.Authors and title in the list of papers of Fredrik Kullenberg's thesis:Kullenberg, F., Peters, K., Dahlgren, D., Heindryckx, F., Sjöblom, M., &amp; Lennernäs, H.Anakinra and dexamethasone treatment of idarubicin-induced mucositis and diarrhea in rats</p

Orthogonality in Principal Component Analysis Allows the Discovery of Lipids in the Jejunum That Are Independent of Ad Libitum Feeding

Ad libitum feeding of experimental animals is preferred because of medical relevance together with technical and practical considerations. In addition, ethical committees may require ad libitum feeding. However, feeding affects the metabolism so ad libitum feeding may mask the effects of drugs on tissues directly involved in the digestion process (e.g., jejunum and liver). Despite this effect, principal component analysis has the potential of identifying metabolic traits that are statistically independent (orthogonal) to ad libitum feeding. Consequently, we used principal component analysis to discover the metabolic effects of doxorubicin independent of ad libitum feeding. First, we analyzed the lipidome of the jejunum and the liver of rats treated with vehicle or doxorubicin. Subsequently, we performed principal component analysis. We could identify a principal component associated to the hydrolysis of lipids during digestion and a group of lipids that were orthogonal. These lipids in the jejunum increased with the treatment time and presented a polyunsaturated fatty acid as common structural trait. This characteristic suggests that doxorubicin increases polyunsaturated fatty acids. This behavior agrees with our previous in vitro results and suggests that doxorubicin sensitized the jejunum to ferroptosis, which may partially explain the toxicity of doxorubicin in the intestines

Anthracyclins Increase PUFAs : Potential Implications in ER Stress and Cell Death

Metabolic and personalized interventions in cancer treatment require a better understanding of the relationship between the induction of cell death and metabolism. Consequently, we treated three primary liver cancer cell lines with two anthracyclins (doxorubicin and idarubin) and studied the changes in the lipidome. We found that both anthracyclins in the three cell lines increased the levels of polyunsaturated fatty acids (PUFAs) and alkylacylglycerophosphoethanolamines (etherPEs) with PUFAs. As PUFAs and alkylacylglycerophospholipids with PUFAs are fundamental in lipid peroxidation during ferroptotic cell death, our results suggest supplementation with PUFAs and/or etherPEs with PUFAs as a potential general adjuvant of anthracyclins. In contrast, neither the markers of de novo lipogenesis nor cholesterol lipids presented the same trend in all cell lines and treatments. In agreement with previous research, this suggests that modulation of the metabolism of cholesterol could be considered a specific adjuvant of anthracyclins depending on the type of tumor and the individual. Finally, in agreement with previous research, we found a relationship across the different cell types between: (i) the change in endoplasmic reticulum (ER) stress, and (ii) the imbalance between PUFAs and cholesterol and saturated lipids. In the light of previous research, this imbalance partially explains the sensitivity to anthracyclins of the different cells. In conclusion, our results suggest that the modulation of different lipid metabolic pathways may be considered for generalized and personalized metabochemotherapies

Chemotherapeutics Combined with Luminal Irritants : Effects on Small-Intestinal Mannitol Permeability and Villus Length in Rats

Chemotherapy causes intestinal mucositis, which includes villous atrophy and altered mucosal barrier function. However, there is an uncertainty regarding how the reduced small-intestinal surface area affects the mucosal permeability of the small marker probe mannitol (MW 188), and how the mucosa responds to luminal irritants after chemotherapy. The aims in this study were to determine (i) the relationship between chemotherapy-induced villus atrophy and the intestinal permeability of mannitol and (ii) how the mucosa regulate this permeability in response to luminal ethanol and sodium dodecyl sulfate (SDS). This was investigated by treating rats with a single intraperitoneal dose of doxorubicin, irinotecan, or 5-fluorouracil. After 72 h, jejunum was single-pass perfused and mannitol permeability determined at baseline and after 15 min luminal exposure to 15% ethanol or 5 mg/mL SDS. Tissue samples for morphological analyses were sampled from the perfused segment. All three chemotherapeutics caused a similar 30% reduction in villus length. Mannitol permeability increased with irinotecan (1.3-fold) and 5-fluorouracil (2.5-fold) and was reduced with doxorubicin (0.5-fold), suggesting that it is not epithelial surface area alone that regulates intestinal permeability to mannitol. There was no additional increase in mannitol permeability induced by luminal ethanol or SDS in the chemotherapy-treated rats compared to controls, which may be related to the relatively high basal permeability of mannitol compared to other common low-permeability probes. We therefore suggest that future studies should focus on elucidating the complex interplay between chemotherapy in combination with luminal irritants on the intestinal permeability of other probes

Evaluation and validation of chemotherapy‐specific diarrhoea and histopathology in rats

Chemotherapy-induced mucositis is characterized by diarrhoea and villous atrophy. However, it is not well-understood why diarrhoea arises, why it only occurs with some chemotherapeutics and how it is related to villus atrophy. The objectives in this study were to determine (i) the relationship between chemotherapy-induced diarrhoea and villus atrophy and to (ii) establish and validate a rat diarrhoea model with clinically relevant endpoints. Male Wistar Han IGS rats were treated with saline, doxorubicin, idarubicin, methotrexate, 5-fluorouracil, irinotecan or 5-fluorouracil+irinotecan. After 72 h, jejunal tissue was taken for morphological, apoptotic and proliferative analyses, and faecal water content and change in body weight were determined. All treatments except methotrexate caused a similar reduction (≈42%) in villus height, but none of them altered mucosal crypt cell proliferation or apoptosis. Doxorubicin, idarubicin, irinotecan and 5-fluorouracil+irinotecan caused body weight reduction, but only irinotecan and idarubicin caused diarrhoea. No direct correlation between diarrhoea and villus height or body weight loss was observed. Therefore, studies of the mechanisms for chemotherapy-induced diarrhoea should focus on functional factors. Finally, the irinotecan and idarubicin diarrhoea models established in this study will be useful in developing supportive treatments of this common and serious adverse effect in patients undergoing chemotherapy