PEGylation of cationic polyplexes is a promising approach to enhance the stability and reduce unspecific interaction with biological components. Herein, we systematically investigate the impact of ...PEGylation on physical and biological properties of chitosan/siRNA polyplexes. A series of chitosan-PEG copolymers (CS-PEG2k, CS-PEG5k and CS-PEG10k) were synthesized with similar PEG mass content but with different molecular weight. PEGylation with higher molecular weight and less grafting degree resulted in smaller and more compacted nanoparticles with relatively higher surface charge. PEGylated polyplexes showed distinct mechanism of endocytosis, which was macropinocytosis and caveolae-dependent and clathrin-independent. In vitro silencing efficiency in HeLa and H1299 cells was significantly improved by PEGylation and CS-PEG5k/siRNA achieved the highest knockdown efficiency. Efficient silence of ribonucleotide reductase subunit M2 (RRM2) in HeLa cells by CS-PEG5k/siRRM2 significantly induced cell cycle arrest and inhibited cell proliferation. In addition, PEGylation significantly inhibited macrophage phagocytosis and unspecific interaction with red blood cells (RBCs). Significant extension of in vivo circulation was achieved only with high molecular weight PEG modification (CS-PEG10k), whereas all CS/siRNA and CS-PEG/siRNA nanoparticles showed similar pattern of biodistribution with major accumulation in liver and kidney. These results imply that PEGylation with higher molecular weight PEG and less grafting rate is a promising strategy to improve chitosan/siRNA nanocomplexes performance both in vitro and in vivo.
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IJS, KILJ, NUK, PNG, UL, UM
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Growing evidence indicates that autophagy plays a vital role during intracellular DNA delivery mediated by lipoplex and polyplex nanoparticles. However, autophagy in intracellular ...siRNA delivery has not been well understood. In this study, lipofectamine 2000 and chitosan were used to formulate lipoplex and polyplex with siRNA for systematically investigating the interplay between siRNA delivery and autophagy. After transfection of H1299 cells with lipoplex and polyplex, the number of autophagic vacuoles was increased significantly indicated by the accumulation of monodansylcadaverine (MDC) staining. Western blot revealed that the LC3-II expression was significantly increased after transfection, whereas p-mTOR expression was not influenced apparently. In addition, small-molecule autophagy modulators significantly affected transfection efficiency. Specifically, the mTOR-dependent autophagy inducer rapamycin enhanced the knockdown efficiency of both lipoplex and polyplex, whereas mTOR-dependent autophagy inhibitor 3-methyladenine (3-MA) suppressed their silencing efficiency. On the contrary, mTOR-independent autophagy inducer LiBr decreased whereas mTOR-independent autophagy inhibitor thapsigargin (TG) increased the knockdown efficacy. Immunofluorescence staining showed that siRNA was partially co-localized with autophagosomes and the percentage of co-localized siRNA was significantly affected by autophagy modulators in the opposite trend of gene knockdown efficacy. In conclusion, our study suggests that autophagy plays an important role during the intracellular siRNA trafficking mediated by both lipoplex and polyplex. Modulating autophagy process will result in distinct knockdown efficiency, which may be applied as a potential convenient way for improving siRNA delivery efficacy.
Although tremendous effects has been made in the development of non-viral siRNA delivery systems, the intracellular siRNA trafficking has not been elucidated clearly. In this study, we systematically investigated the relationship between autophagy and intracellular siRNA delivery. We found that the non-viral siRNA delivery by both lipoplex and polyplex could induce mTOR-independent autophagy response. More interestingly, knockdown efficiency of both lipoplex and polyplex could be modulated with different autophagy regulators. Specifically, the mTOR-dependent autophagy inducer rapamycin enhances the knockdown efficiency of both lipoplex and polyplex, whereas mTOR-dependent autophagy inhibitor 3-methyladenine suppresses their silencing efficiency. On the contrary, mTOR-independent autophagy inducer lithium bromide decreases, whereas mTOR-independent autophagy inhibitor thapsigargin increases the knockdown efficacy. These findings suggest that the mTOR-dependent and -independent autophagy play a distinct role in the intracellular siRNA trafficking. Furthermore, co-administration with proper autophagy regulators could be potential convenient method to modulate siRNA transfection efficacy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The effect of crystallite size and crystallite on the photoreactivity of anatase was studied by phenol photodecomposition. It was found that the decomposition process was strongly influenced by the ...crystallite size but not by the crystallinity. Increasing the crystallite size led to a significant suppression of undesired hydroquinone-benzoquinone redox reactions thus enhanced the photoreactivity of anatase.
•Synthesis of anatase with well-defined crystallite size and crystallinity.•The photo-reactivity of anatase is independent with the crystallinity.•Increase the crystallite size of anatase enhances the photocatalytic performance.•The crystallite size dictates the phenol decomposition mechanism.
Crystallite size and crystallinity have been recognized as important parameters that influence the photocatalytic performance of pristine TiO2 nanoparticles. But how these two parameters individually affect the photo-reactivity of TiO2 remains unclear and debated due to the difficulties in preparing well-controlled TiO2 photocatalysts that vary crystallite size and crystallinity independently. Here, we have studied the effect of crystallite size and crystallinity on phenol photo-decomposition using well-defined anatase nanoparticles synthesized under supercritical water–isopropanol conditions. The photo-reactivity was found to increase with increasing crystallite size but to be independent of the crystallinity. By tracking the evolution of phenolic intermediates, we explored the reaction kinetics and demonstrated that an increase in the crystallite size of anatase nanoparticles can significantly suppress undesired hydroquinone–benzoquinone redox reactions and thus promote the full decomposition of phenol and phenolic compounds.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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•siRNA transfection is enormously increased in HBSS by different chitosan in various cell types.•Chitosan/siRNA nanoparticles are disturbed in HBSS and cell culture medium, with or ...without serum.•Aggregation of chitosan/siRNA does not influence cell uptake in HBSS.•Transfection medium is more important than chitosan or nanoparticles parameters.
It is well known that the chitosan (CS) properties such as molecular weight (Mw) and deacetylation degree (DD) are crucial for siRNA delivery. In the present study, we have unintentionally found that the transfection medium has much more substantial effect, which has not been reported before. Specifically, the transfection efficiency was considerably increased in Hank’s Balanced Saline Solution (HBSS), with various Mw (30–150 kD) and DD (85–95%) combinations in different cell types. In addition, even though the CS/siRNA nanoparticles physical parameters such as diameter size and polydisperse index were disturbed by cell culture medium or serum, a relatively high knockdown efficiency could still be obtained in HBSS. Moreover, the highly efficient silencing in HBSS even did not require pre-mixing to formulate the CS/siRNA nanoparticles. In conclusion, our findings have confirmed a neglected but crucial important factor of transfection medium in the chitosan mediated siRNA delivery.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
MicroRNAs play a vital role in cancer development and are considered as potential biomarkers for early prognostic assessment. Here, we propose a novel biosensing system to achieve fluorescence ...imaging of miRNA21 (miR21) in cancer cells. This system consists of two components: an optimized “off-on” double-stranded DNA (dsDNA) fluorescent for miR21 sensing by efficient strand-displacement reaction and a potent carrier vesicle, termed niosome (SPN), to facilitate the efficient intracellular delivery of the dsDNA probe. A series of dsDNA probes based on fluorescence energy resonance transfer (FRET) was assembled to target miR21. By optimizing the appropriate length of the reporter strand in the dsDNA probe, high accuracy and sensitivity for miR21 recognition are ensured. To overcome the cellular barrier, we synthesized SPN with the main components of a nonionic surfactant Span 80 and a cationic lipid DOTAP, which could efficiently load dsDNA probes via electrostatic interactions and potently deliver the dsDNA probes into cells with good biosafety. The SPN/dsDNA achieved efficient miR21 fluorescent imaging in living cells, and could discriminate cancer cells (MCF-7) from normal cells (L-02). Therefore, the proposed SPN/dsDNA system provides a powerful tool for intracellular miRNA biosensing, which holds great promise for early cancer diagnosis.
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While chemotherapy is universally recognized as a frontline treatment strategy for breast cancer, it is not always successful; among the leading causes of treatment failure is ...existing and/or acquired multidrug resistance. Cancer stem cells (CSCs), which constitute a minority of the cells of a tumor, are acknowledged to be responsible for increased resistance to chemo-drugs through a combination of increased expression of ATP-binding cassette transporters (ABC transporters), an increased anti-apoptotic defense, and/or the ability for extensive DNA repair like normal stem cells. Consequently, more effective therapy, especially targeted to CSCs, is urgently required. We studied the characteristics of 231-CSCs (CD44+/CD24−) sorted from human MDA-MB-231 breast cancer cells and demonstrated that 231-CSCs exhibited enhanced capacities for proliferation, migration, tumorigenesis and chemotherapy resistance. To address these multifunctional facets of CSCs, we devised a non-ionic surfactant-based vesicle (niosome) co-delivery system to simultaneously deliver siRNAs, targeted to both the ABC transporter (ABCG2) and the anti-apoptosis defense gene (BCL2), and doxorubicin (DOX) to CSCs. The rationale is to sensitize CSCs to DOX by down regulating the drug-resistance gene ABCG2 and simultaneously induce apoptosis by lowering BCL2 expression. The co-delivery system (CDS) successfully delivered siRNAs and DOX to the cytoplasm and nuclei, respectively, and resulted in a down-regulation of ABCG2− and BCL2 mRNAs in CSCs by 60% and 65%, respectively, compared to the control. A corresponding decrease in protein expression was observed using Western blotting. The IC50 of DOX in CSCs concurrently decreased significantly. Our result established CDS as a promising multi-drug delivery platform for cancer treatment.
Cancer stem cells (CSCs) are acknowledged to be responsible for increased resistance to chemo-drugs through a combination of increased expression of ABC transporters, an increased anti-apoptotic defense, and/or the ability for extensive DNA repair like normal stem cells. Consequently, effective therapy, especially to CSCs, is urgently required. In current study, we studied the characteristics of 231-CSCs sorted from human MDA-MB-231 breast cancer cells and found that 231-CSCs possessed enhanced proliferation, migration, tumorigenesis, and DOX resistance. We employed a non-ionic surfactant-based vesicle (niosome) delivery system to simultaneously deliver siRNAs targeted to multi-drug resistance genes, and DOX to kill 231-CSCs. The CDS showed an enhanced therapeutic effect by resensitizing 231-CSCs to DOX and may constitute a promising candidate for cancer chemotherapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Cyclooxygenase type 2 (COX-2) plays a predominant role in the progression of kidney injury in obstructive nephropathy. The aim of this study was to test the efficacy of chitosan/small interfering RNA ...(siRNA) nanoparticles to knockdown COX-2 specifically in macrophages to prevent kidney injury induced by unilateral ureteral obstruction (UUO). Using optical imaging techniques and confocal microscopy, we demonstrated that chitosan/siRNA nanoparticles accumulated in macrophages in the obstructed kidney. Consistent with the imaging data, the obstructed kidney contained a higher amount of siRNA and macrophages. Chitosan-formulated siRNA against COX-2 was evaluated on RAW macrophages demonstrating reduced COX-2 expression and activity after LPS stimulation. Injection of COX-2 chitosan/siRNA nanoparticles in mice subjected to three-day UUO diminished the UUO-induced COX-2 expression. Likewise, macrophages in the obstructed kidney had reduced COX-2 immunoreactivity, and histological examination showed lesser tubular damage in COX-2 siRNA-treated UUO mice. Parenchymal inflammation, assessed by tumor necrosis factor-alpha (TNF-α) and interleukin 6 mRNA expression, was attenuated by COX-2 siRNA. Furthermore, treatment with COX-2 siRNA reduced heme oxygenase-1 and cleaved caspase-3 in UUO mice, indicating lesser oxidative stress and apoptosis. Our results demonstrate a novel strategy to prevent UUO-induced kidney damage by using chitosan/siRNA nanoparticles to knockdown COX-2 specifically in macrophages.
With the construction of the modern power system, power load forecasting is significant to keep the electric Internet of Things in operation. However, it usually needs to collect massive power load ...data on the server and may face the problem of privacy leakage of raw data. Federated learning can enhance the privacy of the raw power load data of clients by frequently transmitting model updates. Concerning the increasing communication burden of resource-heterogeneous clients resulting from frequent communication with the server, a communication-efficient federated learning algorithm based on Compressed Model Updates and Lazy uploAd (CMULA-FL) was proposed to reduce the communication cost. CMULA-FL also integrates the error compensation strategy to improve the model utility. First, the compression operator is used to compress the transmitted model updates, of which large norms are uploaded to reduce the communication cost of each epoch and transmission frequency. Second, by measuring the error of compression and lazy upload, the error is accumulated to the next epoch to improve the model utility. Finally, based on simulation experiments on the benchmark power load data, the results show that the communication cost decreases at least 60% with controlled loss of model prediction compared with baseline.
Ensuring personal safety and preventing accidents are critical aspects of power construction safety supervision. However, current monitoring methods are inefficient and unreliable as most of them ...rely on manual monitoring and transmission, which results in slow detection and delayed warnings regarding violations. To overcome these challenges, we propose an intelligent detection system that can accurately identify instances of illegal wearing of power construction workers in real-time. Firstly, we integrated the squeeze-and-excitation (SE) module into our convolutional neural network to enhance detection accuracy. This module effectively prioritizes informative features while suppressing less relevant ones, resulting in improved overall performance. Secondly, we present an embedded real-time detection system that utilizes Jetson Xavier NX and Edge-YOLO. This system promptly detects and alerts power construction workers of instances of illegal wearing behavior. To ensure a lightweight implementation, we design appropriate detection heads based on target size and distribution, reducing model parameters while enhancing detection speed and minimizing accuracy loss. Additionally, we employed data augmentation to enhance the system’s robustness. Our experimental results demonstrate that our improved Edge-YOLO model achieves high detection precision and recall rates of 0.964 and 0.966, respectively, with a frame rate of 35.36 frames per second when deployed on Jetson Xavier NX. Therefore, Edge-YOLO proves to be an ideal choice for intelligent real-time detection systems, providing superior accuracy and speed performance compared to the original YOLOv5s model and other models in the YOLO series for safety monitoring at construction sites.
RNAi-based strategies provide a great therapeutic potential for treatment of various human diseases including kidney disorders, but face the challenge of in vivo delivery and specific targeting. The ...chitosan delivery system has previously been shown to target siRNA specifically to the kidneys in mice when administered intravenously. Here we confirm by 2D and 3D bioimaging that chitosan formulated siRNA is retained in the kidney for more than 48 hours where it accumulates in proximal tubule epithelial cells (PTECs), a process that was strongly dependent on the molecular weight of chitosan. Chitosan/siRNA nanoparticles, administered to chimeric mice with conditional knockout of the megalin gene, distributed almost exclusively in cells that expressed megalin, implying that the chitosan/siRNA particle uptake was mediated by a megalin-dependent endocytotic pathway. Knockdown of the water channel aquaporin 1 (AQP1) by up to 50% in PTECs was achieved utilizing the systemic i.v. delivery of chitosan/AQP1 siRNA in mice. In conclusion, specific targeting PTECs with the chitosan nanoparticle system may prove to be a useful strategy for knockdown of specific genes in PTECs, and provides a potential therapeutic strategy for treating various kidney diseases.
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