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Formation of PEI/DNA/PIP3 ternary complexes. N/P = (nitrogen groups in PEI)/(phosphate groups in DNA and PIP3).
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The polarized architecture of epithelium presents a barrier to therapeutic drug/gene carriers, which is mainly due to a limited (apical) internalization of the carrier systems. The bacterium Pseudomonas aeruginosa invades epithelial cells by inducing production of apical phosphatidylinositol-3, 4, 5-triphosphate (PIP3), which results in the recruit...
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Context 1
... pleckstrin homology (PH) domain of Akt. For Preparation of PEI/DNA and PEI/DNA/PIP3 polyplexes. Branched PEI 25 kDa is considered as one of the most potent synthetic gene carriers in vitro. Here it was used as the polycation in the formation of a ternary polyplex formulation. Phosphoinositide-containing PEI polyplexes were prepared as shown in Fig. 8. Briefly, 10 μ L 300 μ M phosphoinositides in PBS (pH 7.4) was mixed with 1 μ g of (pEGFP-N1) DNA in 0.1 mL serum-free medium by gentle pipetting. Then branched PEI (200 μ g/mL) was added to the DNA/PIP3 mixture and rapidly mixed by pipetting, to obtain PEI/DNA/PIP3 complexes at an N/P ratio of 6.3, where N represents polymer amino ...
Citations
... Plastic granules with a size smaller than 5 mm are known as Microplastics (MPs) which seems to be the most abundant organic synthetic polymers products with destructive ecological impact all around the ecosystem [24], 21, 22, 23. Indeed, they can act as a transporter of pathogens and toxic pollutants, which are accumulated and moved through food chain to the top consumers and consequence serious health problems occur [14,45]. Several studies has been recently directed to MPs monitoring in riverine ecosystems whether in biotic or abiotic levels [41, 13, 20, 54. ...
Background
Rivers are an increasing source of MPs (microplastics) destined to the marine environments. Gorganroud River is passing through many kinds of land uses; including agriculture, forest and urban areas and could be one of the MPs pollution drivers to the Caspian Sea.Method
The abundance and distribution of MPs in sediments and some fish of the Gorganroud River assessed. Also, characteristics of microplastics with attached elements revealed and confirmed by SEM, EDX, ATR and Raman analysis.ResultsThe average abundance of MPs along the River sediments ranged from 220 ± 66 to 2460 ± 444 items/Kg. The most abundant of MPs reported from stations situated in urban areas, which suggests great amount of MPs in the Gorganroud River originates from cities wastewater effluents. Over 80% of these MPs was less than 2 mm. What's more, the majority shapes of MPs were fiber, followed by fragment and film. Black and gray were the dominant color among sediment MPs. Whereas the most common polymer types of these MPs in sediments were polypropylene (39%), polyethylene (26%), polyamide (16%), polystyrene (14%) and polyester (5%). Main type of polymers found in the digestive tract of fish were polypropylene (37%), polystyrene (29%), polyethylene (25%) and polyester (9%). Regard to the fish, the most abundant MPs detected in Neogobius melanostomus (0.72 n/g) caught mainly from stations situated along areas of agricultural fields. The same as sediment samples, prevalence shapes within digestive tract identified as fibers, followed by fragments and films. Although MPs size ranged from 0.1 to 3 mm, dominant MPs size was less than 1mm. Moreover, the frequent color detected within fish digestive tract was black.Conclusions
This survey demonstrated characteristics of MPs contamination along the Gorganroud River and revealed significance of the River as a sinking MPs pathway to the Caspian Sea ecosystem.Graphical Abstract
... Moreover, some freshwater algae, while interacting with nano-PS, had increased concentration of reactive oxygen species and decreased the rate of photosynthesis (Bhattacharya et al., 2010;Kierdorf and Prinz, 2019). In the case of freshwater and marine algae, alteration of growth had also been observed in the presence of micro-PS (Wang et al., 2016). Lugworm is very susceptible to oxidative stress with PVC, nano-PS can directly interact with brain tissues of freshwater zooplankton organisms and therefore enter into blood-to-brain barrier in the top listed consumer fish causing behavioural disorders (Mattsson et al., 2017). ...
Microplastics (< 5 mm) have become an environmental hazard to natural ecosystems and humanity due to their ubiquitous presence in the environment, and possess detrimental effects on aquatic organisms. Sampling, extraction, and detection of microplastics from sediments, surface water, and biota are quite complex and challenging. Rigorous research on the abundance and distribution of microplastics in terrestrial and aquatic ecosystems is primarily attributed to instinctive anthropogenic actions like land-based activities. Although, the fate and transport of microplastics in the environment are uncertain. Therefore, this paper reviews current research progress on the occurrence and distribution of microplastic pollution in the riverine ecosystem. Detailed potential sources, sampling techniques, and experimental design have been discussed, along with the classification and detection of microplastics in the riverine sediment and surface water. Microplastic ingestion through riverine species is also discussed systematically which emphasizes the extraction of microplastics from different body parts of the organism. Different types of polymeric microplastic have been observed in rivers, for instance, PS, PE, PP, PA, PET, PVC, PDPE, LPDE, nylon, acrylic, etc. using spectroscopy. Fragments, foams, pellets, films, fibers and microbeads are common shapes of microplastic which are abundant in the riverine ecosystem. The ingestion and ecological concern of microplastics, along with adsorption mechanisms of toxic contaminants, such as heavy metals, organic pollutants, nutrients, antibiotics, etc. onto microplastics surface and their exposure to aquatic organisms have also been discussed. Microplastic pollution in riverine ecosystems is under global change stress due to ecotoxicological and geochemical consequences. Further, detailed policies intervention for plastics and microplastics mitigation have been focused which delineate for application of science and policy together with scientific evidence. Lastly, scientific knowledge gaps and future research prospective on microplastic pollution have been given, along with suggestions and policy recommendations.
... Because endocytosis of nanoparticles generally involves cell surface receptors that mediate nanoparticle binding and/or uptake (Zuhorn et al., 2007;ur Rehman et al., 2012;Wang et al., 2016;Matsumoto et al., 2017), we next examined the possible role of cell surface receptors in the interaction between NSC exosomes and hCMEC/D3 cells. Heparan sulfate proteoglycans (HSPGs) are highly sulfated glycoproteins, containing one or more HS chains. ...
Drug delivery to the brain is greatly hampered by the presence of the blood-brain barrier (BBB) which tightly regulates the passage of molecules from blood to brain and vice versa. Nanocarriers, in which drugs can be encapsulated, can move across the blood-brain barrier (BBB) via the process of transcytosis, thus showing promise to improve drug delivery to the brain. Here, we demonstrate the use of natural nanovesicles, i.e., exosomes, derived from C17.2 neural stem cells (NSCs) to efficiently carry a protein cargo across an in vitro BBB model consisting of human brain microvascular endothelial cells. We show that the exosomes are primarily taken up in brain endothelial cells via endocytosis, while heparan sulfate proteoglycans (HSPGs) act as receptors. Taken together, our data support the view that NSC exosomes may act as biological nanocarriers for efficient passage across the BBB. Nanomedicines that target HSPGs may improve their binding to brain endothelial cells and, possibly, show subsequent transcytosis across the BBB.
... We then focused our investigations on the endosomal escape capacity of the LPRi and RNAiMAx, a stringent limiting step for nucleic acid delivery [22,24]. It was previously documented that oligodeoxynucleotides (ODN) passively accumulate in the nucleus, once they reach the cytosol [21,51,52]. We took advantage of this feature to monitor the endosomal escape step controlled by LPRi and RNAiMax. ...
MicroRNA (miRNA) oligonucleotides therapeutics are potent and attractive drugs for cancer treatment, but the kinetics of their intracellular trafficking, RISC processing and interaction with their mRNA targets in the cells are still not well understood. Moreover, the absence of efficient carriers impairs their translation into the clinic. Here, we compare the kinetics of miRNA-133a activity after transfection of U87MG glioblastoma cells with either a home-made lipopolyplexes (LPRi) or with the RNAiMax transfection reagent. For this purpose, we combined miRNA intracellular trafficking studies by confocal microscopy with our previously described RILES miRNA-ON reporter system subcloned here in a lentivirus expression vector (LentiRILES) for longitudinal analysis of miRNA activity in transfected cells. Using the LentiRILES system, we report significant differences in terms of miRNA delivery kinetics performed by these two transfection regents. We decipher the mechanisms of miRNA delivery by LPRi and investigate the main steps of miRNA internalization and cytosolic processing. We demonstrate that LPRi preferentially uses caveolae-mediated endocytosis as the main internalization pathway, releases miRNA into the cytosol after the first 3 h of incubation, and addresses the cytosolic miRNAs to P-bodies, while a fraction of miRNAs are exported to the extracellular space through exosomes which were found fully capable to re-transfect the cells. We implanted the LentiRILES cells in the brain of mice and infused the tumours with LPRi.miRNA using the convection-enhanced delivery method. Bioluminescence imaging of the live mice revealed efficient delivery of miRNAs in glioblastoma tumours, attesting successful miRNA uptake, internalization and RISC activation in vivo. Overall, our study provides a comprehensive overview of miRNA intracellular trafficking and processing in a glioblastoma context and highlights the potential use of LPRi for miRNA-based therapy.
... We then analyzed the effect of PIP3 on iMAEC cell morphology and found no difference between untreated cells and cells treated with PIP3 (Fig. 1I). Last, we performed a literature search and found that 25 M PIP3 doses did not cause toxicity (34). These data did not support our hypothesis that reduced protein expression was due to overt cytotoxicity. ...
The biological pathways that affect drug delivery in vivo remain poorly understood. We hypothesized that altering cell metabolism with phosphatidylinositol (3,4,5)-triphosphate (PIP3), a bioactive lipid upstream of the metabolic pathway PI3K (phosphatidylinositol 3-kinase)/AKT/ mTOR (mammalian target of rapamycin) would transiently increase protein translated by nanoparticle-delivered messenger RNA (mRNA) since these pathways increase growth and proliferation. Instead, we found that PIP3 blocked delivery of clinically-relevant lipid nanoparticles (LNPs) across multiple cell types in vitro and in vivo. PIP3-driven reductions in LNP delivery were not caused by toxicity, cell uptake, or endosomal escape. Interestingly, RNA sequencing and metabolomics analyses suggested an increase in basal metabolic rate. Higher transcriptional activity and mitochondrial expansion led us to formulate two competing hypotheses that explain the reductions in LNP-mediated mRNA delivery. First, PIP3 induced consumption of limited cellular resources, “drowning out” exogenously-delivered mRNA. Second, PIP3 triggers a catabolic response that leads to protein degradation and decreased translation.
... The development of cells into cell barriers reduced the gene expression levels of different protein markers and/or caused their relocation to the abluminal plasma membrane, resulting in lower uptake efficiency of luminally applied nanoparticles. It is known that polarized cell barriers express different uptake pathways and receptors on their apical and basal side, and this can affect nanoparticle uptake (Georgieva et al., 2011;Iversen et al., 2011;Wang et al., 2016). Similarly, because of their phenotypical heterogeneity, endothelial cells of different tissue origins are likely to show very different nanoparticle uptake behavior, and this could be exploited for nanomedicine targeting. ...
Endothelial cells exhibit distinct properties in morphology and functions in different organs that can be exploited for nanomedicine targeting. In this work, endothelial cells from different organs, i.e. brain, lung, liver, and kidney, were exposed to plain, carboxylated, and amino-modified silica. As expected, different protein coronas were formed on the different nanoparticle types and these changed when foetal bovine serum (FBS) or human serum were used. Uptake efficiencies differed strongly in the different endothelia, confirming that the cells retained some of their organ-specific differences. However, all endothelia showed higher uptake for the amino-modified silica in FBS, but, interestingly, this changed to the carboxylated silica when human serum was used, confirming that differences in the protein corona affect uptake preferences by cells. Thus, uptake rates of fluid phase markers and transferrin were determined in liver and brain endothelia to compare their endocytic activity. Overall, our results showed that endothelial cells of different organs have very different nanoparticle uptake efficiency, likely due to differences in receptor expression, affinity, and activity. A thorough characterization of phenotypic differences in the endothelia lining different organs is key to the development of targeted nanomedicine.
... Wang and colleagues [79] exploited this phenomenon to stimulate the uptake of nanoparticles into the polarized epithelium. Delivery of drug-carrying nanoparticles to their site of destination is a major hurdle in modern medicine [80] The uptake of nanoparticles into polarized epithelia via the apical plasma membrane is prohibited, presumably because the integrin receptors that facilitate their uptake are localized at the opposing basolateral plasma membrane domain [81]. ...
... Wang and colleagues therefore produced PtdIns(3,4,5)P3-containing nanoparticles carrying siRNA against a host cell gene product. It was demonstrated that these nanoparticles delivered the PtdIns(3,4,5)P3 into the apical plasma membrane domain, which resulted in a local conversion of the apical membrane into a basolateral domain containing the basolateral integrin receptors, followed by the uptake of the nanoparticles into the cells and silencing of the targeted host gene [79]. ...
Epithelial cells are polarized cells and typically display distinct plasma membrane domains: basal plasma membrane domains face the underlying tissue, lateral domains contact adjacent cells and apical domains face the exterior lumen. Each membrane domain is endowed with a specific macromolecular composition that constitutes the functional identity of that domain. Defects in apical-basal plasma membrane polarity altogether or more subtle defects in the composition of either apical or basal plasma membrane domain can give rise to severe diseases. Lipids are the main component of cellular membranes and mechanisms that control their polarized distribution in epithelial cells are emerging. In particular sphingolipids and phosphatidylinositol lipids have taken center stage in the organization of the apical and basolateral plasma membrane domain. This short review article discusses mechanisms that contribute to the polarized distribution of lipids in epithelial cells.
... The transfection procedure was kept consistent with that used for NIH3T3 cells, but the incubation duration was limited to only 4 h, in view of the trend observed from the experiments with NIH3T3 cells (Section 3.6). Fig. 8 shows that without DCC-RGD, both cells experienced high cytotoxicity and very low transfection efficiency, in line with other reports on MDCK cells using similar polymeric vector [46]. Upon the addition of DCC-RGD, however, both the transfection efficiency and viability were significantly improved. ...
Cellulose nanocrystals (CNCs) are promising bio-derived nanomaterials for the bottom-up fabrication of biomedical constructs. In this report, dicarboxylic acid-functionalized CNC (DCC) was functionalized with arginylglycylaspartic acid (RGD) tripeptide as a motif for improved cell adhesion and targeting. The product (DCC-RGD) self-assembled into a more elongated nanofibrillar structure through lateral and end-to-end association. When added into poly(ethylene imine) (PEI)/pDNA polyplex solution, nanocelluloses interacted electrostatically with positively charged polyplexes without affecting their integrity. The constructs were tested for their potentials as non-viral transfection reagents. Cell viability and transfection efficiency of fibroblast NIH3T3 cells were monitored as a function of CNC concentration where, in general, viability increased as the CNC concentration increased, and transfection efficiency could be optimized. Using wild-type MDCK and αV-knockout MDCK cells, the construct was able to provide targeted uptake of polyplexes. The findings have potential applications, for example, cell-selective in vitro or ex vivo transfection of autologous mesenchymal stem cells for cell therapy, or bottom-up design of future innovative biomaterials.
... Thus, the intracellular behavior of the antisense oligonucleotide polyplexes in this study is reminiscent of the behavior observed for siRNA in large LPs [37]. Trafficking of polyplexes to acidic compartments, including LEs and lysosomes, was also observed in another study [47]. ...
Understanding the cellular uptake and intracellular trafficking of oligonucleotides provides an important basic underpinning for the developing field of oligonucleotide-based therapeutics. Whether delivered as "free" oligonucleotides, as ligand-oligonucleotide conjugates, or in association with various nanocarriers, all forms of oligonucleotide enter cells by endocytosis and are initially ensconced within membrane-limited vesicles. Accordingly, the locus and extent of release to the cytosol and nucleus are key determinants of the pharmacological actions of oligonucleotides. A number of recent studies have explored the intracellular trafficking of various forms of oligonucleotides and their release from endomembrane compartments. These studies reveal a surprising convergence on an early-intermediate compartment in the trafficking pathway as the key locus of release for oligonucleotides administered in "free" form as well as those delivered with lipid complexes. Thus, oligonucleotide release from multivesicular bodies or from late endosomes seems to be the crucial endogenous process for attaining pharmacological effects. This intrinsic process of oligonucleotide release may be amplified by delivery agents such as lipid complexes or small molecule enhancers.
... The purpose of co-incorporating fluorescently labeled ONs into the JetPEI/pDNA complexes is to visualize and quantify endosomal escape according to a recently published dequenching assay. 16,18 When the polyplexes reside in the endosome, the fluorescence of these ONs is effectively quenched. Upon endosomal bursting, the labeled ONs escape from the endosome to the cytoplasm which can be seen as an intense burst of fluorescence, thus allowing to evaluate the number of endosomal escape events in time and space ( Figure 1A). ...
In gene therapy, endosomal escape represents a major bottleneck since nanoparticles often remain entrapped inside endosomes and are trafficked towards the lysosomes for degradation. A detailed understanding of the endosomal barrier would be beneficial for developing rational strategies to improve transfection and endosomal escape. By visualizing individual endosomal escape events in live cells we obtain insight into mechanistic factors that influence proton sponge-based endosomal escape. In a comparative study, we found that HeLa cells treated with JetPEI/pDNA polyplexes have a 3.5-fold increased endosomal escape frequency compared to ARPE-19 cells. We found that endosomal size has a major impact on the escape capacity. The smaller HeLa endosomes are more easily ruptured by the proton sponge effect than the larger ARPE-19 endosomes, a finding supported by a mathematical model based on the underlying physical principles. Still, it remains intriguing that even in the small HeLa endosomes, less than 10% of the polyplex-containing endosomes show endosomal escape. Further experiments revealed that the membrane of polyplex-containing endosomes becomes leaky to small compounds, preventing effective build-up of osmotic pressure, which in turn prevents endosomal rupture. Analysis of H1299 and A549 cells revealed that endosomal size determines endosomal escape efficiency when cells have comparable membrane leakiness. However, at high levels of membrane leakiness build-up of osmotic pressure is no longer possible, regardless of endosomal size. Based on our findings that both endosomal size and membrane leakiness have a high impact on proton sponge-based endosomal rupture, we provide important clues towards further improvement of this escape strategy.
