The fabrication of PLGA-Fe3O4- magnetic nanoparticles for targeted delivery of γ-secretase inhibitors to vascular stents

Cardiovascular disease is the number one killer in Ireland and the wider EU. A hallmark of the disease is the obstruction to blood flow due to the build-up of neointimal vascular smooth muscle (SMCs)-like cells within the vessel wall. Several groups have shown SMC-like cells play important roles in the pathophysiological processes of arteriosclerosis, atherosclerosis, and in-stent restenosis [ISR]. Our lab and others have shown a putative role for re-capitulation of Notch signalling components in arteriosclerosis progression. Lately, multipotent vascular stem cells (MVSCs) have been isolated and shown to proliferate and differentiate into SMCs, causing vascular injury in animal models. Therefore, targeting these cells is an attractive therapeutic strategy for treating vascular remodelling disorders. In-stent restenosis treatment options include percutaneous transluminal coronary angioplasty (PTCA) and intravascular stenting yet a significant number of stented vessels may become re-occluded due to ISR. While polymer-coated drug-eluting stents (DES) have significantly reduced the incidence of ISR, current DESs have limitations. This limitation can be overcome by combining magnetic targeting via a uniform field-induced magnetization effect and a biocompatible magnetic nanoparticle (MNP) formulation designed for efficient entrapment and delivery of a specific drug that targets resident multipotent vascular stem cells (MVSCs). Therefore, the overall aim of this thesis was to develop a method for targeting vascular stents using nanotechnology. Specifically, the aim was to (i) fabricate magnetic nanoparticles (MNP’s) containing magnetite (Fe3O4) and functionalised with poly (DL-lactide-co-glycolide) polyvinyl alcohol [PLGA-PVA], (ii) characterise their functional properties, targeting to vascular stents and drug-release kinetics following the entrapment of two drugs, DAPT and Compound E, both of which are Ɣ-secretase inhibitors (GSI) of Notch target gene expression (iii) determine the effects of MNPs loaded with Ɣ-secretase inhibitors (DAPT and Compound E) on the growth and myogenic differentiation capacity of resident vascular stem cells under non- magnetic and magnetic conditions in vitro. The DAPT-loaded MNPs had an average hydrodynamic diameter of 351 d.nm. Up to 68% and 98% of the drug was incorporated into MNPs after one week under magnetic conditions. The Notch ligand, Jagged1 increased Hey1 mRNA levels and promoted myogenic differentiation of MSCs in vitro by increasing SMC differentiation markers, myosin heavy chain 11 (Myh11) and calponin1 (CNN1) expression, respectively. This effect was significantly attenuated following treatment of cells with both MNP’s loaded with DAPT and MNP’s loaded with Compound E when compared to unloaded MNP’s. These data suggest that Notch GSI loaded magnetic nanoparticles are functional at vascular stem cells in vitr

Diagnostic and prognostic value of free PAPP-A in coronary artery disease – clinical studies with novel immunoassay

Complications of coronary artery disease are the number one cause of death worldwide, so identification of high-risk individuals is crucial. Pregnancy-associated plasma protein A (PAPP-A) is a candidate cardiac-risk related biomarker, which has been linked to poorer outcome in various cardiovascular patients. Especially, the free form of PAPP-A (fPAPP-A) is linked to cardiovascular events, but thus far no direct assay detecting fPAPP-A has been published. Most available assays measure total PAPP-A and are widely used in prenatal screening where high concentration changes occur. Thus, cardiac-related slight fPAPP-A changes might be undetectable with total PAPP-A assays. Also, PAPP-A is released to circulation after heparin treatment, which is a common anticoagulant used in dissolving thrombus. This might question the ability of PAPP-A to be used as a marker for haparinized patients. I n this thesis, first direct immunoassay targeting fPAPP-A was created and its analytical and clinical performance as cardiac-risk related biomarker was evaluated. The analytical performance of the fPAPP-A assay was evaluated against current guidelines. The risk of death or myocardial infarction in patients admitted to hospital due to chest pain was studied and compared to tPAPP-A and indirect fPAPP-A assays. The developed assay was also used in predicting obstructive CAD in suspected chronic CAD patients. The disease extent and severity was determined with hybrid computed tomography and positron emission tomography myocardial perfusion imaging. Correlation of heparin-induced release of fPAPP-A and atherosclerotic burden was studied in suspected chronic CAD patients. The developed assay sensitively measured fPAPP-A. The risk of death or myocardial infarction correlated with increasing circulating fPAPP-A concentration. Also, fPAPP-A in combination with contemporary cTnI further improved the riskpredictive capability. Accordingly, the fPAPP-A level was elevated in chronic CAD patients who were diagnosed with obstructive CAD. As a biomarker, fPAPP-A outperformed traditional risk factors and other evaluated biomarkers in predicting obstructive CAD. Heparin-induced release of fPAPP-A to circulation was not associated with clinical coronary atherosclerotic characteristristics observed with coronary computed tomography angiography.Suoralla menetelmällä mitattu vapaa PAPP-A diagnostisena ja ennustavana merkkiaineena sepelvaltimotaudissa Sepelvaltimotautiin liittyvät komplikaatiot ovat yleisin kuolinsyy maailmassa, jonka vuoksi korkean riskin potilaiden tunnistaminen ajoissa on tärkeää. Raskauteen liittyvä plasmaproteiini A (PAPP-A) on lupaava merkkiaine, joka on yhdistetty erilaisilla sydänpotilailla heikompaan selviytymiseen ilman sydäntapahtumia. Erityisesti PAPP-A:n vapaa muoto (fPAPP-A) on liitetty sydäntapahtumiin, mutta suoraa määritysmenetelmää fPAPP-A:lle ei ole vielä kehitetty. Useimmat määritykset mittaavat kaikkia PAPP-A:n muotoja ja niitä käytetään raskaudenaikaiseen seulontaan, jolloin pitoisuusmuutokset ovat suuria. Tällaisella määrityksellä mitattuna sydäntapahtumien yhteydessä tapahtuvat pienet fPAPP-A -muutokset saattavat jäädä huomioimatta. PAPP-A:ta vapautuu verenkiertoon myös verihyytymien liuottamiseen käytetyn hepariinilääkityksen vuoksi. Tämä saattaa kyseenalaistaa PAPP-A:n käytön merkkiaineena hepariinilääkityillä potilailla. Väitöskirjatyössä kehitettiin ensimmäinen suora fPAPP-A:ta mittaava immunomääritys. Määrityksen analyyttista toimivuutta evaluoitiin kliinisen kemian ohjeistusten mukaisesti. FPAPP-A:n käyttöä sydänkohtaukseen tai kuolemaan liitettynä merkkiaineena tutkittiin rintakivun vuoksi sairaalaan saapuneilta potilailta ja tuloksia verrattiin epäsuoralla menetelmällä mitattuun fPAPP-A:han sekä kaikkia PAPP-A:n muotoja mittaavaan määritykseen. Lisäksi, fPAPP-A:n yhteyttä ahtauttavaan sepelvaltimotautiin tarkasteltiin epäillyillä vakaaoireisilla sepelvaltimotautipotilailla. Sepelvaltimotaudin vakavuuden ja laajuuden määrittäimiseen käytettiin hybridikuvantamista, jossa hyödynnetään tietokonetomografiaa ja isotooppitutkimusta. Samoilla potilailla tutkittiin myös hepariinin vapauttaman fPAPPA:n yhteyttä sepelvaltimotaudin vaikeusasteeseen. Kehitetty määritys oli herkkä mittaamaan fPAPP-A:ta. Kohonnut fPAPP-A oli yhteydessä suurentuneeseen sydänkohtauksen tai kuoleman riskiin. Yhdistettynä sydänperäisen troponiini I:n kanssa, kyky ennustaa riskä parani entisestään. Lisäksi, ahtauttavaa sepelvaltimotautia sairastavilla potilailla oli kohonnut fPAPP-A - pitoisuus ja fPAPP-A toimi paremmin kuin perinteiset riskitekijät tai muut mitatut merkkiaineet. Hepariinin vuoksi verenkiertoon vapautunut fPAPP-A ei ollut yhteydessä selvaltimotaudin vakavuuteen tai laajuuteen

Protheomic and adipo/cytokine biomarker analysis of unstable carotid atheroma plaque

L'arteriosclerosi és un procés patològic que provoca manifestacions clíniques de malalties vasculars. Els objectius daquesta tesi són identificar possibles biomarcadors d?aterosclerosi carotídia (AC) en mostres de secretoma dels pacients amb placa carotídia inestable i estudiar el possible paper d’algunes adipo/citoquines en l’estabilitat de les plaques ateroscleròtiques. També analitzem els nivells de diverses adipes/citocines pro i antiinflamatòries en sèrum i secretoma de placa inestable segons la presència de factors de risc cardiovascular. En el primer estudi, realitzem una anàlisi proteòmica quantitativa a les mostres de secretoma obtingudes de plaques ateroscleròtiques carotídies humanes mitjançant endarterectomia carotídia. En un segon estudi, analitzem els nivells de diverses adipes/citocines pro i antiinflamatòries en sèrum i secretoma de placa inestable mitjançant assaigs immunoabsorbents lligats a enzims. Es van detectar alguns canvis proteòmics en els secretomes de les plaques ateroscleròtiques carotídies (CAP) en comparació amb les artèries mamàries no ateroscleròtiques (AMNE). Les proteïnes augmentades als secretomes de CAP van ser la defensina 1 de neutròfils, l'apolipoproteïna E, la clusterina i la glicoproteïna alfa-2 de zinc. Pel que fa als nivells sèrics de visfatina, no hi va haver diferències entre els grups de CAP inestable i AMNE.La arteriosclerosis es un proceso patológico que provoca manifestaciones clínicas de enfermedades vasculares. Los objetivos de esta tesis son identificar posibles biomarcadores de aterosclerosis carotídea (AC) en muestras de secretoma de pacientes con placa carotídea inestable y estudiar el posible papel de algunas adipo/citoquinas en la estabilidad de las placas ateroscleróticas. También analizamos los niveles de varias adipo/citocinas pro y antiinflamatorias en suero y secretoma de placa inestable según la presencia de factores de riesgo cardiovascular. En el primer estudio, realizamos un análisis proteómico cuantitativo en las muestras de secretoma obtenidas de placas ateroscleróticas carotídeas humanas mediante endarterectomía carotídea. En un segundo estudio, analizamos los niveles de varias adipo/citocinas pro y antiinflamatorias en suero y secretoma de placa inestable mediante ensayos inmunoabsorbentes ligados a enzimas. Se detectaron algunos cambios proteómicos en los secretomas de las placas ateroscleróticas carotídeas (CAP) en comparación con las arterias mamarias no ateroscleróticas (AMNE). Las proteínas aumentadas en los secretomas de CAP fueron la defensina 1 de neutrófilos, la apolipoproteína E, la clusterina y la glicoproteína alfa-2 de zinc.Arteriosclerosis is prevalent pathological process that causes clinical manifestations of vascular diseases such as myocardial infarction, stroke or peripheral arterial occlusive disease. Objectives of this thesis are to identify potential candidate biomarkers for carotid atherosclerosis (CA) in secretome samples of patients with unstable carotid plaque and to study the possible role of some adipo/cytokines in the stability of atherosclerotic plaques. We also analysed the levels of several pro- and anti-inflammatory adipo/cytokines in serum and unstable plaque secretome according to the presence of cardiovascular risk factors. In a first study, we performed proteomic quantitative analysis in secretome samples obtained from human carotid atherosclerotic plaques by carotid endarterectomy. In a second, we analysed the levels of several pro- and anti-inflammatory adipo/cytokines in serum and unstable plaque secretome by enzyme-linked immunosorbent assays (ELISA). Some proteomic changes were detected in the secretomes of carotid atherosclerotic plaques (CAP) compared with non atherosclerotic mammary arteries. The increased proteins in CAP secretomes were neutrophil defensin 1, apolipoprotein E, clusterin and zinc-alpha-2-glycoprotein

Functional Nanomaterials in Biomedicine

The great success of nanotechnology promotes a tremendous revolution in the biomedical field. Functional nanomaterials have been widely applied for the treatment of various diseases, such as cancer, bacterial infection, diabetes, inflammation, and neurodegenerative disorders. Various therapeutic nanoplatforms have been developed with therapeutic functions and intelligent properties. However, the development of nanomedicine suffers from several challenges prior to their clinical applications. For instance, disease detection in an early stage is a critical challenge for nanomedicine. It is difficult to detect disease markers (e.g., proteins, genes, or cancer circulating cells), so nanoprobes with high sensitivity and selectivity are required. Moreover, to overcome drug resistance, it is highly desirable to develop functional nanomedicines with the combination of multiple therapeutic modalities, such as chemotherapy, photothermal therapy, photodynamic therapy, chemodynamic therapy, radiotherapy, starving therapy, and immunotherapy. Additionally, the stability and degradability of most nanomedicines in biofluids should be carefully evaluated before their administration to humans. This book provides researchers with the latest investigations and findings in this field

Mechanical Engineering

The book substantially offers the latest progresses about the important topics of the "Mechanical Engineering" to readers. It includes twenty-eight excellent studies prepared using state-of-art methodologies by professional researchers from different countries. The sections in the book comprise of the following titles: power transmission system, manufacturing processes and system analysis, thermo-fluid systems, simulations and computer applications, and new approaches in mechanical engineering education and organization systems