Functionalized Magnetic Fe3O4 Nanoparticles for Targeted Methotrexate Delivery in Ovarian Cancer Therapy.

Magnetic Fe3O4 nanoparticles (MNPs) functionalized with (3-aminopropylo)trietoksysilan (APTES) or N-carboxymethylchitosan (CMC) were proposed as nanocarriers of methotrexate (MTX) to target ovarian cancer cell lines. The successful functionalization of the obtained nanostructures was confirmed by FT-IR spectroscopy. The nanoparticles were characterized by transmission electron spectroscopy (TEM) and dynamic light scattering (DLS) techniques. Their potential zeta, magnetization, and hyperthermic properties were also explored. MTX was conjugated with the nanocarriers by ionic bonds or by amide bonds. The drug release kinetics were examined at different pH and temperatures. The MTT assay showed no toxicity of the MNPs[APTES] and MNPs[CMC]. Finally, the cytotoxicity of the nanostructures with MTX attached towards the ovarian cancer cells was measured. The sensitivity and resistance to methotrexate was determined in simplistic 2D and spheroid 3D conditions. The cytotoxicity tests of the tested nanostructures showed similar values for inhibiting the proliferation of ovarian cancer cells as methotrexate in its free form. Conjugating MTX with nanoparticles allows the drug to be directed to the target site using an external magnetic field, reducing overall toxicity. Combining this approach with hyperthermia could enhance the therapeutic effect in vivo compared to free MTX, though further research on advanced 3D models is needed.

Pharmacokinetics of magnetic iron oxide nanoparticles for medical applications.

Magnetic iron oxide nanoparticles (MNPs) have been under intense investigation for at least the last five decades as they show enormous potential for many biomedical applications, such as biomolecule separation, MRI imaging and hyperthermia. Moreover, a large area of research on these nanostructures is concerned with their use as carriers of drugs, nucleic acids, peptides and other biologically active compounds, often leading to the development of targeted therapies. The uniqueness of MNPs is due to their nanometric size and unique magnetic properties. In addition, iron ions, which, along with oxygen, are a part of the MNPs, belong to the trace elements in the body. Therefore, after digesting MNPs in lysosomes, iron ions are incorporated into the natural circulation of this element in the body, which reduces the risk of excessive storage of nanoparticles. Still, one of the key issues for the therapeutic applications of magnetic nanoparticles is their pharmacokinetics which is reflected in the circulation time of MNPs in the bloodstream. These characteristics depend on many factors, such as the size and charge of MNPs, the nature of the polymers and any molecules attached to their surface, and other. Since the pharmacokinetics depends on the resultant of the physicochemical properties of nanoparticles, research should be carried out individually for all the nanostructures designed. Almost every year there are new reports on the results of studies on the pharmacokinetics of specific magnetic nanoparticles, thus it is very important to follow the achievements on this matter. This paper reviews the latest findings in this field. The mechanism of action of the mononuclear phagocytic system and the half-lives of a wide range of nanostructures are presented. Moreover, factors affecting clearance such as hydrodynamic and core size, core morphology and coatings molecules, surface charge and technical aspects have been described.

A rare mutation (p.F149del) of the NT5C3A gene is associated with pyrimidine 5'-nucleotidase deficiency.

Pyrimidine 5'-nucleotidase deficiency is a rare erythrocyte enzymopathy. Here we report two cases of hemolytic anemia in brothers of Polish origin that are associated with a very rare mutation. Heterozygous deletion in the NT5C3A gene (c.444_446delGTT), inherited most likely from their asymptomatic mother, resulted in a single amino acid residue deletion (p.F149del) in cytosolic pyrimidine 5'-nucleotidase. However, only the mutated transcript was present in the reticulocyte transcriptome of both patients. Only residual activity of pyrimidine 5'-nucleotidase in the brothers' erythrocytes could be observed when compared with the controls, including their asymptomatic father and sister. Western blot showed no sign of the presence of 5'-nucleotidase protein in the erythrocytes of both studied patients. The 2.5-fold reduction of the purine/pyrimidine ratio observed only in the brothers' erythrocytes confirms the correlation of the results of molecular analysis, including whole-exome sequencing, with the phenotype of the pyrimidine 5'-nucleotidase deficiency. Altogether, our results may substantiate the hypothesis of the heterogeneity of the molecular basis of the defect involving both the mutation presented here and negative regulation of expression of the "normal" allele.

Identification of a Novel Mutation of ß-Spectrin in Hereditary Spherocytosis Using Whole Exome Sequencing.

Hereditary spherocytosis (HS), the most commonly inherited hemolytic anemia in northern Europeans, comprises a group of diseases whose heterogeneous genetic basis results in a variable clinical presentation. High-throughput genome sequencing methods have made a leading contribution to the recent progress in research on and diagnostics of inherited diseases and inspired us to apply whole exome sequencing (WES) to identify potential mutations in HS. The data presented here reveal a novel mutation probably responsible for HS in a single Polish family. Patients with clinical evidence of HS (clinical symptoms, hematological data, and EMA test) were enrolled in the study. The examination of the resulting WES data showed a number of polymorphisms in 71 genes associated with known erythrocyte pathologies (including membranopathies, enzymopathies, and hemoglobinopathies). Only a single SPTB gene variant indicated the possible molecular mechanism of the disease in the studied family. The new missense mutation p.C183Y was identified using WES in the SPTB gene, which is most likely the cause of clinical symptoms typical of hereditary spherocytosis (membranopathy) due to structural and functional impairments of human ß-spectrin. This mutation allows for a better understanding of the molecular mechanism(s) of one of the membranopathies, hereditary spherocytosis.

Deficiency of αII-spectrin affects endothelial cell-matrix contact and migration leading to impairment of angiogenesis in vitro.

BACKGROUND: Precise coordination of cytoskeletal components and dynamic control of cell adhesion and migration are required for crucial cell processes such as differentiation and morphogenesis. We investigated the potential involvement of αII-spectrin, a ubiquitous scaffolding element of the membrane skeleton, in the adhesion and angiogenesis mechanism. METHODS: The cell models were primary human umbilical vein endothelial cells (HUVECs) and a human dermal microvascular endothelial cell line (HMEC-1). After siRNA- and shRNA-mediated knockdown of αII-spectrin, we assessed its expression and that of its partners and adhesion proteins using western blotting. The phenotypes of the control and spectrin-depleted cells were examined using immunofluorescence and video microscopy. Capillary tube formation was assessed using the thick gel Matrigel matrix-based method and a microscope equipped with a thermostatic chamber and a Nikon Biostation System camera. RESULTS: Knockdown of αII-spectrin leads to: modified cell shape; actin cytoskeleton organization with the presence of peripheral actin patches; and decreased formation of stress fibers. Spectrin deficiency affects cell adhesion on laminin and fibronectin and cell motility. This included modification of the localization of adhesion molecules, such as αVß3- and α5-integrins, and organization of adhesion structures, such as focal points. Deficiency of αII-spectrin can also affect the complex mechanism of in vitro capillary tube formation, as demonstrated in a model of angiogenesis. Live imaging revealed that impairment of capillary tube assembly was mainly associated with a significant decrease in cell projection length and stability. αII-spectrin depletion is also associated with significantly decreased expression of three proteins involved in capillary tube formation and assembly: VE-cadherin, MCAM and ß3-integrin. CONCLUSION: Our data confirm the role of αII-spectrin in the control of cell adhesion and spreading. Moreover, our findings further support the participation of αII-spectrin in capillary tube formation in vitro through control of adhesion molecules, such as integrins. This indicates a new function of αII-spectrin in angiogenesis.

Efficient method for isolation of reticulocyte RNA from healthy individuals and hemolytic anaemia patients.

Despite enormous progress and development of high-throughput methods in genome-wide mRNA analyses, data on the erythroid transcriptome are still limited, even though they could be useful in medical diagnostics and personalized therapy as well as in research on normal and pathological erythroid maturation. Although obtaining normal and pathological reticulocyte transcriptome profiles should contribute greatly to our understanding of the molecular bases of terminal erythroid differentiation as well as the mechanisms of the hematological diseases, a basic limitation of these studies is the difficulty of efficient reticulocyte RNA isolation from human peripheral blood. The restricted number of possible parallel experiments primarily concern healthy individuals with the lowest number of reticulocytes in the peripheral blood and a low RNA content. In the present study, an efficient method for reticulocyte RNA isolation from healthy individuals and hemolytic anaemia patients is presented. The procedure includes leukofiltration, Ficoll-Paque gradient centrifugation, Percoll gradient centrifugation, and negative (CD45 and CD61) immunomagnetic separation. This relatively fast and simple four-stage method was successfully applied to obtain a reticulocyte-rich population from healthy subjects, which was used to efficiently isolate the high-quality RNA essential for successful NGS-based transcriptome analysis.

[Liposomes as non-viral carriers for genetic drugs]. / Liposomy jako niewirusowe systemy dostarczania leków genetycznych.

Methods in cancer therapy particularly in recent years, are rapidly changing, due to the need of design of new, more effective therapeutic strategies. Very promising approach to treatment of the neoplastic diseases is antisense gene therapy. Due to the low toxicity of treatment and eliminating not only the symptoms but also the molecular causes of the disease it may represent a breakthrough in cancer therapies. Delivery of a therapeutic DNA or RNA oligonucleotides to the target cells in vivo requires suitable carrier system. Non-viral drug carriers are increasingly used in new systems of targeted gene therapy. This review presents new generation of non-viral carriers, and is focused on immunoliposomes finding potential application in targeted gene therapy.

Spectrins: a structural platform for stabilization and activation of membrane channels, receptors and transporters.

This review focuses on structure and functions of spectrin as a major component of the membrane skeleton. Recent advances on spectrin function as an interface for signal transduction mediation and a number of data concerning interaction of spectrin with membrane channels, adhesion molecules, receptors and transporters draw a picture of multifaceted protein. Here, we attempted to show the current depiction of multitask role of spectrin in cell physiology. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Spectrin and phospholipids - the current picture of their fascinating interplay.

The spectrin-based membrane skeleton is crucial for the mechanical stability and resilience of erythrocytes. It mainly contributes to membrane integrity, protein organization and trafficking. Two transmembrane protein macro-complexes that are linked together by spectrin tetramers play a crucial role in attaching the membrane skeleton to the cell membrane, but they are not exclusive. Considerable experimental data have shown that direct interactions between spectrin and membrane lipids are important for cell membrane cohesion. Spectrin is a multidomain, multifunctional protein with several distinctive structural regions, including lipid-binding sites within CH tandem domains, a PH domain, and triple helical segments, which are excellent examples of ligand specificity hidden in a regular repetitive structure, as recently shown for the ankyrin-sensitive lipid-binding domain of beta spectrin. In this review, we summarize the state of knowledge about interactions between spectrin and membrane lipids.

microRNAs: fine tuning of erythropoiesis.

Cell proliferation and differentiation is a complex process involving many cellular mechanisms. One of the best-studied phenomena in cell differentiation is erythrocyte development during hematopoiesis in vertebrates. In recent years, a new class of small, endogenous, non-coding RNAs called microRNAs (miRNAs) emerged as important regulators of gene expression at the post-transcriptional level. Thousands of miRNAs have been identified in various organisms, including protozoa, fungi, bacteria and viruses, proving that the regulatory miRNA pathway is conserved in evolution. There are many examples of miRNA-mediated regulation of gene expression in the processes of cell proliferation, differentiation and apoptosis, and in cancer genesis. Many of the collected data clearly show the dependence of the proteome of a cell on the qualitative and quantitative composition of endogenous miRNAs. Numerous specific miRNAs are present in the hematopoietic erythroid line. This review attempts to summarize the state of knowledge on the role of miRNAs in the regulation of different stages of erythropoiesis. Original experimental data and results obtained with bioinformatics tools were combined to elucidate the currently known regulatory network of miRNAs that guide the process of differentiation of red blood cells.

In vivo Biodistribution and Clearance of Magnetic Iron Oxide Nanoparticles for Medical Applications.

Magnetic iron oxide nanoparticles (magnetite and maghemite) are intensively studied due to their broad potential applications in medical and biological sciences. Their unique properties, such as nanometric size, large specific surface area, and superparamagnetism, allow them to be used in targeted drug delivery and internal radiotherapy by targeting an external magnetic field. In addition, they are successfully used in magnetic resonance imaging (MRI), hyperthermia, and radiolabelling. The appropriate design of nanoparticles allows them to be delivered to the desired tissues and organs. The desired biodistribution of nanoparticles, eg, cancerous tumors, is increased using an external magnetic field. Thus, knowledge of the biodistribution of these nanoparticles is essential for medical applications. It allows for determining whether nanoparticles are captured by the desired organs or accumulated in other tissues, which may lead to potential toxicity. This review article presents the main organs where nanoparticles accumulate. The sites of their first uptake are usually the liver, spleen, and lymph nodes, but with the appropriate design of nanoparticles, they can also be accumulated in organs such as the lungs, heart, or brain. In addition, the review describes the factors affecting the biodistribution of nanoparticles, including their size, shape, surface charge, coating molecules, and route of administration. Modern techniques for determining nanoparticle accumulation sites and concentration in isolated tissues or the body in vivo are also presented.

Spherocytosis-Related L1340P Mutation in Ankyrin Affects Its Interactions with Spectrin.

Previously, we reported a new missense mutation in the ANK1 gene that correlated with the hereditary spherocytosis phenotype. This mutation, resulting in L1340P substitution (HGMD CM149731), likely leads to the changes in the conformation of the ankyrin ZZUD domain important for ankyrin binding to spectrin. Here, we report the molecular and physiological effects of this mutation. First, we assessed the binding activity of human ß-spectrin to the mutated ZZUDL1340P domain of ankyrin using two different experimental approaches-the study of association and dissociation responses of the spectrin-ankyrin binding domain and a sedimentation assay. In addition, we documented the changes in morphology caused by the overexpressed ankyrin ZZUD domain in human cell models. Our results prove the key role of the L1340 aa residue for the correct alignment of the ZZUD domain of ankyrin, which results in binding the latter with spectrin within the erythrocyte membrane. Replacing L1340 with a proline residue disrupts the spectrin-binding activity of ankyrin.

[Spectrin--variety of functions hidden in the structure]. / Spektryna--bogactwo funkcji ukrytych w strukturze.

Membrane skeleton is a structure that provides strength and elasticity to the erythrocyte membrane. This features stem from the main component of this structure, a multifunctional protein called spectrin. Spectrin forms a network underlying membrane bilayer containing integral membrane proteins which interact with multiple proteins and lipid partners. Although membrane skeleton and spectrin structure have been described before, the latest discoveries show their new details and properties. In this work we summarize recent findings concerning structure and function of spectrin together with its possible role in pathology. We focus our interest on lately published structural data and we make an attempt to combine these findings with possible physiological functions.

[Hereditary stomatocytoses--diagnostic problems and their molecular basis]. / Dziedziczne stomatocytozy--problemy diagnostyczne i podstawy molekularne.

Hereditary stomatocytosis (HSt) is a group of haemolytic anaemias in which the common symptom is an increased permeability of the red cell membrane for monovalent cations. HSt is diagnosed really seldom and the difficulties in diagnosing are connected to the fact that the clinical presentation of individual subtypes of HSt is very diverse. Many cases are characterised by unique phenotypes. Nevertheless, the number of diagnosed HSt cases is increasing each year. The aim of this review was the presentation of current information and an attempt to systematize it, what might be helpful in clinical diagnostic of the new cases of this anaemia. The most frequent mistake is to classify a case of HSt as the most common haemolytic anaemia--hereditary spherocytosis (HS), in which to improve patient condition a splenectomy is often recommended. Most cases of HSt no positive response to splenectomy and often thromboembolic complications are observed. It is interesting that commonly present in blood film stomatocytes and in many cases absent or severely reduced stomatin in HSt red cell membrane are not correlated with nucleotide sequence changes of the gene encoding stomatin. Many diagnosed cases are related to mutations in SLC4A1 and RHAG genes. Extensive research carried out on HSt in the entire world will certainly permit to know the molecular basis of the disease, the diversify of its subtypes and to estimate the real incidence of HSt.

αII-spectrin in T cells is involved in the regulation of cell-cell contact leading to immunological synapse formation?

T-lymphocyte activation after antigen presentation to the T-Cell Receptor (TCR) is a critical step in the development of proper immune responses to infection and inflammation. This dynamic process involves reorganization of the actin cytoskeleton and signaling molecules at the cell membrane, leading to the formation of the Immunological Synapse (IS). The mechanisms regulating the formation of the IS are not completely understood. Nonerythroid spectrin is a membrane skeletal protein involved in the regulation of many cellular processes, including cell adhesion, signaling and actin cytoskeleton remodeling. However, the role of spectrin in IS formation has not been explored. We used molecular, imaging and cellular approaches to show that nonerythroid αII-spectrin redistributes to the IS during T-cell activation. The redistribution of spectrin coincides with the relocation of CD45 and LFA-1, two components essential for IS formation and stability. We assessed the role of spectrin by shRNA-mediated depletion from Jurkat T cells and show that spectrin-depleted cells exhibit decreased adhesion and are defective in forming lamellipodia and filopodia. Importantly, IS formation is impaired in spectrin-depleted cells. Thus, spectrin may be engaged in regulation of distinct events necessary for the establishment and maturity of the IS: besides the involvement of spectrin in the control of CD45 and LFA-1 surface display, spectrin acts in the establishment of cell-cell contact and adhesion processes during the formation of the IS.

The YJL185C, YLR376C and YJR129C genes of Saccharomyces cerevisiae are probably involved in regulation of the glyoxylate cycle.

The ER24 aci (acidification) mutant of Saccharomyces cerevisiae excreting protons in the absence of glucose was transformed with a multicopy yeast DNA plasmid library. Three different DNA fragments restored the wild-type phenotype termed Aci- because it does not acidify the complete glucose medium under the tested conditions. Molecular dissection of the transforming DNA fragments identified two multicopy suppressor genes YJL185C, YJR129C and one allelic YLR376C. Disruption of either of the three genes in wild-type yeast strain resulted in acidification of the medium (Aci+ phenotype) similarly to the original ER24 mutant. These data indicate the contribution of the ER24 gene product Ylr376Cp and of the two suppressor gene products Yjl185Cp and Yjr129Cp to a complex regulation of the glyoxylate cycle in yeast.

AlphaII-spectrin is critical for cell adhesion and cell cycle.

Spectrins are ubiquitous scaffolding components of the membrane skeleton that organize and stabilize microdomains on both the plasma membrane and the intracellular organelles. By way of their numerous interactions with diverse protein families, they are implicated in various cellular functions. Using small interfering RNA strategy in the WM-266 cell line derived from human melanoma, we found that alphaII-spectrin deficiency is associated with a defect in cell proliferation, which is related to a cell cycle arrest at the G1 phase (first gap phase), as evaluated by DNA analysis and Rb phosphorylation. These observations coincided with elevated expression of the cyclin-dependent kinase inhibitor, p21Cip. Concomitantly, spectrin loss impaired cell adhesion and spreading. These cell adhesion defects were associated with modifications of the actin cytoskeleton, such as loss of stress fibers, alterations of focal adhesions, and modified expression of some integrins. Our results provide novel insights into spectrin functions by demonstrating the involvement of alphaII-spectrin in cell cycle regulation and actin organization.