RESUMO
Aim: To quantitatively evaluate the inhibition of human DNA repair proteins APE1 and MTH1 by dextran-coated γ-Fe2O3 ultrasmall superparamagnetic iron oxide nanoparticles (dUSPIONs). Materials & methods: Liquid chromatography-tandem mass spectrometry with isotope-dilution was used to measure the expression levels of APE1 and MTH1 in MCL-5 cells exposed to increasing doses of dUSPIONs. The expression levels of APE1 and MTH1 were measured in cytoplasmic and nuclear fractions of cell extracts. Results: APE1 and MTH1 expression was significantly inhibited in both cell fractions at the highest dUSPION dose. The expression of MTH1 was linearly inhibited across the full dUSPION dose range in both fractions. Conclusion: These findings warrant further studies to characterize the capacity of dUSPIONs to inhibit other DNA repair proteins in vitro and in vivo.
Inhibitors of DNA repair proteins are increasingly being utilized as potential anticancer agents to supplement traditional chemotherapy and radiation-based approaches. The present study was focused on investigating the use of iron oxide nanoparticles to inhibit the expression of relevant human DNA repair proteins in a cellular model (MCL-5 cells). The authors utilized liquid chromatographytandem mass spectrometry with isotope dilution to measure the expression levels of two different DNA repair proteins (MTH1 and APE1) in cells after the cells were exposed to increasing levels of the iron oxide nanoparticles. The authors observed significant decreases in DNA repair protein levels that were associated with increasing doses of the iron oxide nanoparticles. The authors' findings warrant more comprehensive studies using other cellular models and suitable animal models.
Assuntos
Dextranos , Nanopartículas Magnéticas de Óxido de Ferro , Humanos , Reparo do DNARESUMO
The research presented here shows QbD implementation for the optimisation of the key process parameters in electrohydrodynamic atomisation (EHDA). Here, the electrosprayed nanoparticles and electrospun fibers consisting of a polymeric matrix and dye. Eight formulations were assessed consisting of 5% w/v of polycaprolactone (PCL) in dichloromethane (DCM) and 5% w/v polyvinylpyrrolidone (PVP) in ethanol. A full factorial DOE was used to assess the various parameters (applied voltage, deposition distance, flow rate). Further particle and fiber analysis using Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), Fourier Transform Infrared Spectroscopy (FTIR), particle/fiber size distribution. In addition to this in vitro release studied were carried out using fluorescein and Rhodamine B as model dyes and in vitro permeation studies were applied. The results show a significant difference in the morphology of resultant structures as well as a more rapid release profile for the PVP particles and fibers in comparison to the sustained release profiles found with PCL. In vitro drug release studies showed 100% drug release after 7 days for PCL particles and showed 100% drug release within 120 min for PVP particles. The release kinetics and the permeation study showed that the MN successfully pierced the membrane and the electrospun MN coating released a large amount of the loaded drug within 6 h. This study has demonstrated the capability of these robust MNs to encapsulate a diverse range drugs within a polymeric matrix giving rise to the potential of developed personalised medical devices.
Assuntos
Microinjeções/instrumentação , Agulhas , Polímeros/química , Pesquisa Qualitativa , Tecnologia Farmacêutica/instrumentação , Liberação Controlada de Fármacos , Microinjeções/normas , Agulhas/normas , Poliésteres/química , Poliésteres/normas , Polímeros/normas , Povidona/química , Povidona/normas , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Tecnologia Farmacêutica/normasRESUMO
Recently, remarkable efforts have focused on research towards enhancing and delivering efficacious and advanced therapeutic agents. Even though this involves significant challenges, innovative techniques and materials have been explored to overcome these. The advantageous properties of mesoporous silica nanoparticles (MSNs), such as unique morphologies and geometries, makes then favorable for use for various drug delivery targeting purposes, particularly in cancer therapy. As we discuss here, MSNs have been utilized over the past few decades to improve the efficiency of anticancer drugs by enhancing their solubility to render them suitable for application, reducing adverse effects, and improving their anticancer cytotoxic efficiency.
Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos/administração & dosagem , Dióxido de Silício/administração & dosagem , Animais , Humanos , PorosidadeRESUMO
BACKGROUND: It is well established that toxicological evaluation of engineered nanomaterials (NMs) is vital to ensure the health and safety of those exposed to them. Further, there is a distinct need for the development of advanced physiologically relevant in vitro techniques for NM hazard prediction due to the limited predictive power of current in vitro models and the unsustainability of conducting nano-safety evaluations in vivo. Thus, the purpose of this study was to develop alternative in vitro approaches to assess the potential of NMs to induce genotoxicity by secondary mechanisms. RESULTS: This was first undertaken by a conditioned media-based technique, whereby cell culture media was transferred from differentiated THP-1 (dTHP-1) macrophages treated with γ-Fe2O3 or Fe3O4 superparamagnetic iron oxide nanoparticles (SPIONs) to the bronchial cell line 16HBE14o-. Secondly construction and SPION treatment of a co-culture model comprising of 16HBE14o- cells and dTHP-1 macrophages. For both of these approaches no cytotoxicity was detected and chromosomal damage was evaluated by the in vitro micronucleus assay. Genotoxicity assessment was also performed using 16HBE14o- monocultures, which demonstrated only γ-Fe2O3 nanoparticles to be capable of inducing chromosomal damage. In contrast, immune cell conditioned media and dual cell co-culture SPION treatments showed both SPION types to be genotoxic to 16HBE14o- cells due to secondary genotoxicity promoted by SPION-immune cell interaction. CONCLUSIONS: The findings of the present study demonstrate that the approach of using single in vitro cell test systems precludes the ability to consider secondary genotoxic mechanisms. Consequently, the use of multi-cell type models is preferable as they better mimic the in vivo environment and thus offer the potential to enhance understanding and detection of a wider breadth of potential damage induced by NMs.
Assuntos
Dano ao DNA , Compostos Férricos/toxicidade , Nanopartículas de Magnetita/toxicidade , Testes de Mutagenicidade/métodos , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Brônquios/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados , Citocinas/biossíntese , Endocitose/efeitos dos fármacos , Humanos , Técnicas In Vitro , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Células THP-1RESUMO
The liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20⯵l drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8â¯g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24â¯h resulted in a 6-fold increase in CYP1A1 enzyme activity (3⯵M B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5⯵M PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures.
Assuntos
Técnicas de Cultura de Células/métodos , Ensaios de Triagem em Larga Escala/métodos , Fígado/patologia , Testes de Mutagenicidade/métodos , Mutagênicos/efeitos adversos , Esferoides Celulares/patologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Testes para MicronúcleosRESUMO
Encapsulation of poorly water-soluble drugs into mesoporous materials (e.g. silica) has evolved as a favorable strategy to improve drug solubility and bioavailability. Several techniques (e.g. spray drying, solvent evaporation, microwave irradiation) have been utilized for the encapsulation of active pharmaceutical ingredients (APIs) into inorganic porous matrices. In the present work, a novel chalcone (KAZ3) with anticancer properties was successfully synthesized by Claisen-Schmidt condensation. KAZ3 was loaded into mesoporous (SBA-15 and MCM-41) and non-porous (fumed silica, FS) materials via two techniques; electrohydrodynamic atomization (EHDA) and solvent impregnation. The effect of both loading methods on the physicochemical properties of the particles (e.g. size, charge, entrapment efficiency, crystallinity, dissolution and permeability) was investigated. Results indicated that EHDA technique can load the active in a complete amorphous form within the pores of the silica particles. In contrast, reduced crystallinity (~79%) was obtained for the solvent impregnated formulations. EHDA engineered formulations significantly improved drug dissolution up to 30-fold, compared to the crystalline drug. Ex vivo studies showed EHDA formulations to exhibit higher permeability across rat intestine than their solvent impregnated counterparts. Cytocompatibility studies on Caco-2 cells demonstrated moderate toxicity at high concentrations of the anticancer agent. The findings of the present study clearly show the immense potential of EHDA as a loading technique for mesoporous materials to produce poorly water-soluble API carriers of high payload at ambient conditions. Furthermore, the scale up potential in EHDA technologies indicate a viable route to enhance drug encapsulation and dissolution rate of loaded porous inorganic materials.
Assuntos
Antineoplásicos/administração & dosagem , Química Farmacêutica/métodos , Portadores de Fármacos/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Células CACO-2 , Cristalização , Liberação Controlada de Fármacos , Humanos , Absorção Intestinal , Masculino , Porosidade , Ratos , Ratos Wistar , Dióxido de Silício/química , Solubilidade , Solventes/química , Tecnologia Farmacêutica/métodos , Água/químicaRESUMO
The liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20⯵l drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8â¯g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24â¯h resulted in a 6-fold increase in CYP1A1 enzyme activity (3⯵M B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5⯵M PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures.
Assuntos
Técnicas de Cultura de Células/métodos , Modelos Biológicos , Esferoides Celulares/citologia , Células Tumorais Cultivadas/citologia , Sobrevivência Celular , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2 , Citocinese , Células Hep G2 , Ensaios de Triagem em Larga Escala , Humanos , Testes de Mutagenicidade , Albumina Sérica Humana/metabolismo , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas/metabolismoRESUMO
Ultrafine superparamagnetic iron oxide nanoparticles (USPION) hold great potential for revolutionising biomedical applications such as MRI, localised hyperthermia, and targeted drug delivery. Though evidence is increasing regarding the influence of nanoparticle physico-chemical features on toxicity, data however, is lacking that assesses a range of such characteristics in parallel. We show that iron redox state, a subtle though important physico-chemical feature of USPION, dramatically modifies the cellular uptake of these nanoparticles and influences their induction of DNA damage. Surface chemistry was also found to have an impact and evidence to support a potential mechanism of oxidative DNA damage behind the observed responses has been demonstrated. As human exposure to ferrofluids is predicted to increase through nanomedicine based therapeutics, these findings are important in guiding the fabrication of USPION to ensure they have characteristics that support biocompatibility.
Assuntos
Compostos Férricos/química , Nanopartículas de Magnetita/química , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Compostos Férricos/efeitos adversos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Nanopartículas de Magnetita/efeitos adversos , Nanopartículas de Magnetita/ultraestrutura , Microscopia Eletrônica de Transmissão , Oxirredução , Espectroscopia FotoeletrônicaRESUMO
Due to the unique physicochemical properties of nanomaterials (NM) and their unknown reactivity, the possibility of NM altering the optical properties of fluorometric/colorimetric probes that are used to measure their cyto- and genotoxicity may lead to inaccurate readings. This could have potential implications given that NM, such as ultrafine superparamagnetic iron oxide nanoparticles (USPION), are increasingly finding their use in nanomedicine and the absorbance/fluorescence based assays are used to assess their toxicity. This study looks at the potential of dextran-coated USPION (dUSPION) (maghemite and magnetite) to alter the background signal of common probes used for evaluating cytotoxicity (MTS, CyQUANT, Calcein, and EthD-1) and oxidative stress (DCFH-DA and APF). In the present study, both forms of dUSPION caused an increase in MTS signal but a decrease in background signal from calcein and 3'-(p-aminophenyl) fluorescein (APF) and no effect on CyQUANT and EthD-1 fluorescence responses. Magnetite caused a decrease in fluorescence signal of DCFH, but it did not decrease fluorescence signal in the presence of the reactive oxygen species-inducer tert-butyl hydroperoxide (TBHP). In contrast, maghemite caused an increase in fluorescence, which was substantially reduced in the presence of the antioxidant N-acetyl cysteine. This study emphasizes the importance of considering and controlling for possible interactions between NM and fluorometric/colorimetric dyes and, most importantly, the oxidation state of dUSPION that may confound their sensitivity and specificity.
Assuntos
Colorimetria/métodos , Corantes/química , Dextranos/química , Compostos Férricos/química , Corantes Fluorescentes/química , Fluorometria/métodos , Nanopartículas de Magnetita/química , Etídio/análogos & derivados , Etídio/toxicidade , Fluoresceínas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , terc-Butil Hidroperóxido/químicaRESUMO
Superparamagnetic iron oxide nanoparticles (SPION) are being widely used for various biomedical applications, for example, magnetic resonance imaging, targeted delivery of drugs or genes, and in hyperthermia. Although, the potential benefits of SPION are considerable, there is a distinct need to identify any potential cellular damage associated with these nanoparticles. Besides focussing on cytotoxicity, the most commonly used determinant of toxicity as a result of exposure to SPION, this review also mentions the importance of studying the subtle cellular alterations in the form of DNA damage and oxidative stress. We review current studies and discuss how SPION, with or without different surface coating, may cause cellular perturbations including modulation of actin cytoskeleton, alteration in gene expression profiles, disturbance in iron homeostasis and altered cellular responses such as activation of signalling pathways and impairment of cell cycle regulation. The importance of protein-SPION interaction and various safety considerations relating to SPION exposure are also addressed.
RESUMO
With the rapid expansion in the nanotechnology industry, it is essential that the safety of engineered nanomaterials and the factors that influence their associated hazards are understood. A vital area governing regulatory health risk assessment is genotoxicology (the study of genetic aberrations following exposure to test agents), as DNA damage may initiate and promote carcinogenesis, or impact fertility. Of late, considerable attention has been given to the toxicity of engineered nanomaterials, but the importance of their genotoxic potential on human health has been largely overlooked. This comprehensive review focuses on the reported abilities of metal nanoparticles, metal-oxide nanoparticles, quantum dots, fullerenes, and fibrous nanomaterials, to damage or interact with DNA, and their ecogenotoxicity is also considered. Many of the engineered nanomaterials assessed were found to cause genotoxic responses, such as chromosomal fragmentation, DNA strand breakages, point mutations, oxidative DNA adducts and alterations in gene expression profiles. However, there are clear inconsistencies in the literature and it is difficult to draw conclusions on the physico-chemical features of nanomaterials that promote genotoxicity, largely due to study design. Hence, areas that require that further attention are highlighted and recommendations to improve our understanding of the genotoxic potential of engineered nanomaterials are addressed.
Assuntos
Dano ao DNA/efeitos dos fármacos , Nanoestruturas/toxicidade , Animais , Humanos , Nanopartículas Metálicas/toxicidade , Nanotecnologia , Nanotubos/toxicidade , Pontos QuânticosRESUMO
Monocyte hyperactivation as seen in diabetes results in increased cytoskeletal rigidity and reduced cell deformability leading to microchannel occlusions and microvascular complications. The thiazolidinediones (TZDs) are PPAR-gamma agonists that have been reported to exert beneficial non-metabolic effects on the vasculature. This study demonstrates that the TZD, Rosiglitazone, significantly reduces f-MLP-induced actin polymerisation in human monocytic cells (p < 0.05). Two of the key signalling processes known to be involved in the regulation of cytoskeletal remodelling were investigated: PI(3)K-dependent Akt phosphorylation and intracellular calcium concentration [Ca(2+)](i). The PI(3)K inhibitor, Wortmannin, ameliorated f-MLP-induced actin polymerisation (p < 0.05), while the Ca(2+) sequestration inhibitor, thapsigargin, induced actin depolymerisation (p < 0.05), confirming the involvement of both processes in cytoskeletal remodelling. Rosiglitazone significantly reduced f-MLP activation of Akt (p < 0.05), and significantly increased [Ca(2+)](i) in both resting and f-MLP-stimulated cells (p < 0.05). Therefore, Rosiglitazone interacts with signalling events downstream of occupancy of the f-MLP receptor, to modulate cytoskeletal remodelling in a PPAR-gamma-independent manner. To our knowledge, these results are the first to present evidence that a PPAR-gamma agonist can modulate actin remodelling in monocytes, and may therefore be protective against microvascular damage in diabetes.