Caveolae-dependent Endocytosis Research Articles

A Quantity-Dependent Nonlinear Model of Sodium Cromoglycate Suppression on Beta-Conglycinin Transport.

Understanding the transport mechanism is crucial for developing inhibitors that block allergen absorption and transport and prevent allergic reactions. However, the process of how beta-conglycinin, the primary allergen in soybeans, crosses the intestinal mucosal barrier remains unclear. The present study indicated that the transport of beta-conglycinin hydrolysates by IPEC-J2 monolayers occurred in a time- and quantity-dependent manner. The beta-conglycinin hydrolysates were absorbed into the cytoplasm of IPEC-J2 monolayers, while none were detected in the intercellular spaces. Furthermore, inhibitors such as methyl-beta-cyclodextrin (MβCD) and chlorpromazine (CPZ) significantly suppressed the absorption and transport of beta-conglycinin hydrolysates. Of particular interest, sodium cromoglycate (SCG) exhibited a quantity-dependent nonlinear suppression model on the absorption and transport of beta-conglycinin hydrolysates. In conclusion, beta-conglycinin crossed the IPEC-J2 monolayers through a transcellular pathway, involving both clathrin-mediated and caveolae-dependent endocytosis mechanisms. SCG suppressed the absorption and transport of beta-conglycinin hydrolysates by the IPEC-J2 monolayers by a quantity-dependent nonlinear model via clathrin-mediated and caveolae-dependent endocytosis. These findings provide promising targets for both the prevention and treatment of soybean allergies.

Transport of glycinin, the major soybean allergen, across intestinal epithelial IPEC-J2 cell monolayers.

Soybean allergen entering the body is the initial step to trigger intestinal allergic response. However, it remains unclear how glycinin, the major soybean allergen, is transported through the intestinal mucosal barrier. The objective of this study was to elucidate the pathway and mechanism of glycinin hydrolysatetransport through the intestinal epithelial barrier using IPEC-J2 cell model. Purified glycinin was digested by in vitro static digestion model. The pathway and mechanism of glycinin hydrolysates transport through intestinal epithelial cells were investigated by cellular transcytosis assay, cellular uptake assay, immunoelectron microscopy and endocytosis inhibition assay. The glycinin hydrolysates were transported across IPEC-J2 cell monolayers in a time/dose-dependent manner following the Michaelis equation. Immunoelectron microscopy showed a number of glycinin hydrolysates appeared in the cytoplasm, but no glycinin hydrolysates were observed in the intercellular space of IPEC-J2 cells. The inhibitors, colchicine, chlorpromazine and methyl-β-cyclodextrin,significantly inhibited the cellular uptake of glycinin hydrolysates. The glycinin hydrolysates crossed IPEC-J2 cell monolayers through the transcellular pathway. Both clathrin- and caveolae-dependent endocytosis were involved in the epithelial uptake of the hydrolysates. These findings provided potential targets for the prevention and treatment of soybean allergy.

Tracking the cellular uptake and phototoxicity of Ru(ii)-polypyridyl-1,8-naphthalimide Tröger's base conjugates.

Ruthenium(ii) complexes are attracting significant research attention as a promising class of photosensitizers (PSs) in photodynamic therapy (PDT). Having previously reported the synthesis of two novel Ru(ii)-polypyridyl-1,8-naphthalimide Tröger's base compounds 1 and 2 with interesting photophysical properties, where the emission from either the Ru(ii) polypyridyl centres or the naphthalimide moieties could be used to monitor binding to nucleic acids, we sought to use these compounds to investigate further and in more detail their biological profiling, which included unravelling their mechanism of cellular uptake, cellular trafficking and cellular responses to photoexcitation. Here we demonstrate that these compounds undergo rapid time dependent uptake in HeLa cells that involved energy dependent, caveolae and lipid raft-dependent mediated endocytosis, as demonstrated by confocal imaging, and transmission and scanning electron microscopy. Following endocytosis, both compounds were shown to localise to mostly lysosomal and Golgi apparatus compartments with some accumulation in mitochondria but no localisation was found to the nucleus. Upon photoactivation, the compounds increased ROS production and induced ROS-dependent apoptotic cell death. The photo-activated compounds subsequently induced DNA damage and altered tubulin, but not actin structures, which was likely to be an indirect effect of ROS production and induced apoptosis. Furthermore, by changing the concentration of the compounds or the laser used to illuminate the cells, the mechanism of cell death could be changed from apoptosis to necrosis. This is the first detailed biological study of Ru(ii)-polypyridyl Tröger's bases and clearly suggests caveolae-dependent endocytosis is responsible for cell uptake - this may also explain the lack of nuclear uptake for these compounds and similar results observed for other Ru(ii)-polypyridyl complexes. These conjugates are potential candidates for further development as PDT agents and may also be useful in mechanistic studies on cell uptake and trafficking.

Magnetic graphene oxide nanoflakes for dual RNA interfering delivery and gene knockdown in prostate and liver cancers

The development of synthetic carriers for small interfering RNA (siRNA) and plasmids is crucial for effective gene therapy. In this study, we synthesized magnetic graphene oxide nanoflakes as carriers for siRNA delivery, with the goal of knockdown specific genes such as the green fluorescence protein (GFP). Our approach combined magnetically reduced graphene oxide with polyethylenimine (PEI) crosslinked to its surface using carbonyl diimidazole. To evaluate the adsorption capacity of the PEI-modified nanocomposite, we investigated its ability to bind two types of nucleic acids: short-hairpin (sh)RNA plasmids and siRNA targeting GFP. The nanocomposite exhibited significant adsorption, with maximum capacities of 426 ng/μg for shRNA and 71 ng/μg for siRNA, respectively. Simultaneous delivery of siRNA and shRNA using our designed nanocomposites was successfully achieved in human hepatoma and prostate cancer cells. Under magnetic guidance, the knockdown efficiencies reached 73.5 % in hepatoma cells for dual delivery of siRNA and shRNA. Our findings revealed that the nanocomplexes were internalized by the cells through a caveolae-dependent endocytosis mechanism. The demonstrated ability of the nanoflakes to efficiently transport siRNA and shRNA, with high loading capacity, controlled release, and magnetic targeting, resulted in effective GFP knockdown in vitro. These findings highlight the potential of magnetic graphene oxide nanoflakes as promising carriers for siRNA delivery and gene knockdown in therapeutic applications.

Enhanced intracellular uptake of an albumin fusion protein in cancer cells by its forced cell surface recruitment

Albumin fusion or conjugation is a well-established technique for tumor delivery and is mainly mediated by albumin-induced caveolae-dependent endocytosis. We report that caveolae-dependent endocytic signaling activated by human serum albumin (HSA) is not sufficiently strong to induce cellular uptake, mainly due to its electrostatic repulsion from the negatively charged cell surface sulfated glycosaminoglycans (GAGs), and fusion of the cell-surface-retained protein with HSA is an effective strategy to activate the HSA-induced endocytic signal, thereby improving its intracellular uptake. In this study, human lactoferrin (hLF), a protein that accumulates on the cell surface along with GAGs, was selected for delivery into human lung adenocarcinoma PC-14 cells. When added exogenously, hLF-fused HSA (hLF-HSA) was successfully endocytosed, whereas the simultaneous addition of HSA and hLF did not result in endocytosis, indicating less efficient activation of endocytic signaling by HSA alone and the importance of its fusion. Importantly, the treatment of cells with chlorate, a known inhibitor of GAG sulfation, dramatically suppressed the endocytosis of hLF-HSA owing to the loss of the hLF-GAG interaction. Therefore, the cell-surface localization of HSA imposed by fusion with the cell-surface-retained protein enhances its binding to the relevant receptor, which improves intracellular delivery as an albumin-fusion platform.

Permeation behavior of porphyrin derivatives with different functional group positions across cancer cell membranes

Porphyrin, which shows selective accumulation in cancer cells, has attracted attention as a drug carrier. The influences of the functional porphyrin positions (β- and meso-positions) on porphyrin accumulation must be understood. In this work, we focused on the investigation of the phenyl functional group whose β-position influences cancer cell accumulation through direct membrane permeation and endocytosis. The endocytic pathway, in particular, is influenced by both clathrin-dependent and caveolae-dependent endocytosis.

Osteoblast-Derived Matrix Vesicles Exhibit Exosomal Traits and a Unique Subset of microRNA: Their Caveolae-Dependent Endocytosis Results in Reduced Osteogenic Differentiation.

Matrix vesicles (MVs) are nano-sized extracellular vesicles that are anchored in the extracellular matrix (ECM). In addition to playing a role in biomineralization, osteoblast-derived MVs were recently suggested to have regulatory duties. The aims of this study were to establish the characteristics of osteoblast-derived MVs in the context of extracellular vesicles like exosomes, assess their role in modulating osteoblast differentiation, and examine their mechanism of uptake. MVs were isolated from the ECM of MG63 human osteoblast-like cell cultures and characterized via enzyme activity, transmission electron microscopy, nanoparticle tracking analysis, Western blot, and small RNA sequencing. Osteoblasts were treated with MVs from two different culture conditions (growth media [GM]; osteogenic media [OM]) to evaluate their effects on the differentiation and production of inflammatory markers and on macrophage polarization. MV endocytosis was assessed using a lipophilic, fluorescent dye and confocal microscopy with the role of caveolae determined using methyl-β-cyclodextrin. MVs exhibited a four-fold enrichment in alkaline phosphatase specific activity compared to plasma membranes; were 50-150 nm in diameter; possessed exosomal markers CD63, CD81, and CD9 and endosomal markers ALIX, TSG101, and HSP70; and were selectively enriched in microRNA linked to an anti-osteogenic effect and to M2 macrophage polarization. Treatment with GM or OM MVs decreased osteoblast differentiation. Osteoblasts endocytosed MVs using a mechanism that involves caveolae. These results support the hypothesis that osteoblasts produce MVs that participate in the regulation of osteogenesis.

Low-Density Lipoprotein Receptor (LDLR) Is Involved in Internalization of Lentiviral Particles Pseudotyped with SARS-CoV-2 Spike Protein in Ocular Cells.

Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 μM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR.

ORFV entry into host cells via clathrin-mediated endocytosis and macropinocytosis

Orf virus (ORFV), also known as infectious pustular virus, leads to an acute contagious zoonotic infectious disease. ORFV can directly contact and infect epithelial cells of skin and mucosa, causing damage to tissue cells. So far, the pathway of ORFV entry into cells is unclear. Therefore, finding the internalization pathway of ORFV will help to elucidate the cellular and molecular mechanisms of ORFV infection and invasion, which in turn will provide a certain reference for the prevention and treatment of ORFV. In the present study, chemical inhibitors were used to analyze the mechanism of ORFV entry into target cells. The results showed that the inhibitor of clathrin-mediated endocytosis could inhibit ORFV entry into cells. However, the inhibitor of caveolae-mediated endocytosis cannot inhibit ORFV entry into cells. In addition, inhibition of macropinocytosis pathway also significantly reduced ORFV internalization. Furthermore, the inhibitors of acidification and dynamin also prevented ORFV entry. However, results demonstrated that inhibitors inhibited ORFV entry but did not inhibit ORFV binding. Notably, extracellular trypsin promoted ORFV entry into cells directly, even when the endocytic pathway was inhibited. In conclusion, ORFV enters into its target cells by clathrin-mediated endocytosis and macropinocytosis, while caveolae-dependent endocytosis has little effects on this process. In addition, the entry into target cells by ORFV required an acid environment and the effect of dynamin. Meanwhile, we emphasize that broad-spectrum antiviral inhibitors and extracellular enzyme inhibitors are likely to be effective strategies for the prevention and treatment of ORFV infection.

Cellular Uptake Mechanism of Nucleic Acid Nanocapsules and Their DNA-Surfactant Building Blocks.

Nucleic acid nanocapsules (NANs) are enzyme-responsive DNA-functionalized micelles built for the controlled release of DNA-surfactant conjugates (DSCs) that present sequences with demonstrated therapeutic potential. Here, we investigate the mechanisms by which DSCs gain access to intracellular space in vitro and determine the effects of serum on the overall uptake and internalization mechanism of NANs. Using pharmacological inhibitors to selectively block certain pathways, we show, through confocal visualization of cellular distribution and flow cytometry quantification of total cellular association, that scavenger receptor-mediated, caveolae-dependent endocytosis is the major cellular uptake pathway of NANs in the presence and absence of serum. Furthermore, as NANs can be triggered to release DSCs by external stimuli such as enzymes, we sought to examine the uptake profile of particles degraded by enzymes prior to cell-based assays. We found that while scavenger receptor-mediated, caveolae-dependent endocytosis is still at play, energy-independent pathways as well as clathrin-mediated endocytosis are also involved. Overall, this study has helped to elucidate early steps in the cytosolic delivery and therapeutic activity of DSCs packaged into a micellular NAN platform while shedding light on the way in which DNA functionalized nanomaterials in general can be trafficked into cells both as nanostructures and as molecular entities. Importantly, our study also shows that the NAN design in particular is able to stabilize nucleic acids when delivered in the presence of serum, a critical step for effective therapeutic nucleic acid delivery.

Intracellular Delivery of Micron-Sized Magnetic Particles through a Virus Infection Pathway.

Micron-sized magnetic particles (M-MPs) have low toxicity, strong magnetic signals, and long-term retention capability, which are significant advantages for their application in biomedical imaging. Unfortunately, M-MPs are only internalized by few cell types, such as macrophages and phagocytes, and because of this lack of active intracellular delivery, their applications are restricted. The emergence of self-assembled virus-like particles (VLPs) offers a viable approach to drive M-MPs into cells, although the specific mechanism has not been revealed. In this study, we investigated in detail the intracellular pathway of M-MPs mediated by VLPs using a fluorescence co-localization method. The results indicated that the intracellular movement of M-MPs was consistent with the virus infection pathway, specifically caveolae-dependent endocytosis, transportation through microtubules, and accumulation in the endoplasmic reticulum. This study provides experimental support for the active transport of M-MPs into other cell types, thereby further extending their applications.

DCBLD2 regulates vascular hyperplasia by modulating the platelet derived growth factor receptor-β endocytosis through Caveolin-1 in vascular smooth muscle cells.

DCBLD2 is a neuropilin-like transmembrane protein that is up-regulated during arterial remodeling in humans, rats, and mice. Activation of PDGFR-β via PDGF triggers receptor phosphorylation and endocytosis. Subsequent activation of downstream signals leads to the stimulation of phenotypic conversion of VSMCs and arterial wall proliferation, which are common pathological changes in vascular remodeling diseases such as atherosclerosis, hypertension, and restenosis after angioplasty. In this study, we hypothesized that DCBLD2 regulates neointimal hyperplasia through the regulation of PDGFR-β endocytosis of vascular smooth muscle cells (VSMCs) through Caveolin-1 (Cav-1). Compared with wild-type (WT) mice or control littermate mice, the germline or VSMC conditional deletion of the Dcbld2 gene resulted in a significant increase in the thickness of the tunica media in the carotid artery ligation. To elucidate the underlying molecular mechanisms, VSMCs were isolated from the aorta of WT or Dcbld2-/- mice and were stimulated with PDGF. Western blotting assays demonstrated that Dcbld2 deletion increased the PDGF signaling pathway. Biotin labeling test and membrane-cytosol separation test showed that after DCBLD2 was knocked down or knocked out, the level of PDGFR-β on the cell membrane was significantly reduced, while the amount of PDGFR-β in the cytoplasm increased. Co-immunoprecipitation experiments showed that after DCBLD2 gene knock-out, the binding of PDGFR-β and Cav-1 in the cytoplasm significantly increased. Double immunofluorescence staining showed that PDGFR-β accumulated Cav-1/lysosomes earlier than for control cells, which indicated that DCBLD2 gene knock-down or deletion accelerated the endocytosis of PDGF-induced PDGFR-β in VSMCs. In order to confirm that DCBLD2 affects the relationship between Cav-1 and PDGFR-β, proteins extracted from VSMCs cultured in vitro were derived from WT and Dcbld2-/- mice, whereas co-immunoprecipitation suggested that the combination of DCBLD2 and Cav-1 reduced the bond between Cav-1 and PDGFR-β, and DCBLD2 knock-out was able to enhance the interaction between Cav-1 and PDGFR-β. Therefore, the current results suggest that DCBLD2 may inhibit the caveolae-dependent endocytosis of PDGFR-β by anchoring the receptor on the cell membrane. Based on its ability to regulate the activity of PDGFR-β, DCBLD2 may be a novel therapeutic target for the treatment of cardiovascular diseases.

Arabinogalactan from Ixeris chinensis (Thunb.) Nakai as a stabilizer to decorate SeNPs and enhance their anti-hepatocellular carcinoma activity via the mitochondrial pathway

Arabinogalactan (ICPA) was isolated from the medicinal plant Ixeris chinensis (Thunb.) Nakai, and was used as the reducing agent to stabilize selenium nanoparticles (SeNPs). ICPA-decorated SeNPs (ICPA-SeNPs) were synthesized with Na2SeO3 and ICPA, and their average diameter was 82.6 ± 2.5 nm. ICPA-SeNPs had good dispersity in phosphate-buffered saline (PBS) with a hydrodynamic size of 156.2 ± 2.8 nm. They were stable in PBS solution with a zeta potential of −24.8 ± 0.6 mV. The anti-hepatoma activity of ICPA-SeNPs was investigated in vitro. ICPA-SeNPs significantly suppressed the growth of SMMC-7721 and HepG2 hepatoma cells. They entered SMMC-7721 cells via energy- and caveolae-dependent endocytosis. ICPA-SeNPs were able to increase reactive oxygen species levels, reduce mitochondrial membrane potential, and increase caspase-3 expression in SMMC-7721 cells, which led to the apoptosis of SMMC-7721 cells. They also arrested the cell cycle of SMMC-7721 cells in the S phase. These findings indicate that ICPA could enhance the stability of SeNPs and inhibit SMMC-7721 cell proliferation via the mitochondrial pathway. ICPA-SeNPs could be developed as an antitumor agent for hepatocellular carcinoma treatment.

Caveolae-dependent endocytosis mediates the cellular uptake of CdTe quantum dots in ovarian cancer cell lines.

Quantum dots (QDs) are semiconductor nanocrystals that are widely used in biology due to their good optical properties. QDs, especially cadmium-based QDs, play an important role in the diagnosis and treatment of cancer due to their intrinsic fluorescence. The aim of the present study was the evaluation of the cellular uptake mechanisms of CdTe QDs in ovarian cancer cell lines. In this study, we used CdTe QDs coated with thioglycolic acid. The ovarian cancer cell lines SKOV3 and OVCAR3 were treated with different concentrations of QDs, triamterene, chlorpromazine, and nystatin, and cell viability was evaluated through the MTT test. To find the way of cellular uptake of CdTe QDs, we used the MTT test and interfering compounds in endocytic pathways. Intrinsic fluorescence and cellular internalization of CdTe QDs were assessed using flow cytometry and fluorescence microscopy imaging. The viability of CdTe QDs-treated cells dose-dependently decreased in comparison to untreated cells. To evaluate the cellular uptake pathways of CdTe QDs, in most cases, a significant difference was observed when the cells were pretreated with nystatin. The results of flow cytometry showed the cellular uptake of CdTe QDs was dose- and time-dependent. Nystatin had a measurable effect on the cellular uptake of CdTe QDs. This finding suggests that caveola-mediated endocytosis has a large portion on the internalization of CdTe QDs. According to the results of this study, CdTe QDs may have potential applications in cancer research and diagnosis.

Self-Assembled Oligo-Urethane Nanoparticles: Their Characterization and Use for the Delivery of Active Biomolecules into Mammalian Cells.

Developing safe and effective strategies to deliver biomolecules such as oligonucleotides and proteins into cells has grown in importance over recent years, with an increasing demand for non-viral methods that enable clinical translation. Here, we investigate uniquely configured oligo-urethane nanoparticles based on synthetic chemistries that minimize the release of pro-inflammatory biomarkers from immune cells, show low cytotoxicity in a broad range of cells, and efficiently deliver oligonucleotides and proteins into mammalian cells. The mechanism of cell uptake for the self-assembled oligo-urethane nanoparticles was shown to be directed by caveolae-dependent endocytosis in murine myoblasts (C2C12) cells. Inhibiting caveolae functions with genistein and methyl-β-cyclodextrin limited nanoparticle internalization. The nanoparticles showed a very high delivery efficiency for the genetic material (a 47-base oligonucleotide) (∼80% incorporation into cells) as well as the purified protein (full length firefly luciferase, 67 kDa) into human embryonic kidney (HEK293T) cells. Luciferase enzyme activity in HEK293T cells demonstrated that intact and functional proteins could be delivered and showed a significant extension of activity retention up to 24 h, well beyond the 2 h half-life of the free enzyme. This study introduces a novel self-assembled oligo-urethane nanoparticle delivery platform with very low associated production costs, enabled by their scalable chemistry (the benchwork cost is $ 0.152/mg vs $ 974.6/mg for typical lipid carriers) that has potential to deliver both oligonucleotides and proteins for biomedical purposes.

Nucleosomes enter cells by clathrin- and caveolin-dependent endocytosis.

DNA damage and apoptosis lead to the release of free nucleosomes—the basic structural repeating units of chromatin—into the blood circulation system. We recently reported that free nucleosomes that enter the cytoplasm of mammalian cells trigger immune responses by activating cGMP-AMP synthase (cGAS). In the present study, we designed experiments to reveal the mechanism of nucleosome uptake by human cells. We showed that nucleosomes are first absorbed on the cell membrane through nonspecific electrostatic interactions between positively charged histone N-terminal tails and ligands on the cell surface, followed by internalization via clathrin- or caveolae-dependent endocytosis. After cellular internalization, endosomal escape occurs rapidly, and nucleosomes are released into the cytosol, maintaining structural integrity for an extended period. The efficient endocytosis of extracellular nucleosomes suggests that circulating nucleosomes may lead to cellular disorders as well as immunostimulation, and thus, the biological effects exerted by endocytic nucleosomes should be addressed in the future.

Intracellular Condensates of Oligopeptide for Targeting Lysosome and Addressing Multiple Drug Resistance of Cancer.

Biomolecular condensates have been demonstrated as a ubiquitous phenomenon in biological systems and play a crucial role in controlling cellular functions. However, the spatiotemporal construction of artificial biomolecular condensates with functions remains challenging and has been less explored. Herein, a general approach is reported to construct biomolecular condensates (e.g., hydrogel) in the lysosome of living cells for cancer therapy and address multiple drug resistance induced by lysosome sequestration. Aromatic-motif-appended pH-responsive hexapeptide (LTP) derived from natural insulin can be uptaken by cancer cells mainly through caveolae-dependent endocytosis, ensuring the proton-triggered phase transformation (solution to hydrogel) of LTP inside the lysosome specifically. Lysosomal hydrogelation further leads to enlargement of the lysosome in cancer cells and increases the permeability of the lysosome, resulting in cancer cell death. Importantly, lysosomal assemblies can significantly improve the efficiency of current chemotherapy drugs toward multidrug resistance (MDR) cells in vitro and in xenograft tumor models. As an example of functional artificial condensates in lysosomes, this work provides a new strategy for controlling functional condensates formation precisely in the organelles of living cells and addressing MDR in cancer therapy.

Fixed-Combination Eye Drops Based on Fluorometholone Nanoparticles and Bromfenac/Levofloxacin Solution Improve Drug Corneal Penetration

PurposeThe multi-instillation of three commercially available (CA) eye drops [fluorometholone (FL)-, bromfenac (BF)- and levofloxacin (LV)-eye drops] has been used to manage pain and inflammation post-intraocular surgery. However, the multi-instillation of these three eye drops causes corneal damage, and the FL drops have the disadvantage of low ocular bioavailability. To overcome these problems, we prepared fixed-combination eye drops based on FL nanoparticles (FL-NPs) and BF/LV solution (nFBL-FC), and evaluated the corneal toxicity and transcorneal penetration of the nFBL-FC eye drops.MethodsFL powder was mixed in 2-hydroxypropyl-β-cyclodextrin solution containing benzalkonium chloride, mannitol and methylcellulose, and milled with a Bead Smash 12 (5500 rpm for 30 s×30 times). The BF/LV solution was then added to the milled-dispersions to be used as nFBL-FC. The FL, BF and LV concentrations were measured by HPLC methods, and transcorneal penetration was evaluated in rabbits.ResultsThe FL particle size in nFBL-FC was 40–150 nm, with only 0.0018% in liquid form. No aggregation of FL particles in the nFBL-FC was observed for 1 month. The viability of human corneal epithelial cells treated with nFBL-FC was remarkably higher than that of cells subjected to the multi-instillation of the corresponding three CA-eye drops. In addition, the corneal penetrations (AUC) of the FL, BF and LV in nFBL-FC were 4.9-, 1.8-, and 7.1-fold those of the corresponding CA-eye drops, respectively. Moreover, the caveolae-dependent endocytosis (CavME) inhibitor (nystatin) significantly prevented the transcorneal penetration of these drugs.ConclusionWe prepared fixed-combination eye drops based on FL-NPs and BF/LV solution (nFBL-FC), and show that high levels of FL-NPs and dissolved BF/LV (liquid drugs) can be delivered into the aqueous humor by the instillation of nFBL-FC. Further, we show that CavME is mainly related to the enhancement of transcorneal penetration of both the solid (NPs) and liquid drugs.

Uptake, intracellular dissolution, and cytotoxicity of silver nanowires in cell models

The uptake, intracellular dissolution, and cytotoxicity of silver nanowires (AgNWs) in two cell models (human keratinocytes - HaCaT cells and murine macrophages) were systemically investigated for the first time. Cellular uptake of AgNWs occurred mainly via pathways of clathrin-dependent endocytosis, caveolae-dependent endocytosis, and phagocytosis. AgNWs could be internalized by two types of cells with numerous lysosomal vesicles detected in close vicinity to AgNWs. Meanwhile, AgNWs exposure caused lysosomal permeabilization and release of cathepsisn B into cytoplasm. Furthermore, for the first time, this study found that AgNWs exposure inhibited the transmembrane ATP binding cassette (ABC) efflux transporter activity, which could make AgNWs as chemosensitizers to increase the toxicity of other xenobiotic pollutants. Toxicity assays evaluating reactive oxygen species production and mitochondrial activity indicated that cytotoxicity differed for different cell types and particles. The intracellular presence of AgNWs with different diameters induced similar toxic events but to different extents. AgNWs were absorbed by macrophages more efficiently than HaCaT cells, while AgNWs exhibited only marginal cytotoxicity towards macrophages compared to HaCaT cells. Using an Ag+ fluorescence probe, it was found that a fraction of AgNWs was dissolved inside the lysosomes. A higher amount of released Ag+ was detected in HaCaT cells than in macrophages, which might partially contribute to their higher cytotoxicity in HaCaT cells. The toxicity of AgNWs in HaCaT cells and macrophages is due to the high-aspect nature of the nanowires rather than the extracellular release of Ag+. This study may be useful for risk assessments of AgNWs in their practical applications in the biomedical field.

Overexpression of Toll-like receptor 4 contributes to the internalization and elimination of Escherichia coli in sheep by enhancing caveolae-dependent endocytosis

BackgroundGram-negative bacterial infections have a major economic impact on both the livestock industry and public health. Toll-like receptor 4 (TLR4) plays a crucial role in host defence against Gram-negative bacteria. Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections. In a previous study, we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance. The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood.ResultsIn the present study, we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model. The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection. This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis. Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4, Src signalling, or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria, weakened the ability of the monocytes to eliminate the bacteria, and increased the pH of the endosomes.ConclusionTogether, our results reveal the effects of TLR4 on the control of E. coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model, providing theoretical support for breeding disease resistance in the future. Furthermore, Src and caveolin 1 (CAV1) could be potentially valuable targets for the control of infectious diseases.