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Effect of shape on cell internalization of polymeric hydrogel nanoparticlesAgarwal, Rachit, Ph. D. 11 August 2015 (has links)
Recent progress in drug discovery has enabled us to target specific intracellular molecules to achieve therapeutic effects. These next generation therapeutics are often biologics which cannot enter cells by mere diffusion. Therefore it is imperative that drug carriers are efficiently internalized by cells before releasing their cargo. Nanoscale polymeric carriers are particularly suitable for such intra-cellular delivery. Although size and surface-charge has been the most studied parameters for nanocarriers, it is now well appreciated that particle shape also plays a critical role in their transport across physiological barriers. Hence there is increasing interest in fabricating shape-specific polymeric nano and microparticles for efficient delivery of drugs and imaging agents. Nanoimprint lithography methods, such as Jet-and-flash imprint lithography (J-FIL), provide versatile top-down processes to fabricate shape-specific, biocompatible nanoscale hydrogels that can deliver therapeutic and diagnostic molecules in response to disease-specific cues. However, the key challenges in top-down fabrication of such nanocarriers are scalable imprinting with biological and biocompatible materials, ease of particle-surface modification using both aqueous and organic chemistry as well as simple yet biocompatible harvesting. Here we report that a biopolymer-based sacrificial release layer in combination with improved nanocarrier-material formulation can address these challenges. The sacrificial layer improves scalability and ease of imprint-surface modification due to its switchable solubility through simple ion exchange between monovalent and divalent cations. This process enables large-scale bio-nanoimprinting and efficient, one-step harvesting of hydrogel nanoparticles in both water- and organic-based imprint solutions. We also show that when shape is decoupled from volume, charge and composition, mammalian cells preferentially internalize disc-shaped nanohydrogels of higher aspect ratios over nanorods. Interestingly, unlike nanospheres, larger-sized hydrogel nanodiscs and nanorods are internalized more efficiently. Uptake kinetics, efficiency and internalization mechanisms are all shape-dependent and cell-type specific. Although macropinocytosis is used by all cells, epithelial cells uniquely internalize nanodiscs using caveolae pathway. On the other hand, endothelial cells use clathrin-mediated uptake along with macropinocytosis for all shapes and show significantly higher uptake efficiency compared to epithelial cells. We also study the effect of shape and surface properties for their tissue uptake and penetration using spheroids as a 3D tumor model and show that hydrophobic particles show no difference in penetration inside such models even after 125 fold reduction in volume. These results provide a fundamental understanding of how cell and tissue behavior is influenced by nanoscale shape and surface properties and are critical for designing improved nanocarriers and predicting nanomaterial toxicity. / text
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Controllable cell delivery and chromatin structure observation using DNA nanotechnology / DNAナノテクノロジーを用いた細胞制御法の開拓とクロマチン構造の観察FENG, YIHONG 23 September 2020 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22719号 / 理博第4628号 / 新制||理||1665(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM
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Studies on Tumour Active Compounds with Multiple Metal CentresDaghriri, Hassan January 2004 (has links)
Four tumour active trinuclear complexes: DH4Cl: [{trans-PtCl(NH3)2}2m-{trans-Pd( NH3)2(H2N(CH2)4NH2)2]Cl4, DH5Cl: [{trans-PtCl(NH3)2}2m-{trans-Pd( NH3)2(H2N(CH2)5NH2)2]Cl4, DH6Cl: [{trans-PtCl(NH3)2}2m-{trans-Pd( NH3)2(H2N(CH2)6NH2)2]Cl4, DH7Cl: [{trans-PtCl(NH3)2}2m-{trans-Pd(NH3)2-( H2N(CH2)7NH2)2]Cl4 and one dinuclear complex DHD: [{trans-PtCl(NH3)2}�-{ H2N(CH2)6NH2}{trans-PdCl(NH3)2]Cl(NO3), have been prepared and characterised based on elemental analyses, IR, Raman, mass and 1 H NMR spectral measurements. For the trinuclear complexes, the synthesis has been carried out using a step-up method branching out from the central palladium unit. A purity of about 95% has been obtained by repeated dissolution and precipitation. The activity against human cancer cell lines including ovary cell lines: A2780, A2780 cisR , A2780 ZD0473R , non small lung cell line: NCI-H640 and melanoma: Me-10538 have been determined based on MMT assay. Cell uptakes, DNA-binding have been determined for ovary cell lines: A2780, A2780 cisR . The nature of interaction with pBR322 plasmid DNA and ssDNA has been studied for trinuclear complexes DH4Cl, DH5Cl, DH6Cl and DH7Cl and the dinuclear complex DHD. Interaction of DH6Cl with adenine and guanine has also been studied by HPLC. The compounds are found to exhibit significant anticancer activity against cancer cell lines especially ovarian cancer cell lines: A2780, A2780 cisR and A2780 ZD0473R . DH6Cl in which the linking diamine has six carbon atoms is found to be the most active compound. As the number of carbon atoms in thelinking diamine is changed from the optimum value of six, the activity is found to decrease, illustrating the structure-activity relationship. The increase in uptake of the trinuclear complexes in A2780 cell line with the increase in size of the linking diamine coupled with the low molar conductivity values found for the solutions of the compounds suggest that the compounds would remain in solution as undissociated �molecules� and hence could cross the cell membrane by passive diffusion. Much lower resistance factors for the all the multinuclear compounds including DHD as applied to A2780 cisR cell line, as compared to that for cisplatin, suggest that the compounds are able to overcome multiple mechanisms of resistance operating in the cell line. All of the multinuclear complexes are expected to form long-range interstrand GG adducts with DNA, causing irreversible global changes in the DNA conformation but unlike cisplatin do not cause sufficient DNA bending to be recognized by HMG 1 protein. Increasing prevention of BamH1 digestion with the increase in concentration of the multinuclear compounds also provide support to the idea that the compounds because of the formation of a plethora of interstrand GG adducts are able to cause irreversible changes in DNA conformation. The results of the study show that indeed new trinuclear tumour active compounds can be found by replacing the central platinum unit in BBR3464 with other suitable metal units.
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High-Yield Synthesis and Applications of Anisotropic Gold NanoparticlesVigderman, Leonid 16 September 2013 (has links)
This work will describe research directed towards the synthesis of anisotropic gold nanoparticles as well as their functionalization and biological applications. The thesis will begin by describing a new technique for the high-yield synthesis of gold nanorods using hydroquinone as a reducing agent. This addresses important limitations of the traditional nanorod synthesis including low yield of gold ions conversion to metallic form and inability to produce rods with longitudinal surface plasmon peak above 850 nm. The use of hydroquinone was also found to improve the synthesis of gold nanowires via the nanorod-seed mediated procedure developed in our lab. The thesis will next present the synthesis of novel starfruit-shaped nanorods, mesorods, and nanowires using a modified nanorod-seed mediated procedure. The starfruit particles displayed increased activity as surface-enhanced Raman spectroscopy (SERS) substrates as compared to smooth structures. Next, a method for the functionalization of gold nanorods using a cationic thiol, 16-mercaptohexadecyltrimethylammonium bromide (MTAB), will be described. By using this thiol, we were able to demonstrate the complete removal of toxic surfactant from the nanorods and were also able to precisely quantify the grafting density of thiol molecules on the nanorod surface through a combination of several analytical techniques. Finally, this thesis will show that MTAB-functionalized nanorods are nontoxic and can be taken up in extremely high numbers into cancer cells. The thesis will conclude by describing the surprising uptake of larger mesorods and nanowires functionalized with MTAB into cells in high quantities.
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Studies on Tumour Active Compounds with Multiple Metal CentresDaghriri, Hassan January 2004 (has links)
Four tumour active trinuclear complexes: DH4Cl: [{trans-PtCl(NH3)2}2m-{trans-Pd( NH3)2(H2N(CH2)4NH2)2]Cl4, DH5Cl: [{trans-PtCl(NH3)2}2m-{trans-Pd( NH3)2(H2N(CH2)5NH2)2]Cl4, DH6Cl: [{trans-PtCl(NH3)2}2m-{trans-Pd( NH3)2(H2N(CH2)6NH2)2]Cl4, DH7Cl: [{trans-PtCl(NH3)2}2m-{trans-Pd(NH3)2-( H2N(CH2)7NH2)2]Cl4 and one dinuclear complex DHD: [{trans-PtCl(NH3)2}�-{ H2N(CH2)6NH2}{trans-PdCl(NH3)2]Cl(NO3), have been prepared and characterised based on elemental analyses, IR, Raman, mass and 1 H NMR spectral measurements. For the trinuclear complexes, the synthesis has been carried out using a step-up method branching out from the central palladium unit. A purity of about 95% has been obtained by repeated dissolution and precipitation. The activity against human cancer cell lines including ovary cell lines: A2780, A2780 cisR , A2780 ZD0473R , non small lung cell line: NCI-H640 and melanoma: Me-10538 have been determined based on MMT assay. Cell uptakes, DNA-binding have been determined for ovary cell lines: A2780, A2780 cisR . The nature of interaction with pBR322 plasmid DNA and ssDNA has been studied for trinuclear complexes DH4Cl, DH5Cl, DH6Cl and DH7Cl and the dinuclear complex DHD. Interaction of DH6Cl with adenine and guanine has also been studied by HPLC. The compounds are found to exhibit significant anticancer activity against cancer cell lines especially ovarian cancer cell lines: A2780, A2780 cisR and A2780 ZD0473R . DH6Cl in which the linking diamine has six carbon atoms is found to be the most active compound. As the number of carbon atoms in thelinking diamine is changed from the optimum value of six, the activity is found to decrease, illustrating the structure-activity relationship. The increase in uptake of the trinuclear complexes in A2780 cell line with the increase in size of the linking diamine coupled with the low molar conductivity values found for the solutions of the compounds suggest that the compounds would remain in solution as undissociated �molecules� and hence could cross the cell membrane by passive diffusion. Much lower resistance factors for the all the multinuclear compounds including DHD as applied to A2780 cisR cell line, as compared to that for cisplatin, suggest that the compounds are able to overcome multiple mechanisms of resistance operating in the cell line. All of the multinuclear complexes are expected to form long-range interstrand GG adducts with DNA, causing irreversible global changes in the DNA conformation but unlike cisplatin do not cause sufficient DNA bending to be recognized by HMG 1 protein. Increasing prevention of BamH1 digestion with the increase in concentration of the multinuclear compounds also provide support to the idea that the compounds because of the formation of a plethora of interstrand GG adducts are able to cause irreversible changes in DNA conformation. The results of the study show that indeed new trinuclear tumour active compounds can be found by replacing the central platinum unit in BBR3464 with other suitable metal units.
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Surface-Charge-Dependent Cell Localization and Cytotoxicity of Cerium Oxide NanoparticlesAsati, Atul, Santra, Santimukul, Kaittanis, Charalambos, Perez, J. M. 28 September 2010 (has links)
Cerium oxide nanoparticles (nanoceria) have shown great potential as antioxidant and radioprotective agents for applications in cancer therapy. Recently, various polymer-coated nanoceria preparations have been developed to improve their aqueous solubility and allow for surface functionalization of these nanoparticles. However, the interaction of polymer-coated nanoceria with cells, their uptake mechanism, and subcellular localization are poorly understood. Herein, we engineered polymer-coated cerium oxide nanoparticles with different surface charges (positive, negative, and neutral) and studied their internalization and toxicity in normal and cancer cell lines. The results showed that nanoceria with a positive or neutral charge enters most of the cell lines studied, while nanoceria with a negative charge internalizes mostly in the cancer cell lines. Moreover, upon entry into the cells, nanoceria is localized to different cell compartments (e.g., cytoplasm and lysosomes) depending on the nanoparticles surface charge. The internalization and subcellular localization of nanoceria plays a key role in the nanoparticles cytotoxicity profile, exhibiting significant toxicity when they localize in the lysosomes of the cancer cells. In contrast, minimal toxicity is observed when they localize into the cytoplasm or do not enter the cells. Taken together, these results indicate that the differential surface-charge-dependent localization of nanoceria in normal and cancer cells plays a critical role in the nanoparticles toxicity profile.
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β-Apocarotenoids: Occurrence in Cassava Biofortified with β-Carotene and Mechanisms of Uptake in Caco-2 Intestinal CellsDurojaye, Boluwatiwi Olalekan 09 October 2015 (has links)
No description available.
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Intestinal and Hepatic Metabolism of Selected Apocarotenoids and RetinoidsDurojaye, Boluwatiwi Olalekan January 2020 (has links)
No description available.
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A focus on critical aspects of uptake and transport of milk-derived extracellular vesicles across the Caco-2 intestinal barrier modelRoerig, Josepha, Schiller, Laura, Kalwa, Herrmann, Hause, Gerd, Vissiennon, Cica, Hacker, Michael C., Wölk, Christian, Schulz-Siegmund, Michaela 10 October 2022 (has links)
Bovine milk-derived extracellular vesicles (EVs) hold promises as oral drug delivery systems. Since EV
bioavailability studies are difficult to compare, key factors regarding EV uptake and intestinal
permeability remain little understood. This work aims to critically study uptake and transport
properties of milk-derived EVs across the intestinal barrier in vitro by standardization approaches.
Therefore, uptake properties were directly compared to liposomes in intestinal Caco-2 cells. Reliable
staining results were obtained by the choice of three distinct EV labeling sites, while non-specific dye
transfer and excess dye removal were carefully controlled. A novel fluorescence correction factor was
implemented to account for different labelling efficiencies. Both EV and liposome uptake occurred
mainly energy dependent with the neonatal Fc receptor (FcRn) providing an exclusive active pathway
for EVs. Confocal microscopy revealed higher internalization of EVs whereas liposomes rather
remained attached to the cell surface. Internalization could be improved when changing the liposomal
formulation to resemble the EV lipid composition. In a Caco-2/HT29-MTX co-culture liposomes and EVs
showed partial mucus penetration.
For transport studies across Caco-2 monolayers we further established a standardized protocol
considering the distinct requirements for EVs. Especially insert pore sizes were systematically
compared with 3 µm inserts found obligatory. Obtained apparent permeability coefficients (Papp)
reflecting the transport rate will allow for better comparison of future bioavailability testing.
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N-Terminal Ile-Orn- and Trp-Orn-Motif repeats enhance membrane interaction and increase the antimicrobial activity of Apidaecins against Pseudomonas aeruginosaBluhm, Martina E. C., Schneider, Viktoria A. F., Schäfer, Ingo, Piantavigna, Stefania, Goldbach, Tina, Knappe, Daniel, Seibel, Peter, Martin, Lisandra L., Veldhuizen, Edwin J. A., Hoffmann, Ralf 21 June 2016 (has links) (PDF)
The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api795. The uptake was reduced for both peptides by 50 and 80%, respectively, after inhibiting endocytotic uptake with dynasore. In summary, Api794 and Api795 were highly active against P. aeruginosa in vitro. Both peptides passed across the bacterial membrane efficiently, most likely then disturbing the ribosome assembly, and resulting in further intracellular damage. Api795 with its IOIO-motif, which was particularly active and only slightly toxic in vitro, appears to represent a promising third generation lead compound for the development of novel antibiotics against P. aeruginosa.
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