RESUMO
One of the main challenges in improving the efficacy of conventional chemotherapeutic drugs is that they do not reach the cancer cells at sufficiently high doses while at the same time affecting healthy tissue and causing significant side effects and suffering in cancer patients. To overcome this deficiency, magnetic nanoparticles as transporter systems have emerged as a promising approach to achieve more specific tumour targeting. Drug-loaded magnetic nanoparticles can be directed to the target tissue by applying an external magnetic field. However, the magnetic forces exerted on the nanoparticles fall off rapidly with distance, making the tumour targeting challenging, even more so in the presence of flowing blood or interstitial fluid. We therefore present a computational model of the capturing of magnetic nanoparticles in a test setup: our model includes the flow around the tumour, the magnetic forces that guide the nanoparticles, and the transport within the tumour. We show how a model for the transport of magnetic nanoparticles in an external magnetic field can be integrated with a multiphase tumour model based on the theory of porous media. Our approach based on the underlying physical mechanisms can provide crucial insights into mechanisms that cannot be studied conclusively in experimental research alone. Such a computational model enables an efficient and systematic exploration of the nanoparticle design space, first in a controlled test setup and then in more complex in vivo scenarios. As an effective tool for minimising costly trial-and-error design methods, it expedites translation into clinical practice to improve therapeutic outcomes and limit adverse effects for cancer patients.
Assuntos
Nanopartículas de Magnetita , Nanopartículas , Neoplasias , Humanos , Modelos Teóricos , Simulação por Computador , Sistemas de Liberação de Medicamentos/métodosRESUMO
The folding and export of proteins and hydrolysis of unfolded proteins are disbalanced in the endoplasmic reticulum (ER) of cancer cells, leading to so-called ER stress. Agents further augmenting this effect are used as anticancer drugs including clinically approved proteasome inhibitors bortezomib and carfilzomib. However, these drugs can affect normal cells, which also rely strongly on ER functions, leading, for example, to accumulation of reactive oxygen species (ROS). To address this problem, we have developed ER-targeted prodrugs activated only in cancer cells in the presence of elevated ROS amounts. These compounds are conjugates of cholic acid with N-alkylaminoferrocene-based prodrugs. We confirmed their accumulation in the ER of cancer cells, their anticancer efficacy, and cancer cell specificity. These prodrugs induce ER stress, attenuate mitochondrial membrane potential, and generate mitochondrial ROS leading to cell death via necrosis. We also demonstrated that the new prodrugs are activated in vivo in Nemeth-Kellner lymphoma (NK/Ly) murine model.
Assuntos
Antineoplásicos/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Linfoma/tratamento farmacológico , Pró-Fármacos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Antineoplásicos/química , Retículo Endoplasmático/metabolismo , Humanos , Linfoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Pró-Fármacos/químicaRESUMO
Intracellular concentration of reactive oxygen species (e.g., H2O2) in cancer cells is elevated over 10-fold as compared to normal cells. This feature has been used by us and several other research groups to design cancer specific prodrugs, for example, N-alkylaminoferrocene (NAAF)-based prodrugs. Further improvement of the efficacy of these prodrugs can be achieved by their targeting to intracellular organelles containing elevated reactive oxygen species (ROS) amounts. For example, we have previously demonstrated that lysosome-targeted NAAF-prodrugs exhibit higher anticancer activity in cell cultures, in primary cells and in vivo (Angew. Chem. Int. Ed. 2017, 56, 15545). Mitochondrion is an organelle, where electrons can leak from the respiratory chain. These electrons can combine with O2, generating O2-⢠that is followed by dismutation with the formation of H2O2. Thus, ROS can be generated in excess in mitochondria and targeting of ROS-sensitive prodrugs to these organelles could be a sensible possibility for enhancing their efficacy. We have previously reported on NAAF-prodrugs, which after their activation in cells, are accumulated in mitochondria (Angew. Chem. Int. Ed. 2018, 57, 11943). Now we prepared two hybrid NAAF-prodrugs directly accumulated in mitochondria and activated in these organelles. We studied their anticancer activity and mode of action. Based on these data, we concluded that ROS produced by mitochondria is not available in sufficient quantities for activation of the ROS-responsive prodrugs. The reason for this can be efficient scavenging of ROS by antioxidants. Our data are important for the understanding of the mechanism of action of ROS-activatable prodrugs and will facilitate their further development.
Assuntos
Compostos Ferrosos/química , Metalocenos/química , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Pró-Fármacos/química , Antineoplásicos/farmacologia , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Boro/química , Linhagem Celular Tumoral , Sobrevivência Celular , Ensaios de Seleção de Medicamentos Antitumorais , Elétrons , Humanos , Peróxido de Hidrogênio/química , Concentração Inibidora 50 , Células Jurkat , Lisossomos/química , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Solubilidade , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The critical size for strong interaction of hydrophobic particles with phospholipid bilayers has been predicted to be 10 nm. Because of the wide spreading of nonpolar nanoparticles (NPs) in the environment, we aimed to reveal the ability of living organisms to entrap NPs via formation of neutrophil extracellular traps (NETs). Upon interaction with various cell types and tissues, 10- to 40-nm-sized NPs induce fast (<20 min) damage of plasma membranes and instability of the lysosomal compartment, leading to the immediate formation of NETs. In contrast, particles sized 100-1,000 nm behaved rather inertly. Resulting NET formation (NETosis) was accompanied by an inflammatory reaction intrinsically endowed with its own resolution, demonstrated in lungs and air pouches of mice. Persistence of small NPs in joints caused unremitting arthritis and bone remodeling. Small NPs coinjected with antigen exerted adjuvant-like activity. This report demonstrates a cellular mechanism that explains how small NPs activate the NETosis pathway and drive their entrapping and resolution of the initial inflammatory response.
Assuntos
Armadilhas Extracelulares/metabolismo , Inflamação/patologia , Nanopartículas/química , Tamanho da Partícula , Animais , Membrana Celular/metabolismo , Eritrócitos/metabolismo , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunidade , Pulmão/metabolismo , Camundongos Endogâmicos BALB C , Nanodiamantes/química , Nanodiamantes/ultraestrutura , Nanopartículas/ultraestrutura , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mitochondrial membrane potential is more negative in cancer cells than in normal cells, allowing cancer targeting by delocalized lipophilic cations (DLCs). However, as the difference is rather small, these drugs affect also normal cells. Now a concept of pro-DLCs is proposed based on an N-alkylaminoferrocene structure. These prodrugs are activated by the reaction with reactive oxygen species (ROS) forming ferrocenium-based DLCs. Since ROS are overproduced in cancer, the high-efficiency cancer-cell-specific targeting of mitochondria could be achieved as demonstrated by fluorescence microscopy in combination with two fluorogenic pro-DLCs inâ vitro and inâ vivo. We prepared a conjugate of another pro-DLC with a clinically approved drug carboplatin and confirmed that its accumulation in mitochondria was higher than that of the free drug. This was reflected in the substantially higher anticancer effect of the conjugate.
Assuntos
Compostos Ferrosos/química , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Cátions/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Compostos Ferrosos/farmacologia , Humanos , Mitocôndrias/efeitos dos fármacos , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Rodamina 123/químicaRESUMO
Conjugates based on nanostructured, superparamagnetic particles, a thermolabile linker and a cytotoxic maytansinoid were developed to serve as a model for tumour-selective drug delivery and release. It combines chemo- with thermal therapy. The linker-modified toxin was prepared by a combination of biotechnology and semisynthesis. Drug release was achieved by hyperthermia through an external oscillating electromagnetic field that induces heat inside the particles. Efficacy of this release concept was demonstrated both for cancer cell proliferation in vitro, and for tumour growth in vivo, in a xenograft mouse model. Biocompatibility studies for these magnetic-nanoparticle/ansamitocin conjugates complement this work.
Assuntos
Portadores de Fármacos/química , Nanopartículas de Magnetita/química , Maitansina/análogos & derivados , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reação de Cicloadição , Liberação Controlada de Fármacos , Humanos , Hipertermia Induzida , Antígeno Ki-67/metabolismo , Espectroscopia de Ressonância Magnética , Maitansina/química , Maitansina/uso terapêutico , Maitansina/toxicidade , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Transplante HeterólogoRESUMO
The phototoxic effect of hypericin can be utilized for Photodynamic Therapy (PDT) of cancer. After intravenous application and systemic distribution of the drug in the patient's body, the tumor site is exposed to light. Subsequently, toxic reactive oxygen species (ROS) are generated, inducing tumor cell death. To prevent unwanted activation of the drug in other regions of the body, patients have to avoid light during and after the treatment cycles, consequently impairing quality of life. Here, we characterize toxicity and hypericin-mediated effects on cancer cells in vitro and confirm that its effect clearly depends on concentration and illumination time. To reduce side effects and to increase therapy success, selective accumulation of hypericin in the tumor region is a promising solution. Loading hypericin on superparamagnetic iron oxide nanoparticles (SPIONs) and guiding them to the desired place using an external magnetic field might accomplish this task (referred to as Magnetic Drug Targeting (MDT)). Thus, using a double targeting strategy, namely magnetic accumulation and laser induced photoactivation, might improve treatment effectivity as well as specificity and reduce toxic side effects in future clinical applications.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Perileno/análogos & derivados , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Administração Intravenosa , Antracenos , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Células HT29 , Humanos , Células Jurkat , Leucemia de Células T/tratamento farmacológico , Leucemia de Células T/metabolismo , Nanopartículas de Magnetita/administração & dosagem , Nanopartículas de Magnetita/química , Perileno/farmacocinética , Perileno/farmacologia , Fármacos Fotossensibilizantes/farmacocinética , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
Cancer cells produce elevated levels of reactive oxygen species, which has been used to design cancer specific prodrugs. Their activation relies on at least a bimolecular process, in which a prodrug reacts with ROS. However, at low micromolar concentrations of the prodrugs and ROS, the activation is usually inefficient. Herein, we propose and validate a potentially general approach for solving this intrinsic problem of ROS-dependent prodrugs. In particular, known prodrug 4-(N-ferrocenyl-N-benzylaminocarbonyloxymethyl)phenylboronic acid pinacol ester was converted into its lysosome-specific analogue. Since lysosomes contain a higher concentration of active ROS than the cytoplasm, activation of the prodrug was facilitated with respect to the parent compound. Moreover, it was found to exhibit high anticancer activity in a variety of cancer cell lines (IC50 =3.5-7.2â µm) and inâ vivo (40â mg kg-1 , NK/Ly murine model) but remained weakly toxic towards non-malignant cells (IC50 =15-30â µm).
Assuntos
Antineoplásicos/farmacologia , Lisossomos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Pró-Fármacos/farmacologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Animais , Antineoplásicos/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Lisossomos/metabolismo , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Pró-Fármacos/química , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-AtividadeRESUMO
Nanoparticles have belonged to various fields of biomedical research for quite some time. A promising site-directed application in the field of nanomedicine is drug targeting using magnetic nanoparticles which are directed at the target tissue by means of an external magnetic field. Materials most commonly used for magnetic drug delivery contain metal or metal oxide nanoparticles, such as superparamagnetic iron oxide nanoparticles (SPIONs). SPIONs consist of an iron oxide core, often coated with organic materials such as fatty acids, polysaccharides or polymers to improve colloidal stability and to prevent separation into particles and carrier medium [1]. In general, magnetite and maghemite particles are those most commonly used in medicine and are, as a rule, well-tolerated. The magnetic properties of SPIONs allow the remote control of their accumulation by means of an external magnetic field. Conjugation of SPIONs with drugs, in combination with an external magnetic field to target the nanoparticles (so-called "magnetic drug targeting", MDT), has additionally emerged as a promising strategy of drug delivery. Magnetic nanoparticle-based drug delivery is a sophisticated overall concept and a multitude of magnetic delivery vehicles have been developed. Targeting mechanism-exploiting, tumor-specific attributes are becoming more and more sophisticated. The same is true for controlled-release strategies for the diseased site. As it is nearly impossible to record every magnetic nanoparticle system developed so far, this review summarizes interesting approaches which have recently emerged in the field of targeted drug delivery for cancer therapy based on magnetic nanoparticles.
Assuntos
Antineoplásicos/administração & dosagem , Preparações de Ação Retardada/administração & dosagem , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/efeitos da radiação , Nanocápsulas/química , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/efeitos da radiação , Preparações de Ação Retardada/química , Preparações de Ação Retardada/efeitos da radiação , Humanos , Campos Magnéticos , Nanopartículas de Magnetita/administração & dosagem , Nanocápsulas/administração & dosagem , Nanocápsulas/efeitos da radiaçãoRESUMO
Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted increasing attention in many biomedical fields. In magnetic drug targeting SPIONs are injected into a tumour supplying artery and accumulated inside the tumour with a magnet. The effectiveness of this therapy is thus dependent on magnetic properties, stability and biocompatibility of the particles. A good knowledge of the effect of storage conditions on those parameters is of utmost importance for the translation of the therapy concept into the clinic and for reproducibility in preclinical studies. Here, core shell SPIONs with a hybrid coating consisting of lauric acid and albumin were stored at different temperatures from 4 to 45 °C over twelve weeks and periodically tested for their physicochemical properties over time. Surprisingly, even at the highest storage temperature we did not observe denaturation of the protein or colloidal instability. However, the saturation magnetisation decreased by maximally 28.8% with clear correlation to time and storage temperature. Furthermore, the biocompatibility was clearly affected, as cellular uptake of the SPIONs into human T-lymphoma cells was crucially dependent on the storage conditions. Taken together, the results show that the particle properties undergo significant changes over time depending on the way they are stored.
Assuntos
Materiais Biocompatíveis/química , Armazenamento de Medicamentos , Compostos Férricos/química , Nanopartículas de Magnetita/química , Nanotecnologia/métodos , Albuminas/química , Sobrevivência Celular/efeitos dos fármacos , Coloides/química , Sistemas de Liberação de Medicamentos , Citometria de Fluxo , Humanos , Hidrodinâmica , Células Jurkat/efeitos dos fármacos , Ácidos Láuricos/química , Linfoma de Células T/metabolismo , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Reprodutibilidade dos Testes , Temperatura , Difração de Raios XRESUMO
Nanoparticles that are aimed at targeting cancer cells, but sparing healthy tissue provide an attractive platform of implementation for hyperthermia or as carriers of chemotherapeutics. According to the literature, diverse effects of nanoparticles relating to mammalian reproductive tissue are described. To address the impact of nanoparticles on cyto- and genotoxicity concerning the reproductive system, we examined the effect of superparamagnetic iron oxide nanoparticles (SPIONs) on granulosa cells, which are very important for ovarian function and female fertility. Human granulosa cells (HLG-5) were treated with SPIONs, either coated with lauric acid (SEONLA) only, or additionally with a protein corona of bovine serum albumin (BSA; SEON(LA-BSA)), or with dextran (SEON(DEX)). Both micronuclei testing and the detection of γH2A.X revealed no genotoxic effects of SEON(LA-BSA), SEON(DEX) or SEON(LA). Thus, it was demonstrated that different coatings of SPIONs improve biocompatibility, especially in terms of genotoxicity towards cells of the reproductive system.
Assuntos
Dextranos/toxicidade , Compostos Férricos/toxicidade , Células da Granulosa/efeitos dos fármacos , Nanopartículas de Magnetita/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dextranos/metabolismo , Feminino , Compostos Férricos/metabolismo , Células da Granulosa/metabolismo , Humanos , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Testes de MutagenicidadeRESUMO
Major problems of cancer treatment using systemic chemotherapy are severe side effects. Magnetic drug targeting (MDT) employing superparamagnetic iron oxide nanoparticles (SPION) loaded with chemotherapeutic agents may overcome this dilemma by increasing drug accumulation in the tumor and reducing toxic side effects in the healthy tissue. For translation of nanomedicine from bench to bedside, nanoparticle-mediated effects have to be studied carefully. In this study, we compare the effect of SPION, unloaded or loaded with the cytotoxic drug mitoxantrone (MTO) with the effect of free MTO, on the viability and proliferation of HT-29 cells within three-dimensional multicellular tumor spheroids. Fluorescence microscopy and flow cytometry showed that both free MTO, as well as SPION-loaded MTO (SPION(MTO)) are able to penetrate into tumor spheroids and thereby kill tumor cells, whereas unloaded SPION did not affect cellular viability. Since SPION(MTO) has herewith proven its effectivity also in complex multicellular tumor structures with its surrounding microenvironment, we conclude that it is a promising candidate for further use in magnetic drug targeting in vivo.
Assuntos
Antineoplásicos/farmacologia , Nanopartículas de Magnetita , Mitoxantrona/farmacologia , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mitoxantrona/administração & dosagem , Esferoides Celulares , Células Tumorais CultivadasRESUMO
Sol-gel borate bioactive glasses (BGs) are promising ion-releasing biomaterials for wound healing applications. Here, we report the synthesis of a series of binary B2O3-CaO borate BGs (CaO ranging from 50 to 90 mol%) using a sol-gel-based method. The influence of CaO content in B2O3-CaO borate BG on morphology, structure and ion release behavior was investigated in detail. Reduced dissolution (ion release) and crystallization could be observed in borate BGs when CaO content increased, while the morphology was not significantly altered by increasing CaO content. Our results evidenced that the ion release behavior of borate BGs could be tailored by tuning the B2O3/CaO molar ratio. We also evaluated the in vitro cytotoxicity, hemostatic, antibacterial and angiogenic activities of borate BGs. Cytocompatibility was validated for all borate BGs. However, borate BGs exhibited composition-dependent hemostatic, antibacterial and angiogenic activities. Generally, higher contents of Ca in borate BGs facilitated hemostatic activity, while higher contents of B2O3 were beneficial for pro-angiogenic activity. The synthesized sol-gel-derived borate BGs are promising materials for developing advanced wound healing dressings, given their fast ion release behavior and favorable hemostatic, antibacterial and angiogenic activities.
RESUMO
Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.
Assuntos
Apoptose , Micropartículas Derivadas de Células/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/farmacologia , Caspase 8/farmacologia , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Galactose/metabolismo , Glicoproteínas/metabolismo , Glicosilação/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Manose/metabolismo , Neuraminidase/metabolismoRESUMO
The presence of autoantibodies against immunoregulatory effectors can be relevant for onset and/or the progression of autoimmune disease. Emerging insights into an immunological activity profile including a role as opsonins give reason to systematically monitor sera of patients for immunoglobulin G (IgG) autoantibodies, preferably for several galectins at the same time. Here, we report on a study of chronic inflammatory rheumatic diseases, i.e. systemic lupus erythematosus (SLE; pilot cohort p, n = 40; confirmation cohort c, n = 109), rheumatoid arthritis (RA; p, n = 32; c, n = 25) and primary antiphospholipid syndrome (APS; c, n = 64). Enzyme-linked immunosorbent assay-based series using galectin-1, -2, -3, -4, -7, -8 and -9 and natural processing products, i.e. the truncated version of galectin-3 and the N-terminal domains of galectin-4, -8 and -9, were performed. Normal healthy donors (p, n = 20; c, n = 21) and patients with paraproteins (c, n = 19) served as controls. Highly significant optical density-value readings for IgG autoantibodies were consistently detected for the proto-type galectin-7 (SLE) and the tandem repeat-type galectin-8 and -9 (SLE and RA). Their presence was independent from the autoantibody status against double-stranded DNA (for patients with SLE) or a rheumatoid factor (for patients with RA), respectively. Importantly, anti-galectin-2 autoantibodies highly significantly correlated with the appearance of a secondary APS in patients with SLE so that this parameter may serve as an additional biomarker for APS. Equally of note, the presence of IgG autoantibodies against galectins capable to act as an opsonin may contribute to a sustained immune dysregulation in patients with chronic inflammatory rheumatic diseases.
Assuntos
Síndrome Antifosfolipídica/imunologia , Autoanticorpos/imunologia , Galectinas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Autoanticorpos/sangue , Biomarcadores/sangue , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Fator Reumatoide/sangueRESUMO
Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major 'eat-me' signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.
Assuntos
Anexina A5/metabolismo , Apoptose , Membrana Celular/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fosfatidilserinas/metabolismo , Células Cultivadas , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Ligação ProteicaRESUMO
Apoptotic cell death and the clearance of dying cells play an important and physiological role in embryonic development and normal tissue turnover. In contrast to necrosis, apoptosis proceeds in an anti-inflammatory manner. It is orchestrated by the timed release and/or exposure of so-called 'find-me', 'eat me' and 'tolerate me' signals. Mononuclear phagocytes are attracted by various 'find-me' signals, including proteins, nucleotides, and phospholipids released by the dying cell, whereas the involvement of granulocytes is prevented via 'stay away' signals. The exposure of anionic phospholipids like phosphatidylserine (PS) by apoptotic cells on the outer leaflet of the plasma membrane is one of the main 'eat me' signals. PS is recognized by a number of innate receptors as well as by soluble bridging molecules on the surface of phagocytes. Importantly, phagocytes are able to discriminate between viable and apoptotic cells both exposing PS. Due to cytoskeleton remodeling PS has a higher lateral mobility on the surfaces of apoptotic cells thereby promoting receptor clustering on the phagocyte. PS not only plays an important role in the engulfment process, but also acts as 'tolerate me' signal inducing the release of anti-inflammatory cytokines by phagocytes. An efficient and fast clearance of apoptotic cells is required to prevent secondary necrosis and leakage of intracellular danger signals into the surrounding tissue. Failure or prolongation of the clearance process leads to the release of intracellular antigens into the periphery provoking inflammation and development of systemic inflammatory autoimmune disease like systemic lupus erythematosus. Here we review the current findings concerning apoptosis-inducing pathways, important players of apoptotic cell recognition and clearance as well as the role of membrane remodeling in the engulfment of apoptotic cells by phagocytes.
Assuntos
Apoptose , Fosfatidilserinas/metabolismo , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Humanos , Fagócitos/citologia , Fagócitos/imunologia , Fagócitos/metabolismo , Fagocitose , Fosfatidilserinas/imunologia , Transdução de SinaisRESUMO
Magnetic drug targeting (MDT) improves the integrity of healthy tissues and cells during treatment with cytotoxic drugs. An anticancer drug is bound to superparamagnetic iron oxide nanoparticles (SPION), injected into the vascular supply of the tumor and directed into the tumor by means of an external magnetic field. In this study, we investigated the impact of SPION, mitoxantrone (MTO) and SPIONMTO on cell viability in vitro and the nonspecific uptake of MTO into circulating leukocytes in vivo. MDT was compared with conventional chemotherapy. MTO uptake and the impact on cell viability were assessed by flow cytometry in a Jurkat cell culture. In order to analyze MTO loading of circulating leukocytes in vivo, we treated tumor-bearing rabbits with MDT and conventional chemotherapy. In vitro experiments showed a dose-dependent MTO uptake and reduction in the viability and proliferation of Jurkat cells. MTO and SPIONMTO showed similar cytotoxic activity. Non-loaded SPION did not have any effect on cell viability in the concentrations tested. Compared with systemic administration in vivo, MDT employing SPIONMTO significantly decreased the chemotherapeutic load in circulating leukocytes. We demonstrated that MDT spares the immune system in comparison with conventional chemotherapy.
Assuntos
Proliferação de Células/efeitos dos fármacos , Citotoxinas , Sistemas de Liberação de Medicamentos/métodos , Leucócitos/metabolismo , Campos Magnéticos , Nanopartículas de Magnetita/química , Neoplasias Experimentais , Animais , Citotoxinas/química , Citotoxinas/farmacologia , Feminino , Humanos , Células Jurkat , Leucócitos/patologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , CoelhosRESUMO
Immune cell therapies, such as adoptive T cell therapies, are an innovative and powerful treatment option for previously non-treatable diseases. Although immune cell therapies are thought to be very specific, there is still the danger of developing severe to life-threatening side effects due to the unspecific distribution of the cells throughout the body (on-target/off-tumor effects). A possible solution for the reduction of these side effects and the improvement of tumor infiltration is the specific targeting of the effector cells (e.g., T cells) to the desired destination (e.g., tumor region). This can be achieved by the magnetization of cells with superparamagnetic iron oxide nanoparticles (SPIONs) for spatial guidance via external magnetic fields. A prerequisite for the use of SPION-loaded T cells in adoptive T cell therapies is that cell viability and functionality after nanoparticle loading are preserved. Here, we demonstrate a protocol to analyze cell viability and functionality such as activation, proliferation, cytokine release, and differentiation at a single cell level using flow cytometry.
Assuntos
Nanopartículas de Magnetita , Nanopartículas , Linfócitos T , Sobrevivência Celular , Citocinas , Linhagem Celular Tumoral , Campos MagnéticosRESUMO
Purpose: Magnetic separation of microbes can be an effective tool for pathogen identification and diagnostic applications to reduce the time needed for sample preparation. After peptide functionalization of superparamagnetic iron oxide nanoparticles (SPIONs) with an appropriate interface, they can be used for the separation of sepsis-associated yeasts like Candida albicans. Due to their magnetic properties, the magnetic extraction of the particles in the presence of an external magnetic field ensures the accumulation of the targeted yeast. Materials and Methods: In this study, we used SPIONs coated with 3-aminopropyltriethoxysilane (APTES) and functionalized with a peptide originating from GP340 (SPION-APTES-Pep). For the first time, we investigate whether this system is suitable for the separation and enrichment of Candida albicans, we investigated its physicochemical properties and by thermogravimetric analysis we determined the amount of peptide on the SPIONs. Further, the toxicological profile was evaluated by recording cell cycle and DNA degradation. The separation efficiency was investigated using Candida albicans in different experimental settings, and regrowth experiments were carried out to show the use of SPION-APTES-Pep as a sample preparation method for the identification of fungal infections. Conclusion: SPION-APTES-Pep can magnetically remove more than 80% of the microorganism and with a high selective host-pathogen distinction Candida albicans from water-based media and about 55% in blood after 8 minutes processing without compromising effects on the cell cycle of human blood cells. Moreover, the separated fungal cells could be regrown without any restrictions.