Route Of Uptake Research Articles

Phosphatidylserine-mediated uptake of extracellular vesicles by hepatocytes ameliorates liver ischemia-reperfusion injury.

Compelling evidence suggests that mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) promote regeneration in animal models of liver injury by delivering signaling molecules. However, their target cells and uptake mechanism remain elusive. In this study, MSC-EVs were intravenously administered in a mouse model of liver ischemia-reperfusion injury (IRI). Our results revealed that MSC-EVs exhibit enhanced liver targeting in IRI mice, and injured hepatocytes display a greater capacity for MSC-EV uptake. We found that phosphatidylserine (PS) displayed on the exterior of injured hepatocytes promotes MSC-EV internalization, possibly by binding to MFGE8, a protein expressed on the MSC-EV membrane. Furthermore, the therapeutic effect of MSC-EVs on liver IRI is highly dependent on this PS-mediated uptake pathway. Our findings provide evidence that MSC-EVs preferentially target injured hepatocytes, relying on a PS-dependent uptake route to exert hepatoprotective effects, which are critical for the future design of EV-based therapeutic strategies for liver IRI.

Swine wastewater co-exposed with veterinary antibiotics enhanced the antibiotic resistance of endophytes in radish (Raphanus sativus L.)

The widespread utilization of antibiotics in livestock has promoted the accumulation and diffusion of antibiotics and antibiotic resistance in agricultural soils and crops. Here we investigated the mechanisms of antibiotic uptake and accumulation in swine wastewater (SW)-treated radish (Raphanus sativus L.) and subsequent impacts on endophyte antibiotic resistance. Under SW treatments, exposure to 500 μg/L sulfamethazine (SMZ) and enrofloxacin (EFX) significantly affected radish biomass, with SMZ causing 63.0% increases and EFX causing 36.3% decreases relative to the untreated control. EFX uptake by radish were from 5 to 100-folds over SMZ. Passive diffusion through anion channel proteins on cell membranes was an important route for SMZ uptake, while both passive diffusion and energy-dependent processes contributed to the uptake of EFX. Bacterial community was time-dependent as a function of both antibiotics and SW, the bacterial alpha diversity in liquid solution co-treated with antibiotics and SW increased over time. The abundance of antibiotic resistance genes (ARGs) in the roots was positively correlated with ARGs in the Hoagland's solution under antibiotic-alone treatments. EFX co-exposure with SW enhanced the dissemination of ARGs from swine wastewater into plant roots, and significant correlations existed between ARGs and integrons in both Hoagland's solution and roots. These findings increased our understanding of the fate of antibiotics in crops and their subsequent impacts on antibiotic resistance of endophytic bacteria.

Natural 14C abundances and stable isotopes suggest discrete uptake routes for carbon and nitrogen in cold seep animals

Cold seeps, where geofluids containing methane and other hydrocarbons originating from the subseafloor seeps through the sediment surface, play important roles in the elemental and energy flux between sediment and seawater. These seep sites often harbor communities of endemic animals supported by chemolithoautotrophic bacteria, either through symbiosis or feeding. Despite these animal communities being intensively studied since their discovery in the 1980’s, the contribution of carbon from seep fluid to symbiotic microbes and subsequently host animals remains unclear. Here, we used natural-abundance radiocarbon to discern carbon sources: the ambient bottom water or the seeping geofluid. The 14C concentrations were measured for vesicomyid clams, a parasitic calamyzine polychaete, and a siboglinid tubeworm species from four different cold seep sites around Japan. We found most vesicomyid clams exhibiting 14C concentrations slightly lower than that of the ambient bottom water, suggesting up to 9% of C for chemolithoautotrophy originates from geofluid DIC. The different extent of fluid contribution across species may be explained by different routes to incorporate DIC and/or different DIC concentrations in the geofluid at each seep site. Stable nitrogen isotopic compositions further suggested N incorporation from geofluids in these clams, where the burrowing depth may be a key factor in determining their δ15N values. The siboglinid tubeworm showed a clear dependency for geofluid DIC, with a contribution of > 40%. Our results demonstrate the effectiveness of 14C analyses for elucidating the nutritional ecology of cold seep animals and their symbionts, as was previously shown for hydrothermal vent ecosystems.

PEGylation-Dependent Cell Uptake of Lipid Nanoparticles Revealed by Spatiotemporal Correlation Spectroscopy.

Polyethylene glycol (PEG) is a common surface modification for lipid nanoparticles (LNPs) to improve their stability and in vivo circulation time. However, the impact of PEGylation on LNP cellular uptake remains poorly understood. To tackle this issue, we systematically compared plain and PEGylated LNPs by combining dynamic light scattering, electrophoretic light scattering, and synchrotron small-angle X-ray scattering (SAXS) that unveils a striking similarity in size and core structure but a significant reduction in surface charge. Upon administration to human embryonic kidney (HEK 293) cells, plain and PEGylated LNPs were internalized through different endocytic routes, as revealed by spatiotemporal correlation spectroscopy. An imaging-derived mean square displacement (iMSD) analysis shows that PEGylated LNPs exhibit a significantly stronger preference for caveolae-mediated endocytosis (CAV) and clathrin-mediated endocytosis (CME) pathways compared to plain LNPs, with these latter being better tailored to MCR-dependent internalization and trafficking. This suggests that PEG plays a crucial role in directing LNPs toward specific cellular uptake routes. Further studies should explore how PEG-mediated endocytosis impacts intracellular trafficking and ultimately translates to therapeutic efficacy, guiding the design of next-generation LNP delivery systems.

A baseline assessment of contamination in the Sacramento deep water ship channel

The Sacramento Deep Water Ship Channel (SDWSC) in the San Francisco Estuary, which is an active commercial port, is critical habitat for pelagic fish species including delta smelt (Hypomesus transpacificus), longfin smelt (Spirinchus thaleichthys), and Sacramento perch (Archoplites interruptus). Pelagic organism decline has been attributed to covarying factors such as manipulation of habitat, introduction of invasive species, decrease in food production, and contaminant exposure. Quantification of bioavailable toxicant loads in the SDWSC is limited despite previous surveys that have detected elevated contaminant concentrations in the sediments. Therefore, the focus of the present study was to characterize the bioavailability of the contaminants in the SDWSC from six sites along the channel. At each site, organochlorine pesticides (OCPs), pyrethroid insecticides, polyaromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs) were quantified in sediment, zooplankton, and suspended solids. In addition, Tenax extraction was used to measure the bioaccessible fraction of sediment-associated contaminants freely dissolved in the water. Bioaccessible contaminants in the sediment provided an uptake route for these stressors into invertebrates and fish with bioaccessible OCPs being found at all sites, particularly 4,4′-dichlorodiphenyldichloroethylene (DDE). Bifenthrin was the only pyrethroid detected in the chosen matrices and it was found at concentrations below levels of concern. Bioaccessible PAHs were found at all sites, with highest detections for phenanthrene and pyrene. No PCBs were detected in sediments, but were detected in both suspended solids and zooplankton. Contaminant concentrations overall were significantly higher in suspended solids, followed by zooplankton and sediments. The highest sediment concentrations of DDE, fluoranthene, pyrene, and dibenzo[a,h]anthracene exceeded sediment quality benchmarks indicating potential risk to sediment-dwelling species. Finally, elevated contaminant levels were found in both suspended solids and zooplankton, suggesting additional risk to pelagic species in the SDWSC.

Parameterization of Physiologically Based Biopharmaceutics Models: Workshop Summary Report.

This Article shares the proceedings from the August 29th, 2023 (day 1) workshop "Physiologically Based Biopharmaceutics Modeling (PBBM) Best Practices for Drug Product Quality: Regulatory and Industry Perspectives". The focus of the day was on model parametrization; regulatory authorities from Canada, the USA, Sweden, Belgium, and Norway presented their views on PBBM case studies submitted by industry members of the IQ consortium. The presentations shared key questions raised by regulators during the mock exercise, regarding the PBBM input parameters and their justification. These presentations also shed light on the regulatory assessment processes, content, and format requirements for future PBBM regulatory submissions. In addition, the day 1 breakout presentations and discussions gave the opportunity to share best practices around key questions faced by scientists when parametrizing PBBMs. Key questions included measurement and integration of drug substance solubility for crystalline vs amorphous drugs; impact of excipients on apparent drug solubility/supersaturation; modeling of acid-base reactions at the surface of the dissolving drug; choice of dissolution methods according to the formulation and drug properties with a view to predict the in vivo performance; mechanistic modeling of in vitro product dissolution data to predict in vivo dissolution for various patient populations/species; best practices for characterization of drug precipitation from simple or complex formulations and integration of the data in PBBM; incorporation of drug permeability into PBBM for various routes of uptake and prediction of permeability along the GI tract.

The physiological basis of renal nuclear medicine.

Renal physiology underpins renal nuclear medicine, both academic and clinical. Clearance, an important concept in renal physiology, comprises tissue uptake rate of tracer (tissue clearance), disappearance rate from plasma (plasma clearance), appearance rate in urine (urinary clearance) and disappearance rate from tissue. In clinical research, steady-state plasma clearances of para-amino-hippurate and inulin have been widely used to measure renal blood flow (RBF) and glomerular filtration rate (GFR), respectively. Routinely, GFR is measured at non-steady state as plasma clearance of a filtration agent, such as technetium-99m diethylenetriaminepentaacetic acid. Scaled to three-dimensional whole body metrics rather than body surface area, GFR in women is higher than in men but declines faster with age. Age-related decline is predominantly from nephron loss. Tubular function determines parenchymal transit time, which is important in renography, and the route of uptake of technetium-99m dimercaptosuccinic acid, which is via filtration. Resistance to flow is defined according to the pressure-flow relationship but in renography, only transit time can be measured, which, being equal to urine flow divided by collecting system volume, introduces further uncertainty because the volume is also unmeasurable. Tubuloglomerular feedback governs RBF and GFR, is regulated by the macula densa, mediated by adenosine and renin, and can be manipulated with proximal tubular sodium-glucose cotransporter-2 inhibitors. Other determinants of renal haemodynamics include prostaglandins, nitric oxide and dopamine, while protein meal and amino acid infusion are used to measure renal functional reserve. In conclusion, for measuring renal responses to exogenous agents, steady-state para-amino-hippurate and inulin clearances should be replaced with rubidium-82 and gallium-68 EDTA for measuring RBF and GFR.

Cadmium bioaccumulation dynamics during amphibian development and metamorphosis

Cadmium pollution poses a significant threat to aquatic ecosystems due to its propensity to bioaccumulate and cause toxicity. This study assessed the complex dynamics of cadmium uptake, accumulation and distribution across anuran development to provide new insights into the fate of cadmium burdens during metamorphosis and compare the susceptibility of different life stages to cadmium accumulation. Tadpoles of various developmental stages were exposed to dissolved 109-cadmium and depurated in clean water in a series of experiments. Temporal changes in whole-body and tissue concentrations were analysed using gamma spectroscopy, and anatomical distributions were visualised using autoradiography. Results showed that animals exposed at the onset of metamorphic climax (forelimb emergence) retained significantly less cadmium than animals exposed through larval stages. After exposure, cadmium partitioned predominantly in the skin, gills and remains of metamorphs, whereas larvae accumulated cadmium predominately through their gut. This shows a shift in the primary route of uptake at the onset of climax, which relates to the structural and functional changes of uptake sites through metamorphosis. During climax, some cadmium was redistributed in tissues developing de novo, such as the forelimbs, and concentrated in the regressing tail. Our findings highlight the need for stage-specific considerations in assessing exposure risks.

Engineered microbubbles decorated with red emitting carbon nanoparticles for efficient delivery and imaging

Altering the route of uptake by the cells is an attractive strategy to overcome drug-receptor adaptation problems. Carbon nanoparticles (CNPs) with emission beyond tissue autofluorescence for imaging biological tissues were used to study the phenomenon of uptake by the cells. In this regard, red-emitting carbon nanoparticles (CNPs) were synthesized and incorporated onto lipid microbubbles (MBs). The CNPs showed red emissions in the range of 640 nm upon excitation with 480 nm wavelength of light. Atomic force microscopic and confocal microscopic images showed the successful loading of CNPs onto the MB. Carbon nanoparticle loaded microbubbles (CNP-MBs) were treated with NIH 3 T3 cells at different concentrations. Confocal microscopic imaging studies confirm the presence of CNPs inside the treated cells. Cytotoxicity studies revealed that the CNPs showed minimal toxicity towards cells after loading onto MBs. The CNPs are usually taken up by the cells through the clathrin-mediated (CME) pathway, but when loaded onto MBs, the mechanism of uptake of CNPs is altered, and the uptake by the cells was observed even in the presence of inhibitors for the CME pathway. Loading CNPs onto MBs resulted in the uptake of CNPs by the cell through micropinocytosis and sonophoresis in the presence of ultrasound. The in vivo uptake CNP-MBs were performed in Danio rerio (Zebrafish larvae). This study provides insights into altering the uptake pathway through reformulation by loading nanoparticles onto MBs.

Abstract 1126: Extracellular vesicle mediated radioresistance in H3K27M-diffuse midline glioma

Abstract H3K27M-Diffuse Midline Gliomas (DMGs) are a subset of uncurable malignant pediatric gliomas and the deadliest form of brain tumors in children worldwide. Radiation therapy, the current standard of care, is initially effective but all tumors become resistant within 4-6 months. H3K27M-DMGs are heterogenous, comprised of an average of 6 genetically distinct subclones. Some subclones harbor mutations in genes previously linked to radiation resistance, such as TP53. We have found subclones are also functionally distinct, with some clones harboring intrinsic radiation resistance. Extracellular signaling between clones is linked with therapy resistance and tumor progression in other tumors, and we hypothesized that radioresistant H3K27M-DMG release small extracellular vesicles (sEVs) that impact the radiosensitivity of neighboring tumor cells. We found that all patient-derived H3K27M-DMG samples examined (n=6) produced sEVs (< 220nm), indicating this is a common feature in this tumor type. We isolated sEVs from a radioresistant (RR) patient-derived H3K27M-DMG cell line and used them to treat radiosensitive (RS) DMG cells. Using flow cytometry to profile sEV uptake dynamics, we found that RR-sEVs are internalized by RS cells within 2 hours of exposure and are retained at 18 hours (p<0.001, compared to control). This uptake can be significantly abrogated by proteinase K treatment of the sEVs, (p<0.001), indicating that uptake involves sEV surface proteins. We then applied a biosensor approach to assess single-cell phenotypic changes in RS DMG after RR-sEV treatment on radiation-induced cell death, DNA damage, and cell senescence, three dynamic processes. Using a genetically encoded death indicator (GEDI), we found that RR-sEVs significantly enhanced the survival of RS cells after acute radiation exposure (p<0.001). This was due to enhanced DNA damage repair as measured by 53BP1 foci (p<0.01). Small RNA sequencing of sEV cargo demonstrated that RR-sEVs express multiple miRNAs that are involved in oncogenic signaling such as the miR-7 family, miR-1246, and miR-1290 at several log fold higher than RS-sEVs. Overall, this data reveals the first functional role for sEVs in the context of radiation induced tumor transformation in DMG. We plan to further investigate the primary route of sEV uptake and define the mechanism through which sEV cargo alters radiosensitivity. This study provides new insight into the impact of heterogeneity and sEVs in mediating radioresistance in H3K27M-DMG and may lead to the development of novel radiosensitizers, which are critically needed for all patients. Citation Format: Viral Oza, Shilpa Sampathi, Yelena Chernyavskava, Kenan Flores, Jessica Blackburn. Extracellular vesicle mediated radioresistance in H3K27M-diffuse midline glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1126.

Determination of Residual Presence of Toxic Heavy Metal in the Liver of LABEO BATA (HAMILTON, 1822) Using Rain Water Harvest System

Abstract: Fish are special among vertebrates because they may absorb metal from the water through their gills and from their food through their guts (direct and trophic absorption methods). Since the fish's gills are the primary organ targeted by metal toxicity, the direct uptake route is more significant. Most research on the toxicity and accumulation of metals in fish focuses on individual metals. But in the natural world, fish are exposed to mixes of many metals, whose toxicity levels typically differ from those of individual metals because of their additive, sometimes less than additive (antagonistic) effects, or higher than additive (synergistic) effects. This article discusses the build-up of heavy metals in fish organs and tissues, particularly in relation to the intake of water-borne metals via the trophic pathway. The trophic route is important because it is related with “Biological Magnification”. The fish we eat, usually comes from rivers or pond or bheri, in Kolkata. These are also contaminated with heavy metal toxicity. The rain water is contamination free from heavy metal. We collected live fish from market and kept them in “Rain Water Harvest System” for 24 hours. The accumulated heavy metal was expected to be eliminated from fish liver within 24 hours through the process of detoxification. We found that, even after 24 hours, fish liver contain certain heavy metal as residue. The model fish was Labeo bata (Hamilton,1822). Far more studies on metal interactions should be performed to reach the level of predictability

Development and validation of an analytical pyrolysis method for detection of airborne polystyrene nanoparticles

Microplastic is ubiquitous in the environment. Recently it was discovered that microplastic (MP, 1 μm−5 mm) contamination is present in the atmosphere where it can be transported over long distances and introduced to remote pristine environments. Sources, concentration levels, and transportation pathways of MP are still associated with large uncertainties. The abundance of atmospheric MP increases with decreasing particle size, suggesting that nanoplastics (NP, <1μm) could be of considerable atmospheric relevance. Only few analytical methods are available for detection of nanosized plastic particles. Thermoanalytical techniques are independent of particle size and are thus a powerful tool for MP and NP analysis. Here we develop a method for analysis of polystyrene on the nanogram scale using pyrolysis gas chromatography coupled to mass spectrometry. Pyrolysis was performed using a slow temperature ramp, and analytes were cryofocused prior to injection. The mass spectrometer was operated in selected ion monitoring (SIM) mode. A lower limit of detection of 1±1 ng and a lower limit of quantification of 2±2 ng were obtained (for the trimer peak). The method was validated with urban ▪ matrices of low (7 μg per sample) and high (53 μg per sample) aerosol mass loadings. The method performs well for low ▪ loadings, whereas high ▪ loadings seem to cause a matrix effect reducing the signal of polystyrene. This effect can be minimized by introducing a thermal desorption step prior to pyrolysis. The study provides a novel analysis method for qualitative and semi-quantitative analysis of PS on the nanogram scale in an aerosol matrix. Application of the method can be used to obtain concentration levels of polystyrene in atmospheric MP and NP. This is important in order to improve the understanding of the sources and sinks of MP and NP in the environment and thereby identify routes of exposure and uptake of this emerging contaminant.

Understanding the Role of Scavenger Receptor A1 in Nanoparticle Uptake by Murine Macrophages.

Nanoparticles can be cleared from the circulation and taken up by tissue-resident macrophages. This property can be beneficial when drug or antigen delivery to macrophages is desired; however, rapid clearance of nanoparticles not intended for delivery to immune cells may reduce nanoparticle circulation time and affect the efficacy of nanoparticle-formulated drug products. Therefore, understanding nanoparticles' uptake by macrophages is an essential step in the preclinical development of nanotechnology-based drug products. Understanding the route of nanoparticle uptake by macrophages may also provide mechanistic insights into the immunotoxicity of nanomaterials. The protocol described herein can be used to assess the nanoparticles' uptake by macrophages and understand the involvement of scavenger receptor A1 to inform mechanistic studies.

Mechanistic characterization of waterborne selenite uptake in the water flea, Daphnia magna, indicates water chemistry affects toxicity in coal mine-impacted waters.

Concentrations of selenium that exceed regulatory guidelines have been associated with coal mining activities and have been linked to detrimental effects on aquatic ecosystems and the organisms therein. Although the major route of selenium uptake in macroinvertebrates is via the diet, the uptake of waterborne selenite (HSeO3-), the prominent form at circumneutral pH, can be an important contributor to selenium body burden and thus selenium toxicity. In the current study, radiolabelled selenite (Se75) was used to characterize the mechanism of selenite uptake in the water flea, Daphnia magna. The concentration dependence (1-32μM) of selenite uptake was determined in 1-hour uptake assays in artificial waters that independently varied in bicarbonate, chloride, sulphate, phosphate and selenate concentrations. At concentrations representative of those found in highly contaminated waters, selenite uptake was phosphate-dependent and inhibited by foscarnet, a phosphate transport inhibitor. At higher concentrations, selenite uptake was dependent on waterborne bicarbonate concentration and inhibited by the bicarbonate transporter inhibitor DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid). These findings suggest that concentrations of phosphate in coal mining-affected waters could alter selenite uptake in aquatic organisms and could ultimately affect the toxic impacts of selenium in such waters.

Nanocarrier foliar uptake pathways affect delivery of active agents and plant physiological response.

Layered double hydroxide (LDH) nanoparticles enable foliar delivery of genetic material, herbicides, and nutrients to promote plant growth and yield. Understanding the foliar uptake route of nanoparticles is needed to maximize their effectiveness and avoid unwanted negative effects. In this study, we investigated how delivering layered double hydroxide (d = 37 ± 1.5 nm) through the adaxial (upper) or abaxial (lower) side of leaves affects particle uptake, nutrient delivery, and photosynthesis in tomato plants. LDH applied on the adaxial side was embedded in the cuticle and accumulated at the anticlinal pegs between epidermal cells. On the abaxial side, LDH particles penetrated the cuticle less, but the presence of the stomata enables penetration to deeper leaf layers. Accordingly, the average penetration levels of LDH relative to the cuticle were 2.47 ± 0.07, 1.25 ± 0.13, and 0.75 ± 0.1 μm for adaxial, abaxial with stomata, and abaxial without stomata leaf segments, respectively. In addition, the colocalization of LDH with the cuticle was ∼2.3 times lower for the adaxial application, indicating the ability to penetrate the cuticle. Despite the low adaxial stomata density, LDH-mediated delivery of magnesium (Mg) from leaves to roots was 46% higher for the adaxial than abaxial application. In addition, adaxial application leads to ∼24% higher leaf CO2 assimilation rate and higher biomass accumulation. The lower efficiency from the abaxial side was, at least partially, a result of interference with the stomata functionality which reduced stomatal conductance and evapotranspiration by 28% and 25%, respectively, limiting plant photosynthesis. This study elucidates how foliar delivery pathways through different sides of the leaves affect their ability to deliver active agents into plants and consequently affect the plants' physiological response. That knowledge enables a more efficient use of nanocarriers for agricultural applications.

Ingestion and translocation of microplastics in tissues of deposit-feeding crabs (Grapsoidea, Ocypodoidea) in Kochi estuary, Japan

Estuaries contain some of the highest concentrations of accumulated microplastics (MPs) that can be ingested by abundant deposit-feeding crabs. We investigate MPs in gill, hepatopancreatic, and gastrointestinal tissues of seven intertidal crab species in Kokubu River, Kochi, Japan. By applying a reliable method that considers limits of detection and quantification, we report MPs in 63 of 116 crabs (>50%), with a mean of 3.2 MPs individual−1. Concentrations are greatest in gastrointestinal tracts (62.15%), suggesting that feeding is the main route for MP uptake. PET is the dominant polymer (44%), and fragments are the dominant shape (50%–77%). A greater MPs burden g−1 body weight is reported for deposit-feeding small ocypodid crabs than for larger herbivorous/omnivorous grapsoid crabs. Factors possibly influencing MP uptake by crabs include feeding habit, crab size, and ambient MP composition.

Efficient drug supply in stem cell cytosol via pore-forming saponin nanoparticles promotes in vivo osteogenesis and bone regeneration

Directional differentiation of stem cells is a key step in stem cell therapy. In this study, we developed saponin-based nanoparticles (Ad-SNPs) containing dexamethasone (Dex) and alpha-lipoic acid (ALA) to promote osteogenic differentiation of human mesenchymal stem cells (hMSCs) and bone regeneration. The Ad-SNPs can achieve rapid cellular uptake through a pore-forming effect without cytotoxic cationic charges. They also provide extended retention in cell cytosol due to their uptake route. These properties are advantageous in efficiently supplying drugs to the hMSCs. The combination of Dex and ALA facilitated mitochondrial fusion and prevented reactive oxygen species–induced DNA damage. It also helped to preserve mitochondrial dynamics, and the efficient supply of it provided by the Ad-SNPs induced differentiation of hMSCs into osteoblasts. The Ad-SNPs showed outstanding performance in osteoblast differentiation, maturation, and mineralization in 3D culture compared with NPs without saponin and with free drugs. When Ad-SNP-treated hMSCs were tested in a rat femoral bone defect model, they showed the fastest regeneration of bones and complete repair in the shortest period among all groups. To the best of our knowledge, this study is the first application of pore-forming saponin-based NPs with rapid cellular uptake and extended retention to stem cell therapy, and we demonstrated their promising potential in bone regeneration and efficient delivery of Dex and ALA.

Chondroitin sulfate modified calcium phosphate nanoparticles for efficient transfection via caveolin-mediated endocytosis

Efficient transfection remains a challenge for gene delivery in both cell biological scientific research and gene therapeutic fields. Existing transfection strategies rarely pay attention to altering the endocytosis pathway of nanocarriers for transfection efficiency improvement. In this work, we innovatively postulated that calcium phosphate nanoparticles coated with glycosaminoglycan could be internalized by cells mainly through caveolin-mediated endocytosis pathway allowing genes to bypass lysosome route, and hence enhance the transfection efficiency. To achieve this, we developed calcium phosphate nanoparticles (CP-ALN-CS) coated with chondroitin sulfate (CS) and alendronate (ALN) in a modular manner. The CP-ALN-CS had a hydrodynamic size of 131.0 ± 8.7 nm and exhibited favorable dispersity, stability, and resistance to nuclease degradation. Unlike conventional calcium phosphate and PEI-based transfection, CP-ALN-CS exhibited efficient cellular uptake with co-localization in Golgi apparatus and endoplasmic reticulum. Through bypassing the lysosome involved cellular uptake route, CP-ALN-CS can effectively protect genes from degradation and relieve cytotoxicity. After loading plasmid DNA, CP-ALN-CS showed extraordinary transfection efficiency in HEK 293T cells, outperforming the PEI which is considered as the gold standard. The current work provides a novel and facile approach to improve gene transfection efficiency and is valuable for the design of next-generation in vitro transfection reagents.

Peptide‐Modified AIEgen Probe for Selective Imaging and Killing of Drug‐Resistant Cancer Cells Based on Cell‐Cycle‐Dependent Uptake

AbstractDistinguishing the precise phases of cells in the cell cycle is important for elucidation of their regulatory mechanisms and exploiting cell‐cycle‐targeted drugs. Here, a simple but efficient strategy is established based on a peptide‐modified aggregation‐induced emission luminogen (AIEgen) probe PKK–TTP by utilizing its differential cellular uptake amounts for specific cell cycle recognition and targeted therapy. After incubating the probe in cells with different phases, maximal fluorescence and reactive oxygen species (ROS) generation are observed in S phase, middle in G0/G1 phase, and the minimum in G2/M phase. The cell‐cycle‐dependent fluorescent intensity is proven to be related to variable uptake routes of PKK–TTP. The maximized uptake of PKK–TTP in S phase relies on three simultaneously working endocytic routes, including macro‐pinocytosis and clathrin‐ and caveolin‐dependent endocytosis, while decreased uptake in the G0/G1 and G2/M phases mainly depends on the single route. Furthermore, the uptake of PKK–TTP is further verified in normal cells and drug‐resistant cancer cells (S). Bright fluorescence of PKK–TTP is observed in drug‐resistant cancer cells in S phase, while a little fluorescence is observed in paclitaxel (PTX)‐treated cancer cells due to G2/M phase. Collectively, PKK–TTP can be applied to distinguish cancer cells, evaluating the drug resistance and exerting its therapeutic efficiency downstream.

Arsenic toxicity to earthworms in soils of historical As mining sites: an assessment based on various endpoints and chemical extractions

Eisenia fetida is an earthworm species often used to assess the toxicity of contaminants in soils. Several studies indicated that its response can be unpredictable because it depends both on total concentrations of contaminants and also on their forms that differ in susceptibility to be released from soil solid phase. The issue is complex because two various uptake routes are concurrently involved, dermal and ingestion in guts, where the bioavailability of contaminants can considerably change. The aim of this study was to analyze the toxicity of arsenic (As) in various strongly contaminated meadow and forest soils, representative for former As mining and processing area, to earthworms E. fetida and its accumulation in their bodies. An attempt was made to find relationships between the response of earthworms and chemical extractability of As. In the bioassay, carried out according to the standard ISO protocol, different endpoints were applied: earthworm survival, fecundity measured by the numbers of juveniles and cocoons, earthworm weight and As accumulation in the bodies. The results proved that E. fetida can tolerate extremely high total As concentrations in soils, such as 8000 mg/kg, however, the individual endpoints were not correlated and showed different patterns. The most sensitive one was the number of juveniles. No particular soil factor was identified that would indicate an exceptionally high As susceptibility to the release from one of soils, however, we have demonstrated that the sum of non-specifically and specifically bound As (i.e. fractions F1 + F2 in sequential extraction according to Wenzel) could be a good chemical indicator of arsenic toxicity to soil invertebrates.