RESUMO
The technique of differential dynamic microscopy is extended here, showing that it can provide a powerful and objective method of video analysis for optical microscopy videos of in vitro samples of live human bronchial epithelial ciliated cells. These cells are multiciliated, with motile cilia that play key physiological roles. It is shown that the ciliary beat frequency can be recovered to match conventional analysis, but in a fully automated fashion. Furthermore, it is shown that the properties of spatial and temporal coherence of cilia beat can be recovered and distinguished, and that if a collective traveling wave (the metachronal wave) is present, this has a distinct signature and its wavelength and direction can be measured.
Assuntos
Brônquios/metabolismo , Cílios/metabolismo , Células Epiteliais/metabolismo , Microscopia de Vídeo/métodos , Mucosa Nasal/metabolismo , Automação Laboratorial/métodos , Células Cultivadas , Humanos , Imagem Óptica/métodosRESUMO
Predicting acute clinical events caused by atherosclerotic plaque rupture remains a clinical challenge. Anatomic mapping of the vascular tree provided by standard imaging technologies is not always sufficient for a robust diagnosis. Yet biological mechanisms leading to unstable plaques have been identified and corresponding biomarkers have been described. Nanosystems charged with contrast agents and targeted towards these specific biomarkers have been developed for several types of imaging modalities. The first systems that have reached the clinic are ultrasmall superparamagnetic iron oxides for Magnetic Resonance Imaging. Their potential relies on their passive accumulation by predominant physiological mechanisms in rupture-prone plaques. Active targeting strategies are under development to improve their specificity and set up other types of nanoplatforms. Preclinical results show a huge potential of nanomedicine for cardiovascular diagnosis, as long as the safety of these nanosystems in the body is studied in depth.
Assuntos
Doenças Cardiovasculares/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Sondas Moleculares/química , Nanomedicina/métodos , Nanopartículas/química , Animais , Biomarcadores/análise , Doenças Cardiovasculares/metabolismo , Humanos , Nanopartículas/ultraestruturaRESUMO
Heart failure occurs in over 30% of the worldwide population and most commonly originates from cardiovascular diseases such as myocardial infarction. microRNAs (miRNAs) target and silence specific mRNAs, thereby regulating gene expression. Because the endogenous miR-155-5p has been ascribed to vasculoprotection, loading it onto positively charged, core-shell poly(isobutylcyanoacrylate) (PIBCA)-polysaccharide nanoparticles (NPs) was attempted. NPs showed a decrease (p < 0.0001) in surface electrical charge (ζ potential), with negligible changes in size or shape when loaded with the anionic miR-155-5p. Presence of miR-155-5p in loaded NPs was further quantified. Cytocompatibility up to 100 µg/mL of NPs for 2 days with human coronary artery endothelial cells (hCAECs) was documented. NPs were able to enter hCAECs and were localized in the endoplasmic reticulum (ER). Expression of miR-155-5p was increased within the cells by 75-fold after 4 hours of incubation (p < 0.05) and was still noticeable at day 2. Differences between loaded NP-cultured cells and free miRNA, at days 1 (p < 0.05) and 2 (p < 0.001) suggest the ability of prolonged load release in physiological conditions. Expression of miR-155-5p downstream target BACH1 was decreased in the cells by 4-fold after 1 day of incubation (p < 0.05). This study is a first proof of concept that miR-155-5p can be loaded onto NPs and remain intact and biologically active in endothelial cells (ECs). These nanosystems could potentially increase an endogenous cytoprotective response and decrease damage within infarcted hearts.
RESUMO
Thrombotic diseases rarely cause symptoms until advanced stage and sudden death. Thus, early detection of thrombus by a widely spread imaging modality can improve the prognosis and reduce mortality. Here, polymer microbubbles (MBs) made of degradable poly(IsoButylCyanoAcrylate) and functionalized with fucoidan (Fucoidan-MBs) were designed as a new targeted ultrasound contrast agent to image venous thrombus. The physicochemical characterizations demonstrate that the MBs with fucoidan surface exhibit a size of 2-6⯵m and stability in suspension at 4⯰C up to 2 months. MBs exhibit high echogenicity and could be completely burst under high destructive pulse. Flow chamber experiments on activated human platelets show a higher affinity of Fucoidan-MBs than control anionic MBs (CM-Dextran-MBs) under shear stress conditions. In vivo analysis by ultrasound and histological results demonstrate that Fucoidan-MBs are localized in rat venous thrombotic wall, whereas few CM-Dextran-MBs are present. In addition, the binding of Fucoidan-MBs in healthy vein is not observed. Collectively, Fucoidan-MBs appear as a promising functionalized carrier for ultrasound molecular imaging in thrombotic diseases.
Assuntos
Meios de Contraste/química , Microbolhas , Selectina-P/análise , Trombose/diagnóstico por imagem , Células 3T3 , Animais , Bucrilato/química , Masculino , Camundongos , Imagem Molecular/métodos , Polissacarídeos/química , Ratos Wistar , Ultrassonografia/métodosRESUMO
Injection of recombinant tissue plasminogen activator (rt-PA) is the standard drug treatment for thrombolysis. However, rt-PA shows risk of hemorrhages and limited efficiency even at high doses. Polysaccharide-poly(isobutylcyanoacrylate) nanoparticles functionalized with fucoidan and loaded with rt-PA were designed to accumulate on the thrombus. Fucoidan has a nanomolar affinity for the P-selectin expressed by activated platelets in the thrombus. Solid spherical fluorescent nanoparticles with a hydrodynamic diameter of 136 ± 4 nm were synthesized by redox radical emulsion polymerization. The clinical rt-PA formulation was successfully loaded by adsorption on aminated nanoparticles and able to be released in vitro. We validated the in vitro fibrinolytic activity and binding under flow to both recombinant P-selectin and activated platelet aggregates. The thrombolysis efficiency was demonstrated in a mouse model of venous thrombosis by monitoring the platelet density with intravital microscopy. This study supports the hypothesis that fucoidan-nanoparticles improve the rt-PA efficiency. This work establishes the proof-of-concept of fucoidan-based carriers for targeted thrombolysis.
Assuntos
Nanopartículas/química , Selectina-P/antagonistas & inibidores , Polímeros/química , Polissacarídeos/química , Terapia Trombolítica , Animais , Modelos Animais de Doenças , Liberação Controlada de Fármacos , Hemorreologia/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Agregação Plaquetária/efeitos dos fármacos , Distribuição Tecidual/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/farmacologia , Ativador de Plasminogênio Tecidual/uso terapêutico , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios X , Trombose Venosa/patologia , Trombose Venosa/terapiaRESUMO
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.
Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/toxicidade , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Nanopartículas/química , Nanopartículas/toxicidade , Adesão Celular/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Monócitos/citologia , Propriedades de Superfície , Células THP-1RESUMO
New tools for molecular imaging and targeted therapy for cardiovascular diseases are still required. Herein, biodegradable microcapsules (MCs) made of polycyanoacrylate and polysaccharide and functionalized with fucoidan (Fuco-MCs) are designed as new carriers to target arterial thrombi overexpressing P-selectin. Physicochemical characterizations demonstrated that microcapsules have a core-shell structure and that fucoidan is present onto the surface of Fuco-MCs. Furthermore, their sizes range from 2 to 6 µm and they are stable on storage over 30 d at 4 °C. Flow cytometry experiments evidenced the binding of Fuco-MCs for human activated platelets as compared to MCs (mean fluorescence intensity: 12 008 vs. 9, p < 0.001) and its absence for nonactivated platelets (432). An in vitro flow adhesion assay showed high specific binding efficiency of Fuco-MCs to P-selectin and to activated platelet aggregates under arterial shear stress conditions. Moreover, both types of microcapsules reveal excellent compatibility with 3T3 cells in cytotoxicity assay. One hour after intravenous injection of microcapsules, histological analysis revealed that Fuco-MCs are localized in the rat abdominal aortic aneurysm thrombotic wall and that the binding in the healthy aorta is low. In conclusion, these microcapsules appear as promising carriers for targeting of tissues characterized by P-selectin overexpression and for their molecular imaging or treatment.
Assuntos
Aneurisma da Aorta Abdominal , Sistemas de Liberação de Medicamentos/métodos , Imagem Molecular/métodos , Selectina-P/metabolismo , Polissacarídeos , Trombose , Células 3T3 , Animais , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/metabolismo , Cápsulas , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Polissacarídeos/química , Polissacarídeos/farmacocinética , Polissacarídeos/farmacologia , Ratos , Ratos Wistar , Trombose/diagnóstico por imagem , Trombose/metabolismoRESUMO
AIM: We report the physicochemical analysis of nanosystems intended for cardiovascular applications and their toxicological characterization in static and dynamic cell culture conditions. METHODS: Size, polydispersity and ζ-potential were determined in 10 nanoparticle systems including liposomes, lipid nanoparticles, polymeric and iron oxide nanoparticles. Nanoparticle effects on primary human endothelial cell viability were monitored using real-time cell analysis and live-cell microscopy in static conditions, and in a flow model of arterial bifurcations. RESULTS & CONCLUSIONS: The majority of tested nanosystems were well tolerated by endothelial cells up to the concentration of 100 µg/ml in static, and up to 400 µg/ml in dynamic conditions. Pilot experiments in a pig model showed that intravenous administration of liposomal nanoparticles did not evoke the hypersensitivity reaction. These findings are of importance for future clinical use of nanosystems intended for intravascular applications.
Assuntos
Nanopartículas/química , Nanopartículas/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Compostos Férricos/química , Compostos Férricos/toxicidade , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipossomos/química , Lipossomos/toxicidade , Masculino , Polímeros/química , Polímeros/toxicidade , SuínosRESUMO
Polysaccharides are abundant biomolecules in nature presenting important roles in a wide variety of living systems processes. Considering the structural and biological functions of polysaccharides, their properties have raised interest for tissue engineering. Herein, we described the latest advances in cardiac tissue engineering mediated by polysaccharides. We reviewed the data already obtained in vitro and in vivo in this field with several types of polysaccharides. Cardiac injection, intramyocardial in situ polymerization strategies, and scaffold-based approaches involving polysaccharides for heart tissue engineering are thus discussed.
Assuntos
Coração , Polissacarídeos/química , Engenharia Tecidual , AnimaisRESUMO
Angiogenesis, the formation of blood vessels from pre-existing ones, is of vital importance during the early stages of bone healing. Extracellular stiffness plays an important role in regulating endothelial cell behavior and angiogenesis, but how this mechanical cue affects proliferation kinetics, gene regulation, and the expression of proteins implicated in angiogenesis and bone regeneration remains unclear. Using collagen-coated polyacrylamide (PAAm) hydrogels, human umbilical vein endothelial cells (HUVECs) are exposed to an environment that mimics the elastic properties of collagenous bone, and cellular proliferation and gene and protein expressions are assessed. The proliferation and gene expression of HUVECs are not differentially affected by culture on 3 or 30 kPa PAAm hydrogels, henceforth referred to as low and high stiffness gels, respectively. Although the proliferation and gene transcript levels remain unchanged, significant differences are found in the expressions of functional proteins and growth factors implicated both in the angiogenic and osteogenic processes. The down-regulation of the vascular endothelial growth factor receptor-2 protein with concomitant over-expression of caveolin-1, wingless-type 2, bone morphogenic protein 2, and basic fibroblast growth factor on the high stiffness PAAm hydrogel suggests that rigidity has a pro-angiogenic effect with inherent benefits for bone regeneration.
RESUMO
This review highlights the preclinical and clinical research based on the use of nano- and micro-carriers in thrombolytic drug delivery. Ischemic heart and stroke caused by thrombosis are the main causes of death in the world. Because of their inactivation in the blood, high doses of thrombolytics are administered to patients, increasing the risk of intracranial hemorrhage. Preclinical research conducted with lipid, polymer or magnetic nanoparticles loaded with thrombolytic drugs showed an enhancement of thrombolysis and a reduction of undesirable side effects. Targeted nanocarriers exhibited an increased accumulation into clot. Clinical trials were already conducted with lipid-based microbubbles combined with ultrasound and thrombolytic drug and showed thrombolysis improvement. Future validation of nanosystems is awaited in clinic. This research opens new strategies for the management of thrombotic diseases.