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Scatter Enhanced Phase Contrast Microscopy for Discriminating Mechanisms of Active Nanoparticle Transport in Living Cells
摘要： Understanding the uptake and transport dynamics of engineered nanomaterials (ENMs) by mammalian cells is an important step in designing next-generation drug delivery systems. However, to track these materials and their cellular interactions, current studies often depend on surface-bound fluorescent labels which have the potential to alter native cellular recognition events. As a result, there is still a need to develop methods capable of monitoring ENM-cell interactions independent of surface modification. Addressing these concerns, here we show how Scatter Enhanced Phase Contrast (SEPC) microscopy can be extended to work as a generalized label-free approach for monitoring nanoparticle uptake and transport dynamics. To determine which materials can be studied using SEPC, we turn to Lorenz-Mie theory, predicting that individual particles down to ~35 nm can be observed. We confirm this experimentally, demonstrating that SEPC works for a variety of metal and metal oxides, including: Au, Ag, TiO2, CeO2, Al2O3, and Fe2O3 nanoparticles. We then demonstrate that SEPC microscopy can be used in a quantitative, time-dependent fashion to discriminate between distinct modes of active cellular transport, including intracellular transport and membrane assisted transport. Finally, we combine this technique with microcontact printing to normalize transport dynamics across multiple cells, allowing for a careful study of ensemble TiO2 nanoparticle uptake. This revealed three distinct regions of particle transport across the cell, indicating that membrane dynamics play an important role in regulating particle flow. By avoiding fluorescent labels, SEPC allows for a rational exploration of the surface properties of nanomaterials in their native state and their role in endocytosis and cellular transport.
关键词： Scatter Enhanced Phase Contrast,Lorenz-Mie Theory,Endocytosis,Nano-Bio Interface,Nanoparticle
更新于2025-09-23 15:23:52
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Intercellular Trafficking of Gold Nanostars in Uveal Melanoma Cells for Plasmonic Photothermal Therapy
摘要： Efficient plasmonic photothermal therapies (PPTTs) using non-harmful pulse laser irradiation at the near-infrared (NIR) are a highly sought goal in nanomedicine. These therapies rely on the use of plasmonic nanostructures to kill cancer cells while minimizing the applied laser power density. Cancer cells have an unsettled capacity to uptake, retain, release, and re-uptake gold nanoparticles, thus offering enormous versatility for research. In this work, we have studied such cell capabilities for nanoparticle trafficking and its impact on the effect of photothermal treatments. As our model system, we chose uveal (eye) melanoma cells, since laser-assisted eye surgery is routinely used to treat glaucoma and cataracts, or vision correction in refractive surgery. As nanostructure, we selected gold nanostars (Au NSs) due to their high photothermal efficiency at the near-infrared (NIR) region of the electromagnetic spectrum. We first investigated the photothermal effect on the basis of the dilution of Au NSs induced by cell division. Using this approach, we obtained high PPTT efficiency after several cell division cycles at an initial low Au NS concentration (pM regime). Subsequently, we evaluated the photothermal effect on account of cell division upon mixing Au NS-loaded and non-loaded cells. Upon such mixing, we observed trafficking of Au NSs between loaded and non-loaded cells, thus achieving effective PPTT after several division cycles under low irradiation conditions (below the maximum permissible exposure threshold of skin). Our study reveals the ability of uveal melanoma cells to release and re-uptake Au NSs that maintain their plasmonic photothermal properties throughout several cell division cycles and re-uptake. This approach may be readily extrapolated to real tissue and even to treat in situ the eye tumor itself. We believe that our method can potentially be used as co-therapy to disperse plasmonic gold nanostructures across affected tissues, thus increasing the effectiveness of classic PPTT.
关键词： nanoparticle endocytosis,femtosecond pulse laser,nanoparticle exocytosis,gold nanostars,plasmonic photothermal therapy
更新于2025-09-23 15:21:01
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SRRF-stream imaging of optogenetically controlled furrow formation shows localized and coordinated endocytosis and exocytosis mediating membrane remodeling
摘要： Cleavage furrow formation during cytokinesis involves extensive membrane remodeling. In the absence of methods to exert dynamic control over these processes, it has been a challenge to examine the basis of this remodeling. Here we used a subcellular optogenetic approach to induce this at will and found that furrow formation is mediated by actomyosin contractility, retrograde plasma membrane flow, localized decrease in membrane tension and endocytosis. FRAP, 4-D imaging and inhibition or upregulation of endocytosis or exocytosis show that ARF6 and Exo70 dependent localized exocytosis supports a potential model for intercellular bridge elongation. TIRF and Super Resolution Radial Fluctuation (SRRF) stream microscopy show localized VAMP2-mediated exocytosis and incorporation of membrane lipids from vesicles into the plasma membrane at the front edge of the nascent daughter cell. Thus, spatially separated but coordinated plasma membrane depletion and addition are likely contributors to membrane remodeling during cytokinetic processes.
关键词： SRRF-stream,optogenetics,Cleavage furrow,membrane remodeling,endocytosis,exocytosis
更新于2025-09-19 17:13:59
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Investigation of the Internalization of Fluorescently Labeled Lipophilic siRNA into Cultured Tumor Cells
摘要： The attachment of lipophilic molecules of natural origin, which have natural means for cell internalization, to small interfering RNA (siRNA) is an effective way of delivering siRNA to cells for biomedical purposes in vitro and in vivo. Earlier, we showed that the attachment of cholesterol to the 5'-end of the sense strand of nuclease-resistant siRNA through the optimized linker allows it to penetrate the cells and suppress the expression of the target gene. However, the effectiveness of the conjugates is different for cells of different origin, and in hematopoietic cells, they are not active, despite effective accumulation. In this work, we investigated the accumulation of fluorescently labeled cholesterol conjugates of siRNA using endocytosis inhibitors and showed that fluorescently labeled 5'-cholesterol conjugate of siRNAs penetrate KB-3-1 and K562 cells in several ways whose contribution differs depending on cell type and the presence of serum. In a serum-free medium, it was found that macropinocytosis and clathrin-dependent endocytosis contribute to the accumulation of the conjugate in KB-3-1 cells, while clathrin-dependent endocytosis makes the main contribution in K562 cells, while inhibitors of different types of endocytosis do not reduce the biological activity of the conjugate without a fluorescent label.
关键词： cell delivery mechanism,inhibitors of endocytosis,siRNA,cholesterol conjugate,chemical modifications
更新于2025-09-19 17:13:59
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Pharmacokinetic Properties of Fluorescently Labelled Hydroxypropyl-Beta-Cyclodextrin
摘要： 2-Hydroxypropyl-beta-cyclodextrin (HPBCD) is utilized in the formulation of pharmaceutical products and recently orphan designation was granted for the treatment of Niemann–Pick disease, type C. The exact mechanism of HPBCD action and side effects are not completely explained. We used fluorescently labelled hydroxypropyl-beta-cyclodextrin (FITC-HPBCD) to study its pharmacokinetic parameters in mice and compare with native HPBCD data. We found that FITC-HPBCD has fast distribution and elimination, similar to HPBCD. Interestingly animals could be divided into two groups, where the pharmacokinetic parameters followed or did not follow the two-compartment, first-order kinetic model. Tissue distribution studies revealed, that a significant amount of FITC-HPBCD could be detected in kidneys after 60 min treatment, due to its renal excretion. Ex vivo fluorescent imaging showed that fluorescence could be measured in lung, liver, brain and spleen after 30 min of treatment. To model the interaction and cellular distribution of FITC-HPBCD in the wall of blood vessels, we treated human umbilical vein endothelial cells (HUVECs) with FITC-HPBCD and demonstrated for the first time that this compound could be detected in the cytoplasm in small vesicles after 30 min of treatment. FITC-HPBCD has similar pharmacokinetic to HPBCD and can provide new information to the detailed mechanism of action of HPBCD.
关键词： endocytosis,pharmacokinetics,fluorescence,hydroxypropyl-beta-cyclodextrin,HUVECs
更新于2025-09-16 10:30:52
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Rapid Intestinal Uptake and Targeted Delivery to the Liver Endothelium Using Orally Administered Silver Sulfide Quantum Dots
摘要： Quantum dots (QDs) are used for imaging and transport of therapeutics. Here we demonstrate rapid absorption across the small intestine and targeted delivery of QDs with bound materials to the liver sinusoidal endothelial cells (LSECs) or hepatocytes in vitro and in vivo following oral administration. QDs were radiolabeled with 3H-oleic acid, with a ?uorescent tag or 14C-metformin placed within a drug binding site. Three di?erent biopolymer shell coatings were compared (form-aldehyde-treated serum albumin (FSA), gelatin, heparin). Passage across the small intestine into mesenteric veins is mediated by clathrin endocytosis and micropinocytosis. 60% of an oral dose of QDs was rapidly distributed to the liver within 30 min, and this increased to 85% with FSA biopolymer coating. Uptake into LSECs also increased 3-fold with FSA coating, while uptake into hepatocytes was increased from 40% to 85% with gelatin biopolymer coating. Localization of QDs to LSECs was con?rmed with immuno?uorescence and transmission electron microscopy. 85% of QDs were cleared within 24 h of administration. The bioavailability of 14C-metformin 2 h post-ingestion was increased 5-fold by conjugation with QD-FSA, while uptake of metformin into LSECs was improved 50-fold by using these QDs. Endocytosis of QDs by SK-Hep1 cells (an LSEC immortal cell line) was via clathrin- and caveolae-mediated pathways with QDs taken up into lysosomes. In conclusion, we have shown high speci?city targeting of the LSEC or hepatocytes after oral administration of QDs coated with a biopolymer layer of FSA or gelatin, which improved the bioavailability and delivery of metformin to LSECs.
关键词： nano,mice,metformin,biopolymer,endocytosis
更新于2025-09-16 10:30:52
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MicroRNA Quantitation During Dendritic Cell Endocytosis Using Imaging Flow Cytometry: Key Factors and Requirements
摘要： Background/Aims: MicroRNA (miRNA)-induced suppression of dendritic cells (DCs) has been implicated in many diseases. Therefore, accurate monitoring of miRNA endocytosis by DCs is important for understanding the role of miRNAs in many diseases. Recently, a method for measuring the co-localization of Argonaute 2 (AGO2)-associated miRNAs on laser-scanning confocal microscopy method was proposed to localize the miRNAs. But its definition was limited by the number of observed cells through its accuracy. Methods: In this study, a method based on imaging flow cytometry was developed to localize miR-590-5p with fluorescent probes in DCs. miR-590-5p proven to play an important role in tumor immunity. This method enabled the quantification, visualization and localization of the fluorescence intensity in 30,000 individual cells. Results: Using this method, the DCs with different endocytotic ability were distinguished. The behaviour of miR-590-5p during endocytosis under the stimulation of tumor antigen in DCs was observed, binding to its cognate target mRNA and degradation in DCs. Conclusion: This method based on imaging flow cytometry provide an additional method to study miRNA processing in DCs, which makes it a valuable addition to existing miRNA research techniques
关键词： miR-590-5p,miRNA endocytosis,Flow cytometry,Dendritic cells,Argonaute 2 (AGO2),FRET analysis
更新于2025-09-10 09:29:36
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Elucidating the mode of action for thiophene-based organic D-π-A sensitizers for use in photodynamic therapy
摘要： Photodynamic therapy (PDT) is a non-invasive, selective, and cost-effective cancer therapy. The development of readily accessible templates that allow rapid structural modification for further improvement of PDT remains important. We previously reported thiophene-based organic D-π-A sensitizers consisted of an electron-donating (D) moiety, a π-conjugated bridge (π) moiety, and an electron-accepting (A) moiety as valuable templates for a photosensitizer that can be used in PDT. Our preliminary structure-activity relationship study revealed that the structure of the A moiety significantly influences its phototoxicity. In this study, we evaluated the photoabsorptive, cellular uptake, and photo-oxidizing abilities of D-π-A sensitizers that contained different A moieties. The level of phototoxicity of the D-π-A sensitizers was rationalized by considering those three abilities. In addition, we observed the ability of amphiphilic sensitizers containing either a carboxylic acid or an amide in an A moiety to form aggregates that penetrate cells mainly via endocytosis.
关键词： Organic dye,Photodynamic therapy,Sensitizer,Singlet oxygen,Endocytosis
更新于2025-09-09 09:28:46
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Multivalent Biopolymer Capped Gold Nanoparticles as Stable and Non-Specific Endocytosis-Free Cell Labeling Agents
摘要： In this study, we report that carboxymethyl cellulose (CMC) is a stable capping agent for cellular labeling with gold nanoparticles (AuNPs). AuNPs were easily capped with CMC by simply mixing them and removing any unbound CMC by centrifugation. The CMC-capped AuNPs showed high stability in the presence of up to 100 mM of NaCl and (cid:1)-mercaptoethanol. Structural analysis of CMC-capped AuNPs revealed that multiple binding of CMC on AuNPs resulted in the stable protection. For specific cell labeling, anti-CD44 antibody was conjugated to CMC-capped AuNPs. Cellular labeling performance of the antibody-conjugated AuNPs was verified by adding these nanoparticles to HeLa cell containing culture. We observed light scattering spots from AuNPs inside HeLa cells, which were incubated with the antibody-conjugated AuNPs without non-specific endocytosis. However, clusters of citrate-capped AuNPs were found to be entrapped inside the cells through receptor-mediated endocytosis. In addition, no cytotoxicity was observed, confirming that CMC-capped AuNPs are useful as stable and biocompatible cell-labeling agents.
关键词： Endocytosis,Gold Nanoparticle,Darkfield,Carboxymethyl Cellulose,Cancer,Cellular Labeling Agent,CD44
更新于2025-09-04 15:30:14
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Characterization of a DNA Aptamer for Ovarian Cancer Clinical Tissue Recognition and in Vivo Imaging
摘要： Backgrounds/Aims: Ovarian cancer is the most lethal gynaecologic malignancy and is difficult to detect early. The inefficient early diagnosis of ovarian cancer is the main contributor to its high mortality rate. Aptamers, as chemical antibodies, are single-stranded DNA or RNA oligonucleotides that target cells or molecules with high affinity. Methods: Binding ability of R13 was measured by flow cytometry analysis. Stability of R13 was tested in blood serum of an ovarian cancer patient. Internalization of R13 was verified by confocal microscope imaging. 80 cases ovarian cancer tissues, 10 cases normal ovary tissues in a microarray and 6 fallopian tube tissues were prepared for this study. R13’s target ability was further confirmed in vivo tumor models in NOD/SCID mice. Results: In this study, we found aptamer R13 bound to ovarian cancer cells with dissociation constants in the nanomolar range. Moreover, these results were further confirmed by tissue imaging. Next we demonstrated that the targets of R13 are membrane proteins and that its internalization occurs in a caveolae-mediated and clathrin-mediated manner. The target function of R13 was determined by imaging A2780 tumours in mouse models. Conclusion: These findings suggest that R13 is a promising novel tool to diagnose and deliver drugs to treat ovarian cancer.
关键词： Endocytosis,DNA aptamer,Membrane proteins,Ovarian cancer,Nude mouse,Tissue microarray
更新于2025-09-04 15:30:14