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Comprehensive Study on Aptamers and Aptamer-based AssaysTruedson, Axel, Sundström, Márta, Eriksson, Christoffer, Bergfeldt, Andreas, Jägare Lindvall, Matilda, Normann, Caroline January 2022 (has links)
Antibodies are the gold standard molecular recognition elements and a cornerstone of molecular biology. They are used in immunoassays to precisely measure a specific analyte, but certain targets are especially challenging. Difficult targets include small molecules and molecules that do not induce an immune response. Aptamers are short oligonucleotides that can form 3-dimensional structures and bind targets with high specificity. Aptamers are smaller and more flexible than antibodies and could therefore solve this problem. In contrast to antibodies, aptamers are synthetically produced, so they can have affinity for molecules that do not induce an immune response. This also makes them cheaper, faster and more ethical to produce. They are also easily modified and have the ability to renature and can therefore be reused. Our conclusions are that aptamers can outperform antibodies, especially for small molecule targets, and that the synthetic production of aptamers gives them a further advantage over antibodies. Our report compares several different types of detection methods that use aptamers and we conclude that fluorescence-based methods are the most easy to use with basic lab equipment, can be made similar to the ELISA kits in addition to giving highly sensitive detection. We describe a variety of fluorescence-based detection strategies but the optimal method will depend on the specific aptamer and target. The report also includes an ethical analysis where antibodies and aptamers are compared. This report is commissioned by Mercodia AB, a company that develops, manufactures and distributes immunoassays for biomarkers within the field of metabolic disorders. They commissioned this report in order to give an overview of how aptamers interact with their target, and also to compare aptamer-based detection strategies with sensitivity prioritized over selectivity. This was done by literature research.
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Utilisation des aptamères pour le dosage des petites molécules d'intérêt biologique / Use of aptamers for the determination of small molecules of biological interestChovelon, Benoit 21 December 2018 (has links)
La biochimie médicale est une discipline en constante évolution. L’enjeu du développement de nouvelles techniques est de permettre l’analyse à haut débit, de manière spécifique et à faible coût. Les techniques d’immunoanalyse omniprésentes en laboratoire de biologie médicale (LBM) répondent convenablement à ces critères, mais sont cependant perfectibles en ce qui concerne l’analyse des petites molécules d’intérêt biologique. L’objectif de ce travail est de développer des méthodes de dosage innovantes, pour les petites molécules, en utilisant les aptamères comme nouveaux outils de reconnaissance moléculaire. Il s’agit d’oligonucléotides fonctionnels simple brin, capables de reconnaître de manière spécifique une cible, isolés à partir d’une banque de candidats par une approche combinatoire in vitro nommée SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Ils sont en concurrence avec les anticorps, en particulier dans le domaine du diagnostic. Nous avons dans un premier temps travaillé sur le développement de systèmes de dosage à double reconnaissance pour petite molécule, impliquant la formation d’un complexe boucle-boucle. L’adénosine et la théophylline ont tout d’abord servi de cibles modèles pour le développement en phase hétérogène d’une technique de dosage colorimétrique avec amplification enzymatique du signal. Le développement a ensuite été axé sur un analyte ayant un réel intérêt biologique, l’arginine-vasopressine. Le système a été développé en phase homogène en utilisant la technique d’anisotropie de fluorescence. L’application en milieu biologique a été facilitée par l’utilisation d’oligonucléotides non naturels en série L. Enfin nous avons décrit une méthode particulièrement innovante, sans marquage (« label-free »), permettant l’analyse des cibles de petite taille. Cette méthode basée sur l’utilisation du SYBR Green en solution couplée à la technique d’anisotropie de fluorescence, permet également l’étude des ligands de l’ADN. / Clinical chemistry is a constantly evolving discipline. The challenge of developing new techniques is to enable high throughput analysis specific and at low cost. Immunoassay techniques respond appropriately to these criteria, but are nevertheless perfectible with regards to the detection of small molecules of biological interest. The aim of this work is to develop innovative assay methods for small molecules of biological interest, using aptamers as alternative molecular recognition tools. They are single-stranded functional nucleic acids that are isolated from a very large library of candidates through an in vitro combinatorial approach (SELEX) for their ability to bind a peculiar species. They compete with antibodies, particularly in the area of diagnosis. First, we focused our work on the design of small molecule dual recognition assay systems that involved the formation of a loop-loop complex. Adenosine and theophylline served as model targets for the heterogeneous phase development of a colorimetric assay with enzymatic signal amplification. Subsequent works were performed by using arginine-vasopressin, an analyte with a real biological interest as target. A homogeneous phase fluorescence anisotropy detection system was constructed. Applications in complex matrix were facilitated by the use of non-natural L-oligonucleotides. Finally, a particularly innovative SYBR Green-based fluorescence anisotropy method, was reported allowing the detection of both small targets and DNA ligands.
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Using aptamers to regulate rolling circle amplificationBialy, Roger January 2021 (has links)
The work described in this dissertation focuses on developing simple yet effective assays integrating nucleic acid (NA) aptamers with rolling circle amplification (RCA) for the detection of non-NA biomarkers. The first project, a comprehensive literature review, highlights the current state of the art in functional NA-based RCA applications, and identifies shortcomings in the detection of non-NA targets with RCA. Biosensor design is critically evaluated from four key perspectives: regulation, efficiency, and detection of RCA, and the integration of all three components for point of care (POC) applications. The second project investigates how target-binding to a linear aptamer can be utilized to regulate RCA in a simple and inexpensive format. Phi29 DNA polymerase (DP) exhibits difficulty processing DNA strands that are bound to non-NA materials such as proteins. The work uses this restriction of phi29 DP as a feature by utilizing protein-binding aptamers as primer strands (aptaprimers) for RCA. The simplicity is showcased by adapting the method to a cellulose paper-based device for the real-time detection and quantification of PDGF or thrombin within minutes. As the second project is a turn-off sensor, the third project exploits the inherent 3’-exonuclease activity of phi29 DP to generate a simple turn-on assay instead. As target-bound aptamers were shown to be resistant to exonuclease activity, the phi29 DP preferentially digests target-free aptaprimers instead of target-bound aptaprimers. The target-bound aptaprimer could be liberated by a circular template (CT) by incorporating toehold-mediated strand displacement (TMSD), and used for RCA. Sensitivity was improved relative to project two, though the dynamic range was narrow owing to difficulty liberating target-bound aptaprimer at high target concentrations. Project four instead used RecJ, which has 5’-exonuclease activity, to modulate aptaprimer availability. Similarly to project three, target-binding conferred protection on the aptaprimer from 5’-exonuclease digestion by RecJ. By including a free 3’ terminus on the aptaprimer, inhibition of RCA due to target binding was avoided and CT-mediated TMSD was not needed, simplifying the assay. As well, this approach was generalizable as it was demonstrated using both a protein (thrombin) and a small molecule (ochratoxin A) target. This turn-on method further improved the assay compared to project three with a 100-fold enhancement in sensitivity and a restoration of the dynamic range. In sum, this work contributed multiple simple and sensitive approaches for the real-time fluorescent detection of proteins and small molecules with the RCA of linear aptamers. / Thesis / Doctor of Science (PhD)
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Erzeugung funktionaler Schichten auf Basis von bakteriellen HüllproteinenWeinert, Ulrike 05 July 2013 (has links)
Die hier vorliegende Arbeit beschäftigt sich mit Eignung bakterieller Hüllproteine als Bindungsmatrix für die Kopplung funktionaler Moleküle mit dem Ziel, sensorische Schichten zu erzeugen. Bakterielle Hüllproteine sind biologische SAMs, anderen Oberfläche sich modifizierbare COOH-, NH2- und OH-Gruppen befinden. Die Ausbildung polymerer Strukturen erfolgt dabei in wässrigen Systemen und auf Oberflächen. Im Zuge der boomenden Entwicklung von Biosensoren werden insbesondere Biotemplate gesucht, die zwischen biologischer Komponente und Sensoroberfläche vermitteln. Bakterielle Hüllproteine stellen eine solche Zwischenschicht dar. Als Anwendungsbeispiel wurden die Proteine daher mit einem FRET-Paar und Thrombin und Kanamycin-Aptameren modifiziert. Hierbei wurden das FRET-Paar H488 und H555 an die bakteriellen Hüllproteine der beiden Haldenisolate A12 und B53 mittels EDC mit einer Modifizierungsrate von 0,54 molFarbstoff/molProtein kovalent gebunden. Bei der vorhandenen p4-Symmetrie bedeutet dies, dass ein FRET-Paar pro Einheitszelle vorhanden war. Der Nachweis eines Energietransfers zwischen den beiden am Protein gebundenen Fluoreszenzfarbstoffen H488 und H555 erfolgte mittels statischer und zeitaufgelöster Fluoreszenzmessung.
Die Ergebnisse zeigten, dass ein Energietransfer nur möglich war, wenn die Proteine in polymerer Form vorlagen, unabhängig davon, ob sich die Proteine immobilisiert an einer Oberfläche oder in wässriger Lösung befanden. Mittels Variieren des Donor-Akzeptor-Verhältnisses konnte ein maximaler Energietransfer von 40 % generiert werden, wenn das Verhältnis der Fluoreszenzfarbstoffe von Donor und Akzeptor 4 betrug. Die Fluoreszenzintensität der Fluorophore wurde durch die Bindung an die Proteine nicht verringert oder gelöscht. Dies legt nahe, dass die Farbstoffe in den hydrophoben Poren immobilisiert wurden und die Poren die Fluoreszenzfarbstoffe schützen. Um weitere Aussagen über die Lage der gebundenen Fluoreszenzfarbstoffe zu erhalten, wurden die bakteriellen Hüllproteine der Stämme A12 und B53 enzymatisch verdaut und die Fragmente mittels SEC und SDS-PAGE untersucht. Dabei zeigten sich je nach Enzym und Protein unterschiedliche Bandenmuster bezüglich modifizierter und nativer Hüllproteine. Dies belegt, dass die Fluoreszenzfarbstoffe an NH2-und COOH-Gruppen der Proteine gebunden wurden und so teilweise den enzymatischen Verdau hinderten. Die SEC deutet an, dass die Fluoreszenzfarbstoffe an verschiedenen Stellen am Protein gebunden wurden.
In einem zweiten Beispiel wurde das bakterielle Hüllprotein von A12 mit einem Aptamer modifiziert. Aptamere sind kurze einzelsträngige Oligonukleotide, die u.a. mittels ihrer ausgebildeten 3D-Struktur spezifisch Zielstrukturen reversibel binden können. Die hier verwendeten Aptamere binden spezifisch Thrombin und Kanamycin. Die Aptamere wurden mit Hilfe einer der beiden Vernetzer PMPI oder Sulfo-SMCC an die bakteriellen Hüllproteine kovalent gebunden. Nach dem Modifizieren der Proteine wurden diese auf entsprechenden Sensorchips immobilisiert und die Aktivität des gekoppelten Aptamers mittels Affinitätsmessungen, SPR-Spektroskopie und QCM-D-Messungen analysiert. Die Funktion des gebundenen Thrombinaptamers konnte mittels Affinitätsmessungen und QCM-D nachgewiesen werden und entspricht in beiden Fällen einer Bindung von 2 nmol Thrombin pro Quadratzentimeter. Die Funktionalität des Kanamycinaptamers sollte mittels SPR bestimmt werden, jedoch konnte keine Funktionalität des gekoppelten Kanamycinaptamers nachgewiesen werden. Alle Messungen bestätigten jedoch, dass die Bindungsmatrix aus bakteriellen Hüllproteinen keinerlei oder nur ein sehr geringes Hintergrundsignal liefert.
Werden nun beide Komponenten, FRET-Paar und Aptamere, an das Protein gebunden, ist es möglich, eine sensorische Schicht zu erzeugen. Die Zielstruktur, welche detektiert werden soll, wird an das Aptamer gebunden und so in räumliche Nähe zur Sensorfläche gebracht. Stell die Zielstruktur einen Fluoreszenzlöscher dar, so wird der Energietransfer durch die räumliche Nähe des Fluoreszenzlöscher gestört. Die Detektion des Zielmoleküls erfolgt nun über die Änderung von Fluoreszenzintensitäten. Die hier vorgelegte Arbeit soll einen Grundstein legen für die Entwicklung eines solchen Sensors und insbesondere die Detektion eines Energietransfers optimieren und Schwachstellen in der Detektion nachweisen. Die systematische Untersuchung der Fluoreszenzfarbstoffe auf dem Protein ermöglichen es, in zukünftigen Arbeiten einen FRET zweifelsfrei zu detektieren. Die Modifizierung von bakteriellen Hüllproteinen von A12 mit Aptameren und die Detektion der Funktionalität der Aptamere mittels verschiedener Methoden zeigte auf, dass die bakteriellen Hüllproteine als universelle Bindungsmatrix für sensorische Moleküle dienen können, bei denen Affinitätsmessungen, SPR- oder QCM-D-Messungen genutzt werden. Besonders hervorzuheben ist, dass bakterielle Hüllproteine nahezu kein Hintergrundsignal liefern und aufgrund ihrer dünnen Monolage von etwa 6 - 9 nm die Sensitivität der Messungen nur gering beeinträchtigen.
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Novel electrochemical aptamer-based sensing mechanism inspired by selection strategiesLyalina, Tatiana 01 1900 (has links)
Des millions de patients souffrant d’insuffisance cardiaque bénéficieraient d’analyses sanguines hebdomadaires pour surveiller l’évolution de leur état de santé comme c’est le cas avec les personnes atteintes du diabète. Cependant, il n’existe pas de technologies d’analyses sanguines rapides et efficaces pour détecter des marqueurs d’insuffisance cardiaque, telle que la créatinine, la NT-proBNP et la troponine I par exemple. La possibilité pour les patients de surveiller leurs taux de créatinine régulièrement, du confort de chez soi, améliorerait largement leur qualité de vie ainsi que leur taux de survie. En suivant leur taux de créatinine, le patient pourrait prédire des signes d’insuffisance cardiaque, et ainsi faire ajuster leur plan de traitement en conséquence. Pour y arriver, les biocapteurs électrochimiques, dont un exemple est le glucomètre, représentent une classe prometteuse de dispositifs d’analyse sanguine puisqu’ils sont faciles à utiliser, rapides, peu coûteux, sensibles, stables et potentiellement universels. Les biocapteurs électrochimiques à base d’ADN pourraient potentiellement être adaptés en biocapteur de créatinine, par l’entremise d’aptamères. Le but de cette recherche est de développer un nouveau mécanisme de détection universel et efficace pouvant être adapté directement à partir des stratégies de sélection des aptamères. Pour ce faire, nous avons identifié et caractérisé un élément de bioreconnaissance sélectif pour la créatinine. Ensuite, nous avons conçu une nouvelle stratégie de détection et nous avons validé cette nouvelle stratégie par spectroscopie de fluorescence avant de l’adapter pour une détection électrochimique. Par la suite, nous avons optimisé les performances du biocapteur en modulant des paramètres analytiques tels que sa gamme linéaire et son gain de signal, tout en validant ses performances dans une matrice complexe comme le sérum. Les résultats de cette recherche suggèrent que la stratégie de conception du nouveau biocapteur électrochimique à base d’aptamère est prometteuse pour la détection efficace de biomarqueurs sanguins. Ce type de mécanisme pourrait être facilement adapté pour détecter d'autres molécules cliniquement pertinentes en modifiant simplement la stratégie de sélection de l'aptamère. / Millions of patients suffering from heart failure would greatly benefit from weekly blood analysis to help them manage their disease state like patients suffering from diabetes. However, no simple blood monitoring technologies detecting heart failure biomarkers, such as creatinine, NT-proBNP, and troponin I, are available. The ability to determine and regularly monitor the creatinine level in the home setting would greatly improve the patient’s quality of life and survival rate. Knowing the concentration of creatinine help to predict heart failure and to revise the treatment plan if the concentration of creatinine is abnormal. To achieve this, electrochemical sensors, like a glucometer, represent a promising class of blood analysis devices due to their ease of use, fast response, low cost, inherent sensitivity and stability, and potential universality. More specifically, DNA-based electrochemical biosensors could potentially be adapted into a creatinine sensor by using aptamers specific to a biomarker. To achieve this goal, we identified a selective biorecognition element for creatinine detection and characterized it. We also designed a novel sensing aptamer-based strategy and validated this strategy by fluorescent spectroscopy before transposing it into the electrochemical format. We then optimized the performance of the sensor by tuning its signal gain and characterizing the dynamic range while also validating its performance in serum. The results of this work suggest that the electrochemical aptamer-based strategy represents a promising sensing mechanism. We believe this mechanism could be easily adapted to detect other clinically relevant molecules by simply relying on the aptamer’s selection strategy.
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Discovery of DNA Aptamers Targeting SARS-CoV-2 Proteins and Protein Binding Epitopes Identification for Label-Free COVID-19 DiagnosticsPoolsup, Suttinee 05 September 2023 (has links)
No description available.
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In Vitro Selection of DNA Aptamers Against Prostate Cancer Peptide BiomarkersKuguoglu, Elif 01 January 2014 (has links)
This project is aimed toward finding DNA aptamers against prostate cancer peptide antigens. DNA aptamers can function to find and indicate the presence of certain molecules in a specimen. These aptamers will be obtained through the process of evolutionary selection, a specific process called SELEX which stands for Systemic Evolution of Ligands by Experimental Enrichment. By conducting several rounds of SELEX, a DNA aptamer will be selected to bind to a known peptide antigen. A biotinylated column will be utilized to stabilize a random library of DNA aptamers, and those peptides that bind to certain aptamers will cause a conformational change leading to the elution of those specific DNA aptamers. This SELEX process will be conducted again on the eluted aptamers to further select for strong binding DNA aptamers. The DNA aptamers that are obtained can further on be sequenced or used for prostate cancer research studies. Another possible usage of aptamers is to diagnose and determine the stage of various different cancer types. Our prediction is that this research will produce a DNA aptamer that will bind to a specific prostate cancer peptide antigen.
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Aptameric Sensors: In Vitro Selection of DNA that Binds Bromocresol PurpleMiller, Derek B 01 January 2016 (has links)
Aptamers being used as sensors is an emerging field that has capabilities of being tomorrow’s diagnostic tools. As aptameric sensors have become more popular, their visualization systems have been limited. The majority of today’s aptameric sensors require expensive machinery such as a fluorometer in order to visualize results. We propose a system that will cut the need for instrumentation and be detected via the naked eye. With the selection of an aptamer to bind the pH indicating dye bromocresol purple (BCP) this may be achieved. When rendered active, the binding towards BCP will facilitate a color change from yellow to purple or vice versa. Previous studies have shown albumin contains the ability to facilitate this role and we now intend to use a DNA aptamer to achieve this as well. The BCP aptamer has the potential to serve as a signaling domain to any already selected aptamer thus making it a universal tool for both research and diagnostic measures. We have found that an alternative structure-switching systematic evolution of ligands by exponential enrichment (SELEX) method which left the dye unaltered was not sufficient for selecting an aptamer. We believe that a classical SELEX will enable us to select an aptamer that may be used to accomplish this role as a universal visual detector.
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The Role of Liposomal Hybrids and Gold Nanoparticles in the Efficacious Transport of Nucleic Acids and Small Molecular Drugs for Cancer NanomedicineKumar, Krishan January 2015 (has links) (PDF)
The thesis entitled “The Role of Liposomal Hybrids and Gold Nanoparticles in the Efficacious Transport of Nucleic Acids and Small Molecular Drugs for Cancer Nanomedicine” elucidates the preparation of various liposomal formulations of cationic monomeric and gemini lipids where hydrophobic domains were consisted of tocopherol, cholesterol and pseudoglyceryl backbone for the cellular transport of nucleic acids. The thesis continues while elucidating the role of various pH sensitive molecules and gold nanoparticles in liposomes to improve the delivery efficacy levels. This thesis also elucidates the role of gold nanoparticles stabilized with natural pH sensitive molecules for efficacious drug delivery applications. Additionally, the role of such pH sensitive gold nanoparticles in association with liposomes for the co-delivery of drug and gene has been discussed. The work has been divided into six chapters.
Chapter 1A: Dimeric Lipids Derived from α-Tocopherol as Efficient Gene Transfection Agents. Mechanistic Insights into Lipoplex Internalization and Therapeutic Induction of Apoptotic Activity
In this chapter, we present cationic dimeric (gemini) lipids for significant plasmid DNA (pDNA) delivery to different cell lines without any marked toxicity in the presence of serum. The six gemini lipids possess α-tocopherol as their hydrophobic backbone and differ from each other in terms of their spacer chain lengths. Each of these gemini lipids mixed with a helper lipid 1, 2-dioleoyl phosphatidyl ethanolamine (DOPE), was capable of forming stable aqueous suspensions. These co-liposomal systems were examined for their potential to transfect pEGFP-C3 plasmid DNA in to nine cell lines of different origins. The transfection efficacies noticed in terms of EGFP expression levels using flow cytometry were well corroborated using independent fluorescence microscopy studies. Significant EGFP expression levels were reported using the gemini co-liposomes which counted significantly better than one well known commercial formulation lipofectamine 2000 (L2K). Transfection efficacies were also analyzed in terms of the degree of intracellular delivery of labeled plasmid DNA
(pDNA) using confocal microscopy which revealed an efficient internalization in the presence of serum. The cell viability assays performed using optimized formulations demonstrated no significant toxicity towards any of the cell lines used in the study. We also had a look at the lipoplex internalization pathway to profile the uptake characteristics. A caveolae/lipid raft route was attributed to their excellent gene transfection capabilities. The study was further advanced by using a therapeutic p53-EGFP-C3 plasmid and the apoptotic activity was observed using FACS and growth inhibition assay.
Figure 1. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of p53-EGFP-C3 plasmid DNA that induces significant apoptotic response.
Chapter 1B: Efficacious Gene Silencing in Serum and Significant Apoptotic Activity Induction by Survivin Downregulation Mediated by Cationic Gemini Tocopheryl Lipids
Non-viral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding the unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We discuss in this chapter about six tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient co-liposomal formulations derived from each of these geminis and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE) were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized co-liposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz. HEK 293T, HeLa and Caco-2 significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and presence of serum (FBS). Notably, the knockdown activity of co-liposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR and western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines.
Figure 2. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of siRNA against survivin that induces significant apoptotic response.
Chapter 2: Efficacious in Vitro EGFP Expression and Silencing in Serum by Cationic Pseudoglyceryl Gemini Lipids
To elicit the desirable efficacy levels in cationic liposome mediated nucleic acid therapeutics has been part of extensive scientific efforts. This chapter describes three cationic gemini lipids and application of their co-liposomes with DOPE as potent pDNA (plasmid DNA) and siRNA (small interfering RNA) cytofectins for remarkably advanced efficacy levels in numerous cell lines in the presence of serum. The hydrophobic structural lineament of cationic gemini lipids is made up of pseudoglyceryl backbone linked to the hydrocarbon chains via oligo-oxyethylene units. The stable aqueous co-liposomal suspensions of gemini lipids showed an efficient binding to pDNA or siRNA and their significant intracellular delivery in various cell lines. The transfection capabilities of different co-liposomal formulations were profiled based on EGFP expression (pEGFP-C3 pDNA transfection) and EGFP knockdown (anti-GFP siRNA transfections) in EGFP expressing cell lines. The cellular EGFP expression levels and intracellular delivery of labeled nucleic acids were thoroughly studied using flow cytometry (FACS), fluorescence and confocal microscopy. The MTT based cell viability assay revealed no loss in cell viabilities for all of the transfection optimized lipoplexes of siRNA or pDNA. The transfection profile of gemini co-liposomes was noted to be significantly much better than a commercial lipofection reagent, Lipofectamine 2000 used for pDNA and siRNA applications in each of the cell lines studied. The co-liposomes and their transfection optimized lipoplexes were physiochemically characterized extensively by means of zeta potential, dynamic light scattering (DLS) and atomic force microscopy (AFM). In brief, these new gemini co-liposomal formulations seem to offer a great opportunity for successful nucleic acid (DNA and siRNA) delivery in a practical scenario.
Figure 3. Efficacious EGFP expression (pDNA transfection) and EGFP silencing (anti GFP siRNA transfection) mediated by co-liposomes of pseudoglyceryl gemini lipids and DOPE.
Chapter 3: Efficient Elicitation of Liposomal Nucleic acid delivery through the Eminence of Gold Nanoparticles Stabilized with pH Responsive Short Tripeptide Derived from Tyrosine Kinase NGF Receptors
The prerequisite in the area of gene therapy today is to serve transfection efficient formulations nullifying the enduring key issues. To this end, we discuss in this chapter, the role of hybrid liposomal formulations derived from structurally distinct cationic lipids, a neutral lipid (DOPE) and pH responsive short tripeptide (KFG, Lys-Phe-Gly) capped gold nanoparticles (PAuNPs). The hybrid liposomes are presented to be efficient enough to transfect pDNA leading to remarkably high gene expression levels in various cell lines of different origins in the presence of serum (FBS). Hybrid liposomes could deliver pDNA more effectively than the native liposomes and commercial standard lipofectamine 2000 (L2K) across the entire range of N/P ratios studied under the influence of intracellular pH response and gold nanoparticles prominence. The gene transfection capabilities are profiled based on transfections performed using two different plasmids (pGL3, luciferase activity and p-EGFP-C3, green fluorescent protein expression). pDNA cellular internalization and subsequent gene expression levels are studied using flow cytometry, fluorescence microscopy and confocal microscopic
studies. The extensive physiochemical characterization of hybrid liposomal formulation and their complexes with pDNA in comparison with respective native liposomes was performed using AFM, TEM, Zeta, DLS, gel retardation assay, U.V. and fluorescence emission measurements. The hybrid liposomes are shown to possess significantly higher fusion activity at lowered pH of intracellular compartments. These hybrid liposomes are fairly biocompatible across the concentration range used in transfection experiments. Precisely, introduction of these pH responsive tripeptide capped gold nanoparticles in to liposomal formulations straightforwardly must be more advantageous for a practical application in biomedical scenario to achieve therapeutic levels.
Figure 4. The hybrid of liposomes and tri-peptide capped gold nanoparticles for significantly improved gene expression levels.
Chapter 4: RNA Aptamer Decorated pH Sensitive Liposomes for Active Transport of Nucleic Acids in Specific Cancer Cells
This chapter describes the target specific transport of pH sensitive liposomes loaded with a RNA aptamer for promising nucleic acid therapeutics. The pH sensitive liposomes are constructed from a cationic cholesteryl gemini lipid (CGL), neutral helper lipid (DOPE) and gemini analog of a pH sensitive lipid, palmitoyl homocysteine (GPHC). The liposomes are shown to be significantly fusogenic that deliver the cargoes upon lowerin the pH (6.0). The fusogenic behaviour of the liposomes was thoroughly studied by means of dynamic light scattering (DLS), zeta potential, lipid mixing, calcein dequenching and atomic force microscopy (AFM). The facile integration of cholesterol
conjugated RNA aptamer in liposomes derived from cholesteryl gemini lipids was exploited for their delivery to specific cancer cells. The RNA aptamer specifically binds to epithelial cell adhesion molecule (EpCAM) with high affinity which is a cell surface marker in various solid cancers such as colorectal and breast carcinoma. These aptamer decorated pH sensitive liposomes could efficiently enter the EpCAM expressing COLO-205, Caco-2, MCF-7 and MDA-MB-231 cell lines while no such noticeable liposome transport was observed in EpCAM negative HEK 293T cells as evidenced by flow cytometry and confocal microscopy. Additionally, the liposomes are shown to be actively transported inside the cells, i.e., receptor mediated endocytosis. These liposomes could complex the nucleic acids (pDNA) in an efficient manner. The MTT based cell viability assay accounted no noticeable loss in cell viabilities for liposome treatments. Concisely, we have formulated RNA aptamer loaded pH sensitive liposomes that would certainly be promising tool in target based cancer nanomedicine.
Figure 5. (A) Cellular internalization of DY-647 labeled aptamer loaded pH sensitive liposomes. (B) The liposomes were actively internalized through receptor mediated endocytosis. Each panel (A and B) represents (from left to right) bright field image, aptamer fluorescence, DAPI stained nuclei and merge of previous three impressions.
Chapter 5: Natural Tri-peptide Capped Gold Nanoparticles for Efficacious Doxorubicin Delivery in Vitro and in Vivo
Nanotechnology has gained ever increasing interest for the successful implementation of chemotherapy based treatment of cancer. This chapter describes the role of gold nanoparticles (AuNPs) capped with a natural pH responsive short tri-peptide (Lys-Phe–Gly or KFG) for significant intracellular delivery of an anti-cancer drug, doxorubicin (DOX). A significantly increased apoptotic response was noted for DOX treatments mediated by KFG-AuNPs in comparison with drug alone treatments in various cell lines (BT-474, HeLa, HEK 293T and U251) in vitro. Furthermore, KFG-AuNPs mediated DOX treatment significantly decreased cell proliferation and tumor growth in BT-474 cell xenograft model in nude mice. In addition, KFG-AuNPs showed efficacious drug delivery in DOX-resistant HeLa cells (HeLa-DOXR) in comparison with drug alone treatments.
Figure 6. Representative images of excised tumors after doxorubicin treatment mediated by pH responsive tri-peptide capped gold nanoparticles (DOX-KFG-AuNPs) (C) in comparison with doxorubicin alone treatments (B) and untreated tumors (A). Extensive cell death as observed under Hematoxylin/eosin (H&E) (D) and TUNEL (E) staining of DOX-KFG-AuNPs treated tumor sections.
Chapter 6: Significant Apoptotic Activity Induction by Efficacious Co-delivery of p53 Gene and Doxorubicin Mediated by the Combination of Co-liposomes of Cationic Gemini lipid and pH Responsive Tri-peptide
Combining chemotherapy with gene therapy has appeared as an efficient tool to treat complex biological disorder like cancer. Herein, we show efficient co-delivery of DNA
and an anti-cancer drug, doxorubicin (DOX) by means of gemini cationic liposome (GCL) based lipoplex nanoaggregates that are coated with DOX encapsulated pH responsive tripeptide nanovesicles. The lipoplex, tripeptide vesicles and their association was thoroughly studied using dynamic light scattering (DLS), zeta potential, atomic force microscopy (AFM). Flow cytometry, fluorescence and confocal microscopic analysis revealed that the GCL-tripeptide association could significantly co-deliver the p53 expression plasmid (p53-EGFP-C3) and DOX in HeLa and HEK 293T cells in the presence of serum. A synergistic increase in gene expression level and DOX internalization was observed in co-delivery which was even substantially higher than individual lipoplex transfection and DOX treatment. The apoptosis induced due to p53 expression and DOX was profiled with the help of annexin-V positivity analysis under flow cytometry and nuclear damage analysis by DAPI nuclei counterstaining under confocal microscopy which noted to be significantly higher in cells during co-delivery. The MTT based cell viability assay revealed a significantly increased loss in cell viability counts for co-delivery treatments. Such a system delivering synergistically increased significant efficacy levels in combinatorial drug and nucleic acid therapeutics would be certainly advantageous for practical biomedical applications.
Figure 7. The co-delivery of pDNA and drug (doxorubicin) mediated by GCL-tripeptide association as observed under (A) confocal microscopy (pDNA; green and doxorubicin; red) and (B) flow cytometry.
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Implicações translacionais de uma nova ferramenta de detecção de célula: tumorais circulantes no monitoramento do câncer de próstataOliveira, Leandro Alves de 17 July 2017 (has links)
Introdução: O diagnóstico precoce de câncer de próstata (CaP) é essencial para aumentar a sobrevida dos pacientes, mas os marcadores e métodos atuais não possuem sensibilidade e especificidade suficientes, tornando o diagnóstico ainda muito impreciso. Recentemente, as células tumorais circulantes (CTCs) têm surgido não como método de rastreio do CaP, mas sim como marcadores de prognóstico utilizando um arsenal de diversos alvos para a captura dessas células. Contudo, a busca por um método ou marcadores comuns para o rastreio, diagnóstico, prognóstico e monitoramento da doença ainda se apresenta com um dos principais objetivos técnico-científicos a ser alcançado. Objetivo: apresentar um novo marcador, o aptâmero A4 selecionado previamente por 3DCell SELEX na linhagem PC3, e avaliar sua capacidade de detectar CTCs por citometria de fluxo no sangue de pacientes com CaP virgens de tratamento e sob diferentes regimes terapêuticos. Material e métodos: o estudo avaliou 34 homens com CaP e 16 homens sem alterações prostáticas. Foi coletado o sangue em tubo com EDTA, e após proceder a lise de hemácias, as células nucleadas de cada paciente foram incubadas com o aptâmero A4 conjugado à biotina, e em seguida lavadas e incubadas com estreptoavidina-FITC para posterior análise em citometria de fluxo. Os percentuais de CTCs foram comparados entre os dois grupos de pacientes e correlacionados com idade, níveis de PSA, estadiamento e procedimentos terapêuticos adotados (bloqueio hormonal, radioterapia e cirurgia). O limite de detecção acima de 1% de CTCs foi considerado positivo, utilizando como base o percentual observado em todos os 16 controles negativos. Resultados: todos os pacientes foram diagnosticados como positivos independentemente do tempo de terapia ou do estadiamento, exceto um paciente sob bloqueio hormonal que não apresentou CTCs. O percentual de CTCs apresentou alta correlação com idade (R=0,75) e com os níveis de PSA (R=0,80) de forma exponencial, embora seis pacientes com altos índices de células circulantes apresentaram PSA<0,02ng/mL, considerados como falha bioquímica. Conclusão: nossos resultados preliminares indicam uma acurácia elevada de 98% e demonstra um grande potencial de aplicação dessa nova tecnologia diagnóstica tanto no rastreamento, quanto no monitoramento do tratamento do CaP, o qual deverá ser melhor investigado em população de risco. / Introduction: prostate cancer (PCa) early diagnosis is essential to boost patients’ life expectance. Although, current biomarkers and diagnosis methods do not present reliable sensibility and specificity, making the diagnosis rather imprecise. Recent methodologies have been using circulating tumor cells (CTCs), not for screening of PCa, but as prognosis indicators, employing a vast array of techniques to capture those cells. However, the search for a new biomarkers or diagnosis methods able to screen, diagnosis, assist in prognosis and in the disease monitoring still one of the major technical and scientific objectives to be achieved. Objective: To present a new biomarker for PCa, the aptamer A4, previous screened in the prostate cancer cell line PC3, using 3DCell SELEX. And to able to detect, by flow cytometry, CTCs in blood samples
of PCa patients undergoing various treatment regimen. Material and methods: the study evaluated 34 PCa patients and 16 health controls. Blood samples were collected in EDTA tubes, and after erythrocytes lysis, nucleated cells were incubated with A4 aptamer conjugated with biotin, them the cells were washed and incubated with streptavidin-FITC for later flow cytometer analysis. Percentage of CTCs were compared between patient’s groups and correlated against age, PSA levels, staging and treatment regimen (hormonal blockade, radiotherapy and surgery). Detection limit above 1% of CTCs was considered positive, based on the percentage observed on all of the 16 negative controls. Results: all patients were positively diagnosed independently of therapy time or staging, except for one patient undergoing hormonal blockade therapy, which does not present detectable CTCs. CTCs percentage presented high correlation against age (R=0.75) and with PSA levels (R=0.80) with exponential behavior, although, six patients with high CTCs count presented PSA levels <0.02 ng/mL, and were considered was biochemical errors. Conclusion: Our preliminary results indicated high accuracy (98%) and demonstrate a potential application of this technology for diagnosis and screening, as well as in the monitoring of PCa evolution, which should be better investigated in the risk population. / Dissertação (Mestrado)
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