Liposomal prednisolone promotes macrophage lipotoxicity in experimental atherosclerosis

Atherosclerosis is a lipid-driven inflammatory disease, for which nanomedicinal interventions are under evaluation. Previously, we showed that liposomal nanoparticles loaded with prednisolone (LN-PLP) accumulated in plaque macrophages, however, induced proatherogenic effects in patients. Here, we confirmed in low-density lipoprotein receptor knockout (LDLr−/−) mice that LN-PLP accumulates in plaque macrophages. Next, we found that LN-PLP infusions at 10 mg/kg for 2 weeks enhanced monocyte recruitment to plaques. In follow up, after 6 weeks of LN-PLP exposure we observed (i) increased macrophage content, (ii) more advanced plaque stages, and (iii) larger necrotic core sizes. Finally, in vitro studies showed that macrophages become lipotoxic after LN-PLP exposure, exemplified by enhanced lipid loading, ER stress and apoptosis. These findings indicate that liposomal prednisolone may paradoxically accelerate atherosclerosis by promoting macrophage lipotoxicity. Hence, future (nanomedicinal) drug development studies are challenged by the multifactorial nature of atherosclerotic inflammation

Fishroesomes as carriers with antioxidant and anti-inflammatory bioactivities

The great diversity of marine habitats and organisms renders them a high-value source to find/develop novel drugs and formulations. Therefore, herein, sardine (Sardina pilchardus) roe was used as a lipidic source to produce liposomes. This fish product presents high nutritional value, being its lipidic content associated with important health benefits. Consequently, it can be advantageously used to produce therapeutically active delivery devices. Roe lipids were extracted using the Matyash method. After lipid film hydration and extrusion, sardine roe-derived large unilamellar liposomes (LUVs), designated as fishroesomes, presented a size of â 330 nm and a significant negative surface charge (â - 27 mV). Radical scavenging assays demonstrated that fishroesomes efficiently neutralized peroxyl, hydroxyl and nitric oxide radicals. Moreover, fishroesomes significantly reduced the expression of pro-inflammatory cytokines and chemokines by LPS-stimulated macrophages at non-toxic concentrations for L929 and THP-1 cells. Consequently, the developed liposomes exhibit unique properties as bioactive drug carriers for inflammatory diseases treatment.This work was supported by FCT/MCTES (Portuguese Foundation for Science and Technology / Ministry of Science, Technology and Higher Education) or FSE/POCH (European Social Fund through the Operational Program of Human Capital) (grant numbers PD/169/2013, PD/ BD/113795/2015, PD/BD/135246/2017, PTDC/BTM-SAL/28882/ 2017), and the NORTE 2020 Structured Project, co-funded by Norte2020 (NORTE-01-0145-FEDER-000021). Authors also thank the local fisherman for the donation of the samples

Oxidative stress and phosphatidylserine exposure in red cells from patients with sickle cell anaemia.

Phosphatidylserine (PS) exposure increases as red cells age, and is an important signal for the removal of senescent cells from the circulation. PS exposure is elevated in red cells from sickle cell anaemia (SCA) patients and is thought to enhance haemolysis and vaso-occlusion. Although precise conditions leading to its externalisation are unclear, high intracellular Ca2+ has been implicated. Red cells from SCA patients are also exposed to an increased oxidative challenge, and we postulated that this stimulates PS exposure, through increased Ca2+ levels. We tested four different ways of generating oxidative stress: hypoxanthine and xanthine oxidase, phenazine methosulphate, nitrite and tert-butyl hydroperoxide, together with thiol modification with N-ethylmaleimide (NEM), dithiothreitol and hypochlorous acid (HOCl), in red cells permeabilised to Ca2+ using bromo-A23187. Unexpectedly, our findings showed that the four oxidants significantly reduced Ca2+ -induced PS exposure (by 40-60%) with no appreciable effect on Ca2+ affinity. By contrast, NEM markedly increased PS exposure (by about 400%) and slightly but significantly increased the affinity for Ca2+ . Dithiothreitol modestly reduced PS exposure (by 25%) and HOCl had no effect. These findings emphasise the importance of thiol modification for PS exposure in sickle cells but suggest that increased oxidant stress alone is not important.We thank the British Heart Foundation for generous financial support (grant number 31966)

Red blood cell complement receptor one level varies with Knops blood group, α(+)thalassaemia and age among Kenyan children

Both the invasion of red blood cells (RBCs) by Plasmodium falciparum parasites and the sequestration of parasite-infected RBCs in the microvasculature are mediated in part by complement receptor one (CR1). RBC surface CR1 level can vary between individuals by more than 20-fold and may be associated with the risk of severe malaria. The factors that influence RBC CR1 level variation are poorly understood, particularly in African populations. We studied 3535 child residents of a malaria-endemic region of coastal Kenya and report, for the first time, that the CR1 Knops blood group alleles Sl2 and McC(b), and homozygous HbSS are positively associated with RBC CR1 level. Sickle cell trait and ABO blood group did not influence RBC CR1 level. We also confirm the previous observation that α(+)thalassaemia is associated with reduced RBC CR1 level, possibly due to small RBC volume, and that age-related changes in RBC CR1 expression occur throughout childhood. RBC CR1 level in malaria-endemic African populations is a complex phenotype influenced by multiple factors that should be taken into account in the design and interpretation of future studies on CR1 and malaria susceptibility

Targeted therapeutics in inflammatory atherosclerosis

Atherosclerosis, a chronic inflammatory vascular disease, which has been recently identified in 5000-year mummies, remains undefeated. It is the most common underlying cause of deadly cardiovascular diseases (CVD), including heart attacks, strokes, and peripheral vascular diseases. This tremendous socioeconomic burden calls for further investigation and investment to develop effective, innovative, and clinically viable interventions for the treatment of atherosclerosis. One important initiative in this direction is NanoAthero, a European Consortium that funded the research work presented in this thesis. This program aims to demonstrate the preliminary clinical feasibility of the use of nanosystems for targeted imaging and treatment of advanced atherosclerosis. The enthusiasm generated for the use of nanocarrier drug delivery systems in atherosclerosis is mainly driven by the significant progress made in the field of oncological nanomedicine. Capitalizing on the achievements in the nanomedicine field, the main aim of this thesis is to contribute to the development and use of targeted nanomedicines in atherosclerosis. To this end, we adopted a ‘disease first’ approach to develop efficient targeted nanomedicines, in which particular attention is paid to the underlying pathophysiological processes in atherosclerosis. Macrophages are key players in these processes that affect atherosclerotic plaque inflammation and vulnerability to rupture. Moreover, their phagocytic capacity makes macrophages ideal targets for nanomedicine-based approaches. Understanding the role of plaque-associated macrophages and their interactions with the different nanocarriers is crucial for the successful development of efficacious, clinically relevant nanotherapeutics for atherosclerotic cardiovascular diseases

Docosahexaenoic acid liposomes for targeting chronic inflammatory diseases and cancer: an in vitro assessment

Amr Alaarg,1,2 Nan Yeun Jordan,1 Johan JF Verhoef,1 Josbert M Metselaar,2,3 Gert Storm,1,2 Robbert J Kok1 1Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, 2Department of Biomaterials Science and Technology, Institute for Biomedical Technology and Technical Medicine (MIRA), University of Twente, Enschede, the Netherlands; 3Department of Experimental Molecular Imaging, University Clinic and Helmholtz Institute for Biomedical Engineering, RWTH-Aachen University, Aachen, Germany Abstract: Inflammation, oxidative stress, and uncontrolled cell proliferation are common key features of chronic inflammatory diseases, such as atherosclerosis and cancer. ω3 polyunsaturated fatty acids (PUFAs; also known as omega3 fatty acids or fish oil) have beneficial effects against inflammation upon dietary consumption. However, these effects cannot be fully exploited unless diets are enriched with high concentrations of fish oil supplements over long periods of time. Here, a nanomedicine-based approach is presented for delivering effective levels of PUFAs to inflammatory cells. Nanoparticles are internalized by immune cells, and hence can adequately deliver bioactive lipids into these target cells. The ω3 FA docosahexaenoic acid was formulated into liposomes (ω-liposomes), and evaluated for anti-inflammatory effects in different types of immune cells. ω-Liposomes strongly inhibited the release of reactive oxygen species and reactive nitrogen species from human neutrophils and murine macrophages, and also inhibited the production of the proinflammatory cytokines TNFα and MCP1. Moreover, ω-liposomes inhibited tumor-cell proliferation when evaluated in FaDu head and neck squamous carcinoma and 4T1 breast cancer cells in in vitro cultures. We propose that ω-liposomes are a promising nanonutraceutical formulation for intravenous delivery of fish oil FAs, which may be beneficial in the treatment of inflammatory disorders and cancer. Keywords: nanomedicine, PUFA, inflammation, cancer, fish oil, deliver

Docosahexaenoic acid liposomes for targeting chronic inflammatory diseases and cancer : an in vitro assessment

Inflammation, oxidative stress, and uncontrolled cell proliferation are common key features of chronic inflammatory diseases, such as atherosclerosis and cancer. ω3 polyunsaturated fatty acids (PUFAs; also known as omega3 fatty acids or fish oil) have beneficial effects against inflammation upon dietary consumption. However, these effects cannot be fully exploited unless diets are enriched with high concentrations of fish oil supplements over long periods of time. Here, a nanomedicine-based approach is presented for delivering effective levels of PUFAs to inflammatory cells. Nanoparticles are internalized by immune cells, and hence can adequately deliver bioactive lipids into these target cells. The ω3 FA docosahexaenoic acid was formulated into liposomes (ω-liposomes), and evaluated for anti-inflammatory effects in different types of immune cells. ω-Liposomes strongly inhibited the release of reactive oxygen species and reactive nitrogen species from human neutrophils and murine macrophages, and also inhibited the production of the proinflammatory cytokines TNFα and MCP1. Moreover, ω-liposomes inhibited tumor-cell proliferation when evaluated in FaDu head and neck squamous carcinoma and 4T1 breast cancer cells in in vitro cultures. We propose that ω-liposomes are a promising nanonutraceutical formulation for intravenous delivery of fish oil FAs, which may be beneficial in the treatment of inflammatory disorders and cancer