1 |
Strategies for microsphere-mediated cellular deliveryCardenas-Maestre, Juan Manuel January 2011 (has links)
Amino-functionalised polystyrene microspheres are promising candidates as delivery systems due to their unique features, tunable surface functionalities, and controllable release of the cargo. Herein several strategies for the conjugation of biologically relevant cargoes to these microspheres and their biological evaluation are described. Firstly, dispersion and suspension polymerisation methods were applied for the synthesis of these devices. Subsequently, these polymeric particles were employed in multistep solid phase synthesis to conjugate a broad range of cargoes. The capability of the resulting constructs to cross the cell membrane and deliver the desired cargo was evaluated by flow cytometry and confocal microscopy. Additionally, the effect of these particles on cell viability was determined. Moreover a chemical strategy for dual fuctionalisation allowed the production of microspheres capable of carrying two cargos simultaneously (e.g. a biologically relevant cargo and a tracking fluorophore). Several strategies were used to transport biomolecules such as peptides and oligonucleotides inside cells. Cell-impermeable peptides with neuroprotective activity were conjugated to microspheres to facilitate their internalisation and they were efficiently delivered into neuroblastom cells (SH-SY5Y) without affecting their therapeutic activity. In addition, several microsphere-mediated oligonucleotide delivery strategies were investigated. As a first approach, siRNA was successfully attached to microspheres via thiol linkage or via electrostatic interaction (by formation of polycationated microspheres-siRNA microplexes). Using both strategies EGFP expression was efficiently down-regulated in cervical cancer cells permanently expressing EGFP (HeLa-EGFP) following beadfection. Additionally embryonic stem (ES) cells were beadfected with siRNA linked to microspheres by amide formation and essential transcription factors implicated in cell renewal and differentiation were successfully silenced, exceeding the silencing capabilities of commercially available lipofection products. Furthermore, a novel approach for the intracellular delivery of plasmid DNA was designed. Following an easy protocol for the linearisation and functionalisation of the plasmid DNA, this was covalently coupled to beads and cells were homogeneously ‘beadfected’. Finally, the coupling of fluorogenic substrates for caspase-3 to microspheres allowed the in situ monitoring and quantification of apoptotic process within cells. In conclusion, these small particles are excellent devices for the efficient intracellular delivery of a broad range of cargoes.
|
2 |
High-throughput intracellular delivery of proteins and plasmidsPark, Seonhee 27 May 2016 (has links)
Intracellular delivery of macromolecules is crucial for the success of many research and clinical applications. Several conventional intracellular delivery methods have been used for many years but are still inadequate for several applications because of the issues associated with toxicity, low-throughput, and/or difficulty to target certain cell types. In this study, we developed and evaluated new high-throughput intracellular delivery methods for the efficient delivery of macromolecules while maintaining high cell viability. First, we studied the feasibility of using an array of nanoneedles, with sharp tip diameters in the range of tens of nanometers, to physically make transient holes in cell membranes for intracellular delivery. Puncture loading and centrifuge loading methods were developed and assessed for the effect of various experimental parameters on cell viability and delivery efficiency of fluorescent molecules. In both methods, high-throughput intracellular delivery was feasible by creating transient holes in cell membranes with the sharp tips of the nanoneedles. The second physical intracellular delivery method we studied was a novel microfluidic device that created transient holes in the cell membrane by mechanical deformation and shear stress to the cell. We observed efficient delivery of fluorescent molecules and studied the effect of device design and flow pressure on the delivery efficiency compared to data in the literature. We accounted for cell loss and clogging in the microfluidic devices and determined the true loss of cell viability associated with this method. Lastly, we investigated the possibility of intracellular delivery using nanoparticles on a leukemia cell line. Among number of materials for nanoparticles tested, mesoporous silica/poly-L-lysine nanoparticles were selected for further intracellular delivery study based on cell viability and intracellular delivery capability. We demonstrated the co-delivery of protein and plasmid by encapsulating into and coating onto the surface of the nanoparticles, respectively, which would be advantageous for certain therapeutic strategies. In summary, this work introduced two new intracellular delivery methods involving nanoneedles and novel nanoparticles, and provided an early, independent assessment of microfluidic delivery, showing the strengths and weaknesses of each method. These methods can be further optimized for a number of laboratory and clinical applications with continued research.
|
3 |
Exploring the Properties of Host-[2]Rotaxanes: From Intracellular Delivery to Molecular MachineryDIALLO, MAMADOU CHERIF 25 August 2008 (has links)
No description available.
|
4 |
Délivrance de molécules dans l'endothélium cornéen par nanoparticules de carbone activées au laser femtoseconde / Delivery of molecules into corneal endothelial cells by carbon nanoparticles activated by femtosecond laserJumelle, Clotilde 10 July 2015 (has links)
Les cellules endothéliales cornéennes (CEC) jouent un rôle essentiel pour le maintien de la transparence de la cornée. Cependant, chez l’homme, elles sont incapables de proliférer en raison d’un arrêt de leur cycle cellulaire en phase G1, ce qui rend la couche endothéliale cornéenne particulièrement vulnérable. La délivrance de molécules thérapeutiques (gènes ou médicaments) représente une solution prometteuse pour maintenir la viabilité des CEC. Néanmoins, la difficulté majeure de cette technique repose sur le fait de traverser la membrane cellulaire, normalement imperméable aux molécules de grande taille. Plusieurs techniques de délivrance de molécules ont déjà été testées sur le tissu cornéen mais aucune d’entre elles ne donnent de résultats suffisamment probants pour être utilisée en applications cliniques. L’objectif de cette thèse est d’adapter et de développer une nouvelle technique de délivrance intracellulaire de molécules, basé sur une perforation cellulaire via un phénomène photoacoustique induite par l’activation de nanoparticules de carbone par laser femtoseconde, sur un modèle d’endothélium cornéen in vitro et ex vivo / Corneal endothelial cells (CEC) are essential for corneal transparency. However, on humans, they are unable of proliferation owing to its arrest of G1 phase of the cell cycle, making corneal endothelial monolayer particularly vulnerable. The gene and drug delivery represents a promising solution to maintain CEC viability. Unfortunately, the major difficulty of this technique is the transport across the cell membrane, normally impermeable to high-size molecules. Several techniques of molecules delivery have already been tested on corneal tissue but none of them gives results sufficiently convincing to be used in clinical applications. The aim of this thesis is to adapt and develop a new technique of intracellular molecules delivery, based on cell perforation via photoacoustic effect induced by the activation of carbon nanoparticles by femtosecond laser, on in vitro and ex vivo models of corneal endothelium
|
5 |
Macropinocytosis-Inducing Peptides: Identification, Utility, and Mechanism-of-Action / 新規マクロピノサイトーシス誘導ペプチドの同定、細胞内送達への有用性と作用様式Arafiles, Jan Vincent Valenzuela 23 September 2020 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22753号 / 薬科博第127号 / 新制||薬科||14(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 二木 史朗, 教授 中山 和久, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM
|
6 |
Structural design of cell-penetrating protein needles toward development of intracellular delivery systems / 細胞内分子輸送システム構築を指向した細胞膜貫通針蛋白質の構造設計Inaba, Hiroshi 23 January 2015 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18693号 / 工博第3971号 / 新制||工||1611(附属図書館) / 31626 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 北川 進, 教授 梅田 眞郷, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM
|
7 |
Development of macrophage-targeted therapy using peptide/protein-loaded extracellular vesicles / ペプチド及びタンパク質搭載細胞外小胞を利用したマクロファージを標的とする疾患治療法の開発Takenaka, Misako 23 March 2023 (has links)
京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24549号 / 薬科博第166号 / 新制||薬科||18(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM
|
8 |
Intracellular Delivery of Functional Cargos Using Cell Penetrating Peptide MotifsSalim, Heba January 2021 (has links)
No description available.
|
9 |
<b>Developing Self-Assembling Peptide Materials and Cell-Penetrating Peptides for Intracellular Delivery</b>Andrew M Encinas (19166128) 18 July 2024 (has links)
<p dir="ltr">In light of new interest in biomaterials for drug formulation and delivery, coiled-coil assemblies have emerged as promising candidates. Characterized by facile synthesis, low toxicity, and biocompatibility, coiled-coil assemblies hold a significant potential for drug encapsulation and cellular delivery. Herein, I will discuss the development of a novel nanoscale metal-mediated coiled-coil assembly with tunable assembly properties, presenting a compelling platform for drug encapsulation and targeted cell delivery, thereby addressing critical challenges in modern pharmaceutical science.</p><p dir="ltr">Moreover, the rise of antimicrobial resistance has contributed to a global health crisis. Many current antibiotics are announced as unviable, and the addition of bacteria that invade the cell further challenges and limits these current drugs. The Chmielewski group has previously shown that cationic amphiphilic polyproline helices (CAPHs) act as dual agents displaying both cell-penetration and antibiotic activity. To further increase these dual properties, new unnatural amino acids and new CAPH peptides were synthesized with new configurations of hydrophobic and hydrophilic moieties. I will discuss the outcomes of these modifications, including cell penetration, subcellular localization, and antibacterial efficacy.</p>
|
10 |
A novel intracellular protein delivery system - Magnesium phosphate nanoparticles with cationic lipid coating for catalase intracellular deliveryFang, Yunzhou 01 January 2014 (has links)
Protein therapeutics have great potential in treating human disease, especially for those caused by alternations in the functions of intracellular proteins. However, clinical use of protein by intracellular delivery has been hampered by the instability due to proteins' physicochemical properties, and some barriers in the delivery pathway. This study was to prepare and test a novel intracellular protein delivery system - magnesium phosphate nanoparticles with cationic lipid coating for catalase intracellular delivery (LP MgP NP-CAT), and investigate whether it can release the encapsulated catalase to cytosol. LP MgP NP-CAT was designed, prepared and characterized, showing that it had an average diameter around 300 nm and zeta potential around +40mV. The pH - triggered catalase release from LP MgP NP-CAT was determined by a hydrogen peroxide degradation assay, where the concentration of remaining hydrogen peroxide was measured by UV-Vis spectroscopy, indicating catalase was released in response to the drop of pH, which was confirmed by the morphology change of LP MgP NP-CAT observed by transmission electron microscopy. The in vitro catalase release behavior was conducted on MCF-7 cells and EA.hy926 cells. LP MgP NP-CAT was delivered into MCF-7 cells and the release behavior was determined by the resultant resistance of the cells against hydrogen peroxide using MTS cell viability assay. The delivery of LP MgP NP-CAT into EA.hy926 cells was determined by the decrease of the reactive oxygen species level. Both of the studies showed that catalase was successfully delivered and released which is supported by the reduction of hydrogen peroxide.
|
Page generated in 0.061 seconds