Amr Alaarg

Red blood cell vesiculation in hereditary hemolytic anemia

Hereditary hemolytic anemia encompasses a heterogeneous group of anemias characterized by decreased red blood cell survival because of inherited membrane, enzyme, or hemoglobin disorders. Affected red blood cells are more fragile, less deformable, and more susceptible to shear stress and oxidative damage, and show increased vesiculation. Red blood cells, as essentially all cells, constitutively release phospholipid extracellular vesicles in vivo and in vitro in a process known as vesiculation. These extracellular vesicles comprise a heterogeneous group of vesicles of different sizes and intracellular origins. They are described in literature as exosomes if they originate from multi-vesicular bodies, or as microvesicles when formed by a one-step budding process directly from the plasma membrane. Extracellular vesicles contain a multitude of bioactive molecules that are implicated in intercellular communication and in different biological and pathophysiological processes. Mature red blood cells release in principle only microvesicles. In hereditary hemolytic anemias, the underlying molecular defect affects and determines red blood cell vesiculation, resulting in shedding microvesicles of different compositions and concentrations. Despite extensive research into red blood cell biochemistry and physiology, little is known about red cell deformability and vesiculation in hereditary hemolytic anemias, and the associated pathophysiological role is incompletely assessed. In this review, we discuss recent progress in understanding extracellular vesicles biology, with focus on red blood cell vesiculation. Also, we review recent scientific findings on the molecular defects of hereditary hemolytic anemias, and their correlation with red blood cell deformability and vesiculation. Integrating bio-analytical findings on abnormalities of red blood cells and their microvesicles will be critical for a better understanding of the pathophysiology of hereditary hemolytic anemias.

Immune cell screening of a nanoparticle library improves atherosclerosis therapy

Significance The immune system plays an essential role in the pathophysiology of major diseases such as atherosclerosis, diabetes, and cancer, which has inspired the development of numerous small molecules to modulate immune cells, intending to create immunotherapies for these diseases. Tissue- and cell-specific delivery of these small molecules is the key to transform these compounds to safe, potent immunotherapies. In this study, we present an in vivo nanoparticle screen approach that involves designing and evaluating a library of nanoparticles with distinct immune cell targeting specificity. This study carries out a systematic in vivo immune cell screening to create effective nanoparticle-based immunotherapy for modulating the pathological immune cells in atherosclerosis. Immunological complexity in atherosclerosis warrants targeted treatment of specific inflammatory cells that aggravate the disease. With the initiation of large phase III trials investigating immunomodulatory drugs for atherosclerosis, cardiovascular disease treatment enters a new era. We here propose a radically different approach: implementing and evaluating in vivo a combinatorial library of nanoparticles with distinct physiochemical properties and differential immune cell specificities. The library’s nanoparticles are based on endogenous high-density lipoprotein, which can preferentially deliver therapeutic compounds to pathological macrophages in atherosclerosis. Using the apolipoprotein E-deficient (Apoe−/−) mouse model of atherosclerosis, we quantitatively evaluated the library’s immune cell specificity by combining immunological techniques and in vivo positron emission tomography imaging. Based on this screen, we formulated a liver X receptor agonist (GW3965) and abolished its liver toxicity while still preserving its therapeutic function. Screening the immune cell specificity of nanoparticles can be used to develop tailored therapies for atherosclerosis and other inflammatory diseases.

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 10mg/kg for 2weeks enhanced monocyte recruitment to plaques. In follow up, after 6weeks 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.

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.

Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis

Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe–/– mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received 89Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe–/– mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP–immune cell interactions significantly decreased over the disease progression. 89Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis.

Real-Time Monitoring of Nanoparticle Formation by FRET Imaging

Understanding the formation process of nanoparticles is of the utmost importance to improve their design and production. This especially holds true for self-assembled nanoparticles whose formation processes have been largely overlooked. Herein, we present a new technology that integrates a microfluidic-based nanoparticle synthesis method and Förster resonance energy transfer (FRET) microscopy imaging to visualize nanoparticle self-assembly in real time. Applied to different nanoparticle systems, for example, nanoemulsions, drug-loaded block-copolymer micelles, and nanocrystal-core reconstituted high-density lipoproteins, we have shown the approachs unique ability to investigate key parameters affecting nanoparticle formation.

Multiple pathway assessment to predict anti-atherogenic efficacy of drugs targeting macrophages in atherosclerotic plaques

BACKGROUND Macrophages play a central role in atherosclerosis development and progression, hence, targeting macrophage activity is considered an attractive therapeutic. Recently, we documented nanomedicinal delivery of the anti-inflammatory compound prednisolone to atherosclerotic plaque macrophages in patients, which did however not translate into therapeutic efficacy. This unanticipated finding calls for in-depth screening of drugs intended for targeting plaque macrophages. METHODS AND RESULTS We evaluated the effect of several candidate drugs on macrophage activity, rating overall performance with respect to changes in cytokine release, oxidative stress, lipid handling, endoplasmic reticulum (ER) stress, and proliferation of macrophages. Using this in vitro approach, we observed that the anti-inflammatory effect of prednisolone was counterbalanced by multiple adverse effects on other key pathways. Conversely, pterostilbene, T0901317 and simvastatin had an overall anti-atherogenic effect on multiple pathways, suggesting their potential for liposomal delivery. CONCLUSION This dedicated assay setup provides a framework for high-throughput assessment. Further in vivo studies are warranted to determine the predictive value of this macrophage-based screening approach and its potential value in nanomedicinal drug development for cardiovascular patients.

Applying nanomedicine in maladaptive inflammation and angiogenesis

Inflammation and angiogenesis drive the development and progression of multiple devastating diseases such as atherosclerosis, cancer, rheumatoid arthritis, and inflammatory bowel disease. Though these diseases have very different phenotypic consequences, they possess several common pathophysiological features in which monocyte recruitment, macrophage polarization, and enhanced vascular permeability play critical roles. Thus, developing rational targeting strategies tailored to the different stages of the journey of monocytes, from bone marrow to local lesions, and their extravasation from the vasculature in diseased tissues will advance nanomedicine. The integration of in vivo imaging uniquely allows studying nanoparticle kinetics, accumulation, clearance, and biological activity, at levels ranging from subcellular to an entire organism, and will shed light on the fate of intravenously administered nanomedicines. We anticipate that convergence of nanomedicines, biomedical engineering, and life sciences will help to advance clinically relevant therapeutics and diagnostic agents for patients with chronic inflammatory diseases.

Investigating the Cellular Specificity in Tumors of a Surface-Converting Nanoparticle by Multimodal Imaging

Active targeting of nanoparticles through surface functionalization is a common strategy to enhance tumor delivery specificity. However, active targeting strategies tend to work against long polyethylene glycols shielding effectiveness and associated favorable pharmacokinetics. To overcome these limitations, we developed a matrix metalloproteinase-2 sensitive surface-converting polyethylene glycol coating. This coating prevents nanoparticle-cell interaction in the bloodstream, but, once exposed to matrix metalloproteinase-2, i.e., when the nanoparticles accumulate within the tumor interstitium, the converting polyethylene glycol coating is cleaved, and targeting ligands become available for binding to tumor cells. In this study, we applied a comprehensive multimodal imaging strategy involving optical, nuclear, and magnetic resonance imaging methods to evaluate this coating approach in a breast tumor mouse model. The data obtained revealed that this surface-converting coating enhances the nanoparticles blood half-life and tumor accumulation and ultimately results in improved tumor-cell targeting. Our results show that this enzyme-specific surface-converting coating ensures a high cell-targeting specificity without compromising favorable nanoparticle pharmacokinetics.

Comparative analysis of nanosystems’ effects on human endothelial and monocytic cell functions

Abstract The objective of our work was to investigate the effects of different types of nanoparticles on endothelial (HUVEC) and monocytic cell functions. We prepared and tested 14 different nanosystems comprising liposomes, lipid nanoparticles, polymer, and iron oxide nanoparticles. Some of the tested nanosystems contained targeting, therapeutic, or contrast agent(s). The effect of particles (0–400 µg/mL) on endothelial-monocytic cell interactions in response to TNF-α was investigated using an arterial bifurcation model and dynamic monocyte adhesion assay. Spontaneous HUVEC migration (0–100 µg/mL nanoparticles) and chemotaxis of monocytic cells towards MCP-1 in presence of particles (0–400 µg/mL) were determined using a barrier assay and a modified Boyden chamber assay, respectively. Lipid nanoparticles dose-dependently reduced monocytic cell chemotaxis and adhesion to activated HUVECs. Liposomal nanoparticles had little effect on cell migration, but one formulation induced monocytic cell recruitment by HUVECs under non-uniform shear stress by about 50%. Fucoidan-coated polymer nanoparticles (25–50 µg/mL) inhibited HUVEC migration and monocytic cell chemotaxis, and had a suppressive effect on monocytic cell recruitment under non-uniform shear stress. No significant effects of iron oxide nanoparticles on monocytic cell recruitment were observed except lauric acid and human albumin-coated particles which increased endothelial-monocytic interactions by 60–70%. Some of the iron oxide nanoparticles inhibited HUVEC migration and monocytic cell chemotaxis. These nanoparticle-induced effects are of importance for vascular cell biology and function and must be considered before the potential clinical use of some of the analyzed nanosystems in cardiovascular applications.