C. albicans ureohydrolases arginase (Car1), agmatinase (Agt1), and guanidinobutyrase (Gbu1) can orchestrate an arginase-independent route for polyamine production and that this is important for C. albicans growth and survival in microenvironments of the mammalian host.We show that the  l-arginine, agmatine, and guanidinobutyrate (GB), and these enzymes generate ornithine (by arginase), putrescine (by agmatinase), or GABA (by 4-guanidinobutyrase or GBase). Candida albicans can metabolize and grow on arginine, agmatine, or guanidinobutyrate as the sole nitrogen source. Three related C. albicans genes whose sequences suggested that they were putative arginase or arginase-like genes were examined for their role in these metabolic pathways. Of these, Car1 encoded the only bona fide arginase, whereas we provide evidence that the other two open reading frames, orf19.5862 and orf19.3418, encode agmatinase and guanidinobutyrase (Gbase), respectively. Analysis of strains with single and multiple mutations suggested the presence of arginase-dependent and arginase-independent routes for polyamine production. CAR1 played a role in hyphal morphogenesis in response to arginine, and the virulence of a triple mutant was reduced in both Galleria mellonella and Mus musculus infection models. In the bloodstream, arginine is an essential amino acid that is required by phagocytes to synthesize nitric oxide (NO). However, none of the single or multiple mutants affected host NO production, suggesting that they did not influence the oxidative burst of phagocytes.Amino acid metabolism is crucial for fungal growth and development. Ureohydrolases produce amines when acting on  Mostly characterized in bacteria  encoding a cytosolic arginase, CAR1 (EC 3.5.3.1), and two uncharacterized open reading frames (ORFs), orf19.3418 and orf19.5862, which were predicted to encode secreted proteins that display similarity to arginases  and guanidinobutyrase (Gbu1), respectively. This arginase-like gene family therefore contributes a set of ureohydrolases required for arginine and agmatine utilization. We demonstrate that this gene family is important for virulence in two independent animal infection models. Together, they play a key role in enabling C. albicans growth and survival in microenvironments of the mammalian host.The rapid increase in the use of whole-genome sequencing has enabled identification of putative arginase-like enzymes in many eukaryotes. However, gene identification of these enzymes, based exclusively on sequence homology, does not enable definitive predictions to be made with respect to the catalytic function of the encoded proteins. The approach  and the approach . We iden10.1128/mBio.01845-20.1FIG S1C. albicans genome reveals two ORFs (orf19.5862 and orf19.3418) that show similarity to the annotated arginase gene CAR1. Data represent protein alignment of sequences (Agt1 and Gbu1) found via BLASTp analysis of C. albicans Car1 (A) and percentages of protein similarity (B), showing residues that are fully conserved (*), conservation between groups of strongly similar properties (:), or conservation between groups of weakly similar properties (.). The images were created by the use of Clustal2.1. Comparative genomics analyses resulted in a phylogenetic tree constructed using fungal 18S species from Yarza et al.  (C) that shows the presence (green) or absence (red) of orthologues in fungal species. Three species show an absence (loss or lack of a gain) of the two newly identified ORFs. P. jirovecii lacks orthologues of all these genes. For gene identifier (ID), length, E value, and bit score data for the orthologues, see FIG S1, TIF file, 0.2 MB.The Copyright © 2020 Schaefer et al.2020Schaefer et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Candida albicans, one that is annotated as an arginase (Car1) and two products of ORFs (orf19.5862 and orf19.3418) with similarity to arginase enzymes 2SO4 as the nitrogen source served as positive-growth controls.We first assessed whether orf19.5862 and orf19.3418 are required for the utilization of arginine and agmatine and their derivatives  or act i10.1128/mBio.01845-20.2FIG S2C. albicans ureohydrolase mutants generated by the mini-URA blaster gene disruption system on media containing intermediate products of the pathway as a sole nitrogen source. Single, double, and triple mutants of C. albicans orf19.3934 (CAR1), orf19.5862 (AGT1), and orf19.3418 (GBU1) were grown on a range of nitrogen sources relevant to the arginine utilization pathway. Red squares highlight reduced growth of mutant strains, confirming the mutant phenotype generated by the CRISP-Cas9 system shown in FIG S2, TIF file, 0.3 MB.Spot assay of Copyright © 2020 Schaefer et al.2020Schaefer et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans wild-type strains were grown on nitrogen-free minimal medium supplemented with (NH4)2SO4, ornithine, urea, arginine, agmatine, 4-guanidinobutyrate (GB), 4-aminobutyric acid (GABA), and putrescine and were shown to be able to utilize all of these as a nitrogen source. All mutant strains were able to grow equally well on ornithine, urea, and GABA  were all affected in growth on minimal medium containing arginine as the sole nitrogen source. These mutants, however, were not affected with respect to their ability to grow on medium containing urea or ornithine, the two products of the arginase reaction. None of the other mutants were affected in growth on arginine, with the single and double mutants of GBU1 and AGT1 (orf19.3418 and orf19.5862) demonstrating clear growth  or CAR1 exhibited wild-type growth on both (GBU1) also displayed a growth defect on GB but not GABA. This suggests that orf19.3418 (GBU1) is likely to encode a GBase, required for the conversion of GB to GABA.The  on both . FurtherAGT1) could utilize all nitrogen sources tested; however, the double mutant lacking both orf19.5862 and orf19.3418 (gbu1Δ agt1Δ) and the triple mutant (gbu1Δ agt1Δ car1Δ) were unable to grow on media containing agmatine as the sole nitrogen source (GBU1 (orf19.3418) and which has recently been reported in A. niger  encoding an agmatinase. In addition, these findings may exclude the possibility of there being an additional alternative route for putrescene biosynthesis, for example, the agmatine deiminase system that converts agmatine into putrescine via N-carbamoyl putrescine in a number of bacterial species  mutants showed any change in arginase activity  were previously shown to be induced strongly in macrophages  and limiting the synthesis of the antimicrobial nitrite oxide. When macrophages were stimulated with wild-type C. albicans, nitrite oxide production was reduced; however, no statistically significant differences in NO reduction were observed in coculturing performed with any of the gene knockout mutants  impacted macrophage arginase activity following their engulfment. Using two methods of cell breakage to release arginase, we prepared either macrophage-derived or total  protein extracts. Increased macrophage arginase activity was seen under conditions of stimulation with wild-type C. albicans cells  pathway , which functions downstream of arginase and hydrolyses urea to NH3 and CO2, has been found to be defective in arginine-induced hypha formation , 0.004% glucose, and a 10 mM concentration of agmatine or GB or GABA. Representative microscope images taken after 150 min of incubation at 37°C are shown. The scale bar is 10 μm. Download FIG S3, TIF file, 0.5 MB.Copyright © 2020 Schaefer et al.2020Schaefer et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicansAGT1, GBU1, and CAR1 are required for virulence, in both the Galleria mellonella and Mus musculus infection models  was detected for the triple mutant (gbu1Δ agt1Δ car1Δ) and the animals demonstrated significantly reduced weight loss relative to controls (P < 0.01), resulting overall in a significantly reduced outcome score (P < 0.01). We have therefore demonstrated that AGT1, GBU1, and CAR1 together contributed to the virulence of C. albicans in both wax moth and mouse infection models.A range of mutants was also tested in a 3-day mouse model of systemic infection . For thie mutant . However10.1128/mBio.01845-20.4FIG S4C. albicans of ureohydrolases is required for virulence in a mouse model of systemic infection. Data represent levels of virulence of the wild-type strain or car1Δ single, gbu1Δ agt1Δ double, and gbu1Δ agt1Δ car1Δ triple mutant and reintegrant strains in a mouse 3 day model of systemic infection. Mice were infected with 3.2 × 104 to 3.4 × 104 CFU/g of the gbu1Δ agt1Δ double mutant strain and associated control strains and with 7.4 × 104 to 7.9 × 104 CFU/g of the car1Δ single and gbu1Δ agt1Δ car1Δ triple mutant strains and associated controls through lateral tail vein infection. Mice were then sacrificed after three days, and kidney burden (log10 CFU/ml), weight loss (%), and outcome score (based on both kidney burden and weight loss) were calculated . Download FIG S4, TIF file, 0.1 MB.A set of Copyright © 2020 Schaefer et al.2020Schaefer et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Candida-host interaction. Previously, we had shown that macrophages that engulf C. albicans, or fungal cell wall chitin, induce host arginase activity . However, these also display sequence similarity to other arginase-like encoding genes, including agmatinase genes; as such, it was necessary to establish what these gene products were and what role they have in C. albicans development and host interaction. We found that none of these enzymes had an impact on host NO production, either directly via substrate depletion or indirectly through host arginase induction or host iNOS repression. Through nitrogen assimilation profiling of mutants with mutations in these genes, we found that only CAR1 encodes a bona fide arginase in C. albicans. We propose here that the other two open reading frames, orf19.5862 and orf19.3418, encode agmatinase (Agt1) and guanidinobutyrase (Gbu1), respectively, based on the ability of our set of mutants to utilize specific nitrogen substrates. While arginase metabolism and arginine metabolism have been studied in a range of fungal species  and it was shown to have an impact on F-actin dynamics and to play an essential role in cell development  in C. albicans, which we describe here as acting as a GBase gene.We show that GB could be used as the sole nitrogen source by queforti  A. nigerA. niger , and K. revisiae . Indeed,revisiae , althougequenced . In K. lC. albicans can utilize agmatine as a sole nitrogen source via two independent pathways . Although the physiological significance of these pathways is not clear, they may provide potential growth advantages under conditions in which C. albicans encounters agmatine as a source of carbon and nitrogen. In fungi that have all three enzymes, agmatinase and guanidinobutyrase are predicted to be secreted enzymes whereas arginase is cytoplasmic. It may be that Car1 is required for central intracellular metabolism, whereas the others may function to scavenge carbon and nitrogen from the environment by breaking down complex nutrients such as agmatine. Alternatively, uptake of agmatine and GB may be limited, and their utilization would consequently be dependent on initial extracellular degradation.We show that pathways . First, −1 wet weight), small intestine (55 ng g−1), and large intestine (28 ng g−1) have the highest agmatine tissue concentrations  and converted by arginine decarboxylase to agmatine, which is then exported to the medium  are important metabolites of intestinal bacteria in humans, and the concentrations of putrescine in the intestinal lumen of a healthy individual range from 0.5 to 1 mM , 54. Fooe medium . Bactericrobiome  has suggcrobiome , 60, indHelicobacter pylori arginase has been proposed where the depletion of arginine attenuates the innate immune response , agmatinase (Agt1), and guanidinobutyrase (Gbu1), represents an enzyme set whose members are collectively essential for C. albicans virulence. This set of ureohydrolases does not affect macrophage arginase or iNOS activity and also does not function in protection from the phagocytic burst but enables arginine and arginine-like substrates to support growth during pathogenesis.The results presented suggest a novel route for polyamine metabolism and agmatine assimilation in the fungal pathogen C. albicans strains used in this work are listed in URA3 recyclable PCR-mediated gene disruption system (URA3 marker was then recycled through selection on SC-U medium plus 5-fluoroorotic acid (2 mg/ml) and uridine (50 μg/ml). For spot assay, cells were grown in minimal medium  washed twice with H2O, and spotted on minimal agar media (2% agar [without NH4SO4]) containing 10 mM concentrations of the indicated nitrogen sources. Solutions of urea , ornithine , arginine , agmatine , 4-guanidinobutyrate , 4-aminobutyrate , and putrescine  were subjected to sterile filtration and added to the media after autoclaving. To monitor alkalinization, the pH indicator bromocresol purple  was added (0.01%) to medium with or without glucose as a carbon source. YPD and minimal medium containing 10 mM NH4SO4 served as the control. Prior to virulence testing, strains were grown in NGY medium  at 30°C for 16 h.n system , transfol-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% (vol/vol) fetal bovine serum, at 37°C and 5% (vol/vol) CO2. After reaching 90% confluence, cells were washed with PBS to remove dead cells and detached using 10 mM EDTA and 4 mg/ml lidocaine–HCl–PBS for 5 to 10 min. Cells were harvested  and either reinoculated for further cultivating or subseeded into 12-well plates at a density of 5 × 105 cells/well in the media described above supplemented with 1% minimum essential medium (MEM) and nonessential amino acids  (100×) and left overnight to adhere. Macrophages were activated with 100 ng/ml gamma interferon (IFN-γ) and lipopolysaccharide (LPS) 4 h prior to coincubation with 2.5 × 106C. albicans cells (macrophage/yeast cell ratio of 1:5) for 3 h. Supernatant was collected and stored at –20°C for arginase activity and nitric oxide measurements.Cells of the RAW 264.7 (ATCC TIB-71) murine macrophage cell line were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma), supplemented with 2 mM C. albicans strain SC5314 (http://osf.io/ARDTX) targeting the C. albicans open reading frame of interest directly upstream of a NGG protospacer-adjacent motif (PAM) site. Overhang sequences of annealed and phosphorylated 20-base forward and complement reverse guide oligonucleotides were ligated into CIP  was then linearized by digestion with KpnI and SacI before transformation was performed for efficient targeting to the ENO1 locus. Transformation was performed along with processing using a purified repair template (3 μg) that was generated with 60-bp oligonucleotide primers containing a 20-bp overlap at their 3′ ends centered on the desired mutation point. Homozygous mutations  were verified by sequencing . Reconstitution of the wild-type genotype from the null mutant background was performed with the CRSIPR-Cas9 transformation protocol described above. Both alleles were restored to the wild-type form . However, the reconstituted wild-type strains included the introduction of a silent PAM mutation. The genotypes of the transformants were verified by sequencing. Details of the primer sequences used are listed in For gene disruption using the CRISPR-Cas9 strategy, n SC5314  was usedn SC5314 , 64. Bri10.1128/mBio.01845-20.6TABLE S2Table S2, TIF file, 0.3 MB.Oligonucleotide sequences used for gene disruption by the CRISP-Cas9 system in this study. Download Copyright © 2020 Schaefer et al.2020Schaefer et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01845-20.7TABLE S3Table S3, TIF file, 0.2 MB.Oligonucleotide sequences used for gene restoration by the CRISP-Cas9 system in this study. Download Copyright © 2020 Schaefer et al.2020Schaefer et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the URA3 marker through selection on 5-fluoroorotic acid. Ultimately, to avoid issues associated with the ectopic expression of URA3, the URA3 marker was integrated into the RPS1 locus of all mutants by transformation with StuI-digested CIp10 plasmid  and 0.004% glucose supplemented with either 20 mM arginine, 20 mM urea and ornithine, 20 mM agmatine, 20 mM 4-guanidinobutyrate (GB), or 20 mM 4-aminobutyrate (GABA).For spot assays, exponentially growing cells were harvested from minimal medium at 30°C, washed twice with deionized water, and adjusted to an optical density (OD) of 0.1. The samples were then serially diluted, and 3 μl was spotted onto the different media. Plates were incubated at 30°C, checked at regular intervals, and scanned after 72 h . For hyphal induction, strains were grown initially in minimal medium  for 24 h; the cells were then washed and inoculated into prewarmed induction media at 1 × 10C. albicans cocultures were washed once with ice-cold PBS before macrophages were lysed for 10 min by adding 200 μl of 0.4% Triton X-100–10 mM Tris-HCl (pH 7.4) supplemented with fresh 1 μM pepstatin A and 1 μM leupeptin in each well. Samples were collected, transferred into centrifugation tubes, and centrifuged for 10 min at 4°C and 13,000 rpm to remove cellular debris. Lysates were transferred into fresh tubes and stored at −20°C until analyzed. For Candida cell lysis, protein lysis buffer  was added to each well and incubated 5 min on ice. Cells were scraped from the surface and transferred into cold centrifugation tubes, snap-frozen in liquid nitrogen, and stored at –20°C before protein extraction. Pellets were thawed at 4°C, washed, and resuspended in 350 μl ice-cold lysis buffer and transferred to a ribolyser tube containing 1 ml chilled glass beads. Samples were disrupted by bead beating (BioSpec) performed twice for 15 s each time. Protein concentrations were determined using Bradford reagent (Bio-Rad standard assay in microplates). Arginase activity and nitric oxide concentrations in supernatants and cell lysates were determined using an arginase activity assay kit (Sigma-Aldrich) and a Griess reagent kit for nitrite determination (Molecular Probes) according to the instructions of the manufacturers.Macrophage and C. auris ortholog that was truncated to ∼0.5 the length of C. albicans CAR1 .Multiple-protein-sequence alignment see , and theG. mellonella and M. musculus virulence assay results . Log rank tests were conducted for the G. mellonella survival assays, and analysis of variance (ANOVA) or Dunnett’s test was used for the M. musculus model to establish statistical significance, which was set at a P value of <0.05.Statistical analyses of  results  were perGalleria mellonella larvae were purchased from the United Kingdom . Groups of 20 healthy larvae (0.25 to 0.35 g) were inoculated through the last left proleg into the hemocoel with 10 μl of cell suspension (8 × 104 cells/larva) using a Hamilton syringe. Following infection, larvae were incubated in the dark at 37°C and survival was monitored daily for 7 days on the basis of responses to physical stimulation and melanization. Larvae inoculated with PBS were used as uninfected controls, and the experiments performed with the uninfected controls resulted in no deaths. Assays were performed at least 3 times independently , percent weight loss, and outcome score .endently . Female me score  (based o


Limited drug loading capacity (LC), mostly below 5% w/w, is a significant drawback of nanoparticulate drug delivery systems (DDS). Squalenoylation technology, which employs bioconjugation of squalenyl moiety and drug, allows self-assemble of nanoparticles (NPs) in aqueous media with significantly high LC (>30% w/w). The synthesis and particle preparation of squalenoylated prodrugs are, however, not facile for molecules with multiple reactive groups. Taking a different approach, we describe the synthesis of amphiphilic squalenyl derivatives (SqDs) as well as the physicochemical and biopharmaceutical characterizations of their self-assembled NPs as DDSs. The SqDs included in this study are (i) cationic squalenyl diethanolamine (ii) PEGylated SqD (PEG 750 Da), (iii) PEGylated SqD , and (iv) anionic squalenyl hydrogen sulfate. All four SqDs self-assemble into NPs in a size range from 100 to 200 nm in an aqueous solution. Furthermore, all NP derivatives demonstrate appropriate biocompatibility and adequate colloidal stability in physiological relevant pH environments. The mucoprotein binding of PEGylated NPs is reduced compared to the charged NPs. Most importantly, this technology allows excellent LC (at maximum of 45% w/w) of a wide range of multifunctional compounds, varying in physicochemical properties and molecular weight. Interestingly, the drug release profile can be tuned by different loading methods. In summary, the SqD-based NPs appear as versatile drug delivery platforms. Nano-sized drug delivery systems (DDS) having the size range from 10 to 1,000 nm have been investigated intensively to improve the treatment efficacy of severe diseases , and 37°C.However, it is challenging to bio-conjugate squalene and drug molecules with multiple functional groups or without functional groups  (THF), ethanol, absolute (HPLC grade) (EtOH), ethyl acetate , and Formic Acid Optima LCMS were purchased from Fisher Scientific. Acetonitrile and methanol (MeOH) were obtained from VWR Chemicals. Yeast extract was obtained from Fluka. Bacto™ Tryptone was obtained from BD Biosciences. Luria Bertani (LB) agar was obtained from Carl Roth. Gibco® HBSS (1x) Hanks' Balanced Salt Solution and Gibco® PBS was obtained from Life Technologies. Purified water was prepared by a Milli-Q water purification system  .The preparation of 1,1′,2-trisnorsqualenic aldehyde (compound 2) and 1,1′,2-trisnorsqualenol (compound 6)  from squvia reductive amination reaction between amine and aldehyde groups, using sodium cyanoborohydride (NaBH3CN) as reducing agent. Briefly, 1 molar equivalent of 1,1′,2-trisnorsqualenic aldehyde (compound 2) was solubilized in a mixed solvent of EtOH:water (95:5 v/v) at a concentration of 10% w/w. Following the addition of 1.2 molar equivalent of amine—methoxyl-PEG750-NH2 , PEG3000-NH2 , or diethanolamine—the reaction was carried out at room temperature for 3 h. NaBH3CN, 1.2 molar equivalent, was subsequently added, and the reaction was kept for an additional 20 h allowing imine reduction. EtOH was then removed under reduced pressure, and water was added, allowing precipitation of the resulted SqD. The product was then collected by centrifugation and washed three times with water. The isolated yield was >90% in all reactions. The cSq product (compound 3) was characterized by 1H-NMR (13C-NMR (1H-NMR (13C-NMR (cSq (compound 3), PEG3000Sq (compound 4), and PEG750Sq (compound 5) were synthesized using the same procedure from 1,1′,2-trisnorsqualenic aldehyde (compound 2) . The cony 1H-NMR , 13C-NMR was slowly dropped into the solution at room temperature. The temperature was then increased to 60°C. Following 16 h of reaction, the resulting system was quenched with an appropriate amount of water. The solvent mixture, DMF and water, was partly removed under reduced pressure. After being diluted in an excess amount of MeOH, NaOH 1 N was added under stirring. MeOH was subsequently removed under reduced pressure, and water was added allowing precipitation of aSq. The aSq product was extracted using ethyl acetate and dried over MgSO4. The solvents were completely removed under reduced pressure at 40°C. The isolated yield was 95%. The aSq product was characterized by 1H-NMR  mixture at a concentration of 1 mg/ml was dropped into 0.75 ml of water. THF was then removed under reduced pressure resulting in the PEGylated SqD–NPs in water at a concentration of 0.5 mg/ml.aSq–NPs, which were prepared as follows: 0.1 ml of aSq solution in THF at a concentration of 10 mg/ml was dropped into 1 ml of water under stirring. THF was then removed under reduced pressure resulting in an aSq–NPs suspension in water at a concentration of 1 mg/ml.The NP characteristics, especially size and polydispersity index (PDI), of the drug-free cSq–NPs and aSq–NPs were studied by varying the initial SqD concentration in THF and the final NP concentration in water. The detailed information is reported in the supplementary information .z-average), PDI, and zeta-potential of the SqD–NPs were determined at 25°C by dynamic and electrophoretic light scattering  using a Zetasizer  equipped with a He–Ne Laser at a 633 nm wavelength, backscattering angle of 173° for DLS. For the measurements, 20 μl of SqD–NPs suspension was diluted into 800 μl of water.The intensity-based hydrodynamic size (reported as 2 radiation level to avoid sample destruction.The morphology of each drug-free SqD–NPs was investigated by cryogenic transmission electron microscopy (cryo-TEM). In brief, after plotting 3 μl of SqD–NPs suspension on a holey carbon grid  for 2 s, the sample was frozen by plunging into −165°C liquid ethane, then transferred to the sample holder under liquid nitrogen conditions. All samples were examined using a JEOL  JEM-2100 LaB6 TEM equipped with a Gatan model 914 cryo-TEM sample holder  and a Gatan Orius SC1000 CCD camera to gain bright-field images. Sample analysis was done at −170°C, under low-dose conditions, meaning conservative settings of ~10 μa/cmThe colloidal stability of the drug-free SqD–NPs was studied in physiological relevant pH milieus, including pH 2, pH 5 (acetate buffer solution), and pH 7.4 (HBSS buffer solution). The tested samples were prepared by adding 25 μl of SqD–NPs suspension into 975 μl of buffer solution. NP characteristics, including size, PDI, and zeta-potential, were determined by DLS and ELS using a Zetasizer after 1, 3, and 24 h at 25°C.4 cells were seeded in each well of the 96-well plates and grown until reaching 80% cell confluence. The SqD–NPs were suspended in HBSS at concentrations ranging from 0.0652 to 1 mg/ml and incubated with cells for 4 h at 37°C and 5% CO2. After the incubation time, cells were washed twice with PBS, and MTT reagent (0.5 mg/ml in HBSS) was added. The cells were then incubated for an additional 4 h, at 37°C, and 5% CO2 allowing the formation of formazan crystals intracellularly  . NTA is able to determine the hydrodynamic diameter of NPs. Under random Brownian motions, NPs scatter light from a laser beam. The scattered light can be visualized by the NTA and captured by a video camera, to enable the software to track the movement of the NPs individually. Using the Stoke–Einstein equation, the hydrodynamic diameter of the particles can be determined.The interaction between fluorescent SqD–NPs and mucin glycoproteins was studied To study the protein–SqD–NP interaction, the Brownian motion of fluorescent NPs in water was compared to their movement in water containing non-fluorescent 0.1% mucin solution . For the measurements, used dilutions of the NPs, either in 0.1% mucin solution or water, were 1:100 (aSq–NPs), 1:200 (cSq–NPs), and 1:50 (PEG750Sq–NPs). To detect only the movement of the SqD–NPs, the particles were fluorescently labeled, and the fluorescence mode of the NTA was used. Therefore, SqD–NPs were loaded with 0.5% Nile red by coprecipitation; a detailed description of the method is in the Preparation of Drug-Loaded SqD–NPs section. As a vehicle control sample, the 0.1% aqueous mucin solution was studied with and without the fluorescence filter, to show the ability of the filter to exclude optical interferences of the proteins . Briefly, hydrophobic compound and SqD were dissolved in THF. Upon NP self-assembling in water by nanoprecipitation, the hydrophobic compound localized in the core of the SqD–NPs by hydrophobic interaction.Solvent evaporation method: In this study, we found that this method worked best for loading hydrophilic compounds having multiple functional groups, especially for small Mw hydrophilic compounds  and amphiphilic compounds . Briefly, the SqD and drug were dissolved completely in the mixture of THF:water (1:1 v/v) at desired concentrations (0.25–0.125 mg NPs/ml of NP suspension). The solution was then stirred for 24 h at room temperature allowing charge interaction of SqD and drug molecules, while the hydrophobic interaction between SqD and drug could be induced upon removal of THF using a rotary evaporator  at 40 mbar, 40°C and 65 rpm.Dropping method: In this study, we found that this method worked best for loading both small  and high Mw hydrophilic compounds  having multiple functional groups. Briefly, the charged drug-free SqD–NPs were prepared in water as described in the Preparation of Drug-Free SqD–NPs section and diluted to a concentration of 0.25 mg/ml. SqD–NPs suspension (1 ml) was then dropped into 1 ml of the prepared drug solution in water under stirring at 200 rpm. The resulting drug-loaded SqD–NPs were further stirred for 1 day at room temperature.With the aim to maximize the LC while maintaining the NP stability, appropriate preparation methods for generating drug-loaded NPs were chosen depending on the drug properties. In brief, we explored three NP preparation methods as follows:The “solvent evaporation method” and “dropping method,” which allowed the loading of drugs in both compartments, core and shell, of the NPs could be used in multiple drug-loading purposes. As an example, Nile red and FITC-albumin co-loaded cSq–NPs are presented in P-values .g for 30 min. Colistin, tigecycline, or FITC-albumin was collected by centrifuging the drug-loaded NPs at 24.400 g for 30 min. The amount of cholesteryl BODIPY, Nile red, tigecycline, or FITC-albumin in the supernatant, respectively, was analyzed by a plate reader (details described in The loading capacity (LC) and encapsulation efficiency (EE) of the drugs loaded to SqD–NPs were determined indirectly using the amount of non-loaded drug in the supernatant of the SqD–NPs suspension. The hydrophobic compound  was extracted from the supernatant using ethyl acetate  at 37°C and constant shaking at 250 rpm. Briefly, the optimal LC sample of the drug-loaded SqD–NPs was concentrated and then diluted in PBS to have a final concentration of the corresponding drug at 10% w/w. The cumulative drug release in percent was evaluated over a 24 h period. Samples were collected after 1, 2, 4, 6, 8, 16, and 24 h, while the release acceptor volume was always kept constant. The drug amount in the acceptor fluid was analyzed. The hydrophobic drugs were extracted using ethyl acetate before further analysis. The drug quantification was done by plate reader or HPLC  Part 6.If not stated otherwise, all procedures were conducted at least in three independent experiments and measured in technical triplicate. Results are presented as mean ± standard deviation (SD). Calculations were done using either Excel, Microsoft 2016 and 2019, or GraphPad Prism 8.0. Physicochemical information of molecules was predicted by ChemDraw Professional 16.0.1H-NMR and 13C-NMR (m/z(%) 465.3 (100) (M-H)−] and cSq [(ESI-) m/z(%) 474.4 (100) (M-H)+], respectively. The synthetic schemes are facile and allowed isolating the final products in high yields (>90%), which, moreover, could be scaled-up easily. Notably, the synthesis of cSq with a diethanolamino polar head was further optimized with one step less compared to that of the previously reported squalene-amine derivative  . The zetWe prepared two PEGylated SqDs having PEG with different chain length and terminal groups. The PEG750Sq terminates with a methyl group, while the PEG3000Sq terminates with a hydroxyl group. The self-assembling of these PEGylated SqDs allowed the formation of NPs with a dense surface layer of PEG. Regardless of the recorded zeta-potential, this PEG layer is expected to stabilize the NPs and minimize surface interactions between NPs and other molecules , skin (pH ~5), and common physiological pH environments at 7.4 , which helped stabilizing the NP suspension , showed positive zeta-potential of 71 ± 11 and 33 ± 4 mV at pH 2 and 5, respectively, reflecting the protonation of the amine groups. The size and PDI of stable cSq–NPs after 24 h of incubation at pH 5 were 120.5 ± 1.5 nm and 0.19 ± 0.01 nm, respectively. Interestingly, a slightly smaller particle size (76.9 ± 17.1 nm) and a larger PDI (0.30 ± 0.07) were recorded when dispersing cSq–NPs at pH 2. The cSq–NPs could be fully charged at pH 2, which enhanced the amphiphilicity of cSq molecules. Consequently, this induced intermolecular forces and hydrophobic interactions between the squalenyl moieties leading to a denser particle packing  holds excellent assembling properties, the monoconjugation with PEG having a Mw of 3,000 Da might result in a better water solubility. Thus, the hydrophobic interaction of squalenyl moieties would not be strong enough to stabilize the PEG3000Sq–NPs. On the complete opposite, SqPEG750–NPs demonstrated excellent colloidal stability in all pH milieus at all time points. The hydrodynamic diameter and PDI were always around 129.7 ± 4.5 nm and lower than 0.23 ± 0.02, respectively. The zeta-potential of SqPEG750–NPs was nearly neutral at pH 5 and 7.4; however, it increased to 40 ± 3 mV at pH 2 due to the protonation of the amino conjugation.PEGylation offers plenty of advantages to nano-sized drug carriers especially to improve the The particles holding appropriate colloidal stability in physiological relevant pH milieus were used in further investigations, including aSq–NPs, cSq–NPs, and PEG750Sq–NPs.via MTT assay  water  . Notably, the optimal LC of dexamethasone was ~33, ~32, and ~24% in aSq–NPs, cSq–NPs, and PEG750Sq–NPs, respectively, while the EE values in all cases were ~90% . The optIsoniazid, colistin, tigecycline, and FITC-albumin, representing different Mw and hydrophilicity, were loaded in either aSq–NPs or cSq–NPs. Accordingly, the loading of these compounds was investigated using solvent evaporation method and/or dropping method , while tIsoniazid—a positively charged and small molecule (Mw 137.14 Da)—was loaded in aSq–NPs. The solvent evaporation method resulted in a significantly higher LC of isoniazid at 27.43 ± 0.79% compared to that of the dropping method at 6.17 ± 0.97%. Using the solvent evaporation method, the preparation of isoniazid and aSq in the solvent mixture (THF:water 1:1 v/v) allowed the maximum charged interaction between the two reagents, thus, increased the EE and LC. In contrast, the preformed aSq–NPs and dropping method limited the interaction with isoniazid molecules. The optimal isoniazid-loaded aSq–NPs had the size of 198.9 ± 8.6 nm and PDI below 0.25.via charged interactions.The molecules with multiple functional groups—tigecycline (Mw 585.66 Da), colistin , and FITC-albumin —could be loaded in NPs using the dropping method. The optimal LC and EE of stable tigecycline-loaded aSq–NPs  were 44.26 ± 2.03 and 85.3 ± 6.7%, respectively. The LC and EE of colistin in aSq–NPs were significantly high at 35.83 ± 0.51 and 83.6 ± 4.1%, respectively, which also produced a stable DDS having the size of 195.6 ± 2.6 nm and PDI lower than 0.2. The reasonable LC and EE of FITC-albumin—a representative of negatively charged protein molecules—in cSq–NPs were 3.52 ± 0.33 and 92.1 ± 3.8%, respectively. The stable protein-loaded cSq–NPs had the size of 205.6 ± 2.8 nm and PDI below 0.2. As shown in via both charged and hydrophobic interactions  were prepared by the coprecipitation, and then further loaded with hydrophilic FTIC-albumin (LC~0.5%) by the dropping method. The colocalization of both fluorescent compounds in NPs was confirmed by confocal laser scanning microscopy images . Such siIn short, the SqD–NPs are capable of loading a broad range of drug molecules differing in physicochemical characteristics, thus, are versatile drug delivery platforms.In vitro release profiles were conducted under the same conditions at physiological pH 7.4 (PBS), 37°C, and over 24 h , FITC-albumin (protein representative) and colistin (comparison of two preparation methods), respectively. The association of hydrophilic drugs loaded onto the NP surface by the dropping method is not as strong as a covalent conjugation, leading to the assumption of a complete drug release. Cumulative release of tigecycline, colistin, and FITC-albumin reached 88 ± 13, 75 ± 11, and 83 ± 13%, respectively, after 24 h in PBS at 37°C . The burPseudomonas aeruginosa strain PA14 wild type and Staphylococcus aureus strain Newman. As shown in Furthermore, IC90 values of tigecycline and colistin-loaded aSq–NPs were determined by the minimum inhibitory concentration (MIC) assay performed on In this study, we explored the use of self-assembling amphiphilic SqDs as DDS, which had started with the aSq. We have further described the straightforward synthesis of cSq and PEGylated SqDs. In addition to the ability of self-assembling to supramolecular colloids, the latter demonstrated that SqD–NPs showed excellent stability in physiological relevant media and biocompatibility. The application of different methods for the preparation of drug-loaded SqD–NPs allowed to modulate not only the LC but also the release rate, as desired. In further studies of carrier properties, the SqD–NPs showed significantly high LC of various cargos. Our findings postulate the self-assembled SqD–NPs as versatile drug delivery platforms.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.in vitro assays and drug release. CDR and MK visualized the NPs. XM conceptualized and validated the mucin–NPs interaction studies. RC and SF performed NP stability and mucin–NPs interaction studies. RC and D-KH analyzed the data, prepared the figures, and wrote the original draft. RC reviewed and edited the final draft. All authors read and revised the manuscript.D-KH, BL, DD, PC, and C-ML conceptualized and initiated the study. PC and C-ML acquired funding and provided resources. XM, US, BL, DD, PC, and C-ML were supervisors. D-KH did chemical synthesis and characterization. D-KH and RC investigated NP self-assembly and drug-loading capacities. CDR, RC, and D-KH validated the drug quantification methods. D-KH conducted The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Crassostrea hongkongensis (Hong Kong oyster) is an ecologically and economically valuable shellfish endemic to South/Southeast Asia. Due to ocean acidification and warming waters, they have become increasingly vulnerable to invading microbes including Vibrio parahaemolyticus, a significant foodborne human pathogen. In recent years, outbreaks of V. parahaemolyticus have emerged as a perennial phenomenon in parts of the world, necessitating to better understand the biology of host-pathogen interactions in this under-examined marine invertebrate. Although an immunologically relevant autophagy apparatus has been identified in Crassostrea gigas, an evolutionarily close mollusk cousin, the precise mechanistic details of C. hongkongensis autophagy during V. parahaemolyticus infection are still wanting. Here, we compellingly demonstrated that in vivo V. parahaemolyticus challenge robustly triggered autophagic signaling in C. hongkongensis hemocytes peaking at 6 h post-infection, which subsequently promoted bacterial clearance and dampened premature apoptosis. Simultaneously, a large surplus of adenosine monophosphate (AMP) and elevations in reactive oxygen species  formation were observed post-infection. Extrinsically applied AMP and ROS could synergistically induce AMP-activated protein kinase (AMPK) phosphorylation to stimulate downstream autophagic events. V. parahaemolyticus infection-induced autophagy was pharmacologically shown to be AMPK-dependent in vivo. Overall, our results establish autophagy as a crucial arm of host defense against Vibrio infections in mollusks, and provide new insights into the underappreciated roles of ROS and AMP as co-regulators of autophagy. Crassostrea hongkongensis), an edible mollusk species with an aquaculture history of 700 years and an annual production exceeding 1.6 million tons  have been implicated as a major cause of mortality and morbidity through seafood-associated gastroenteritis worldwide  was found to present autophagic features similar to mammalian cells  and Vibrio aestuarianus  were collected from Zhanjiang, Guangdong Province, China, and acclimated to laboratory conditions by rearing in aerated sand-filtered seawater at ambient temperature (24 ± 1°C) and appropriate salinity (18‰) for 2 weeks prior to experiments. During the acclimation period, oysters were fed with the microalgae Isochrysis galbana (105 cells/mL) and Chaetoceros calcitrans (2 × 105 cells/mL) twice a day.Two-year-old Vibrio parahaemolyticus used in the study had been isolated from C. hongkongensis following established methods  broth at 37°C to reach OD600nm = 0.6∼0.8, and then centrifuged at 800 × g at 4°C for 10 min. Afterward, bacteria were washed 3 times with PBS . Bacterial pellet was re-suspended in PBS to a density of OD600nm = 1.0. Oysters in the challenged group were injected with 100 μL bacterial suspension into the adductor muscles, while oysters in the untreated control group were injected with an equal volume of PBS. After injection, oysters were returned to separate fiberglass tanks. Subsequently, hemolymph was withdrawn from the pericardial cavity through adductor muscles of C. hongkongensis by means of a sterile 1-mL syringe with an 18 G1/2-inch needle. Hemolymph was kept on ice to prevent cellular aggregation or clotting and centrifuged immediately at 200 × g for 10 min to harvest hemocytes. The supernatant was passed through a 0.22-μm filter paper, as hemolymph serum. Hemocytes were maintained in hemolymph serum at 18°C in petri dishes until subsequent analyses. methods . For in 4Cl  at 20 mM for 6 h in aquariums (3 L of seawater). For chemical induction of autophagy, oysters were injected with 200 μL carbamazepine  into the adductor muscles, and then transferred back to aquariums for 6 h. To test the effects of exogenous AMP  and H2O2  on autophagy induction or APMK phosphorylation, each oyster was injected with 30 mg AMP into the adductor muscles and treated with H2O2 at 10 mM for 6 h in 3L seawater. Additionally, for inhibiting the AMPK pathway, Compound C  was injected at a dose of 20 μg individually. To validate the role of oxidative stress on autophagy induction, mito-TEMPO, as a broadly used mitochondria-specific ROS scavenger, was injected at a dose of 0.5 mg per oyster, by using the same method.To inhibit autophagy, oysters were treated with NHg for 20 min at 4°C. The supernatant was diluted 10× to determine the protein concentrations by using bicinchoninic acid (BCA) protein assay . Lysates were mixed with a 4 × protein loading buffer  and heated to 95°C for 15 min. Then, the homogenate containing approximately 60 μg per lane of total protein was loaded onto a 12% polyacrylamide gel for SDS-PAGE, which were subsequently transferred to 0.2 μm PVDF membranes . The membranes were blocked with QuickBlockTM blocking buffer for Western blot  and then incubated overnight with primary antibodies at 4°C. Later, the membranes were washed with PBST (PBS with 0.1% Tween-20), and incubated for 2 h at room temperature with a 5,000 × diluted anti-rabbit HRP-linked secondary antibody . After washing 3 times with PBST, membranes were visualized following reactions with an ECL luminescence reagent . Relative protein expression was quantified by using the ImageJ software (v1.8.0). The following primary antibodies were used to detect their corresponding protein substrates: rabbit anti-LC3 , rabbit anti-AMPKα , rabbit anti-phospho-threonine 172-AMPKα , and rabbit anti-β-actin . The housekeeping protein β-actin was selected as a loading control, and its blotting was done within its linear range of detection . Lysates were centrifuged at 1,200 × etection .g for 10 min and washed with a 0.1 M phosphate buffer (pH 7.4) for 3 times (15 min each). Pellets were post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer at room temperature for 2 h. After washing with the same buffer for 3 times, pellets were collected and embedded in 2% agar. Following dehydration in a graded series of ethanol and acetone, pellets were embedded in Epon 812  and polymerized at 60°C for 48 h. Ultrathin sections of 60 nm in thickness were mounted onto a copper grid and stained with uranyl acetate and then lead citrate (15 min each). Electron micrographs were acquired by using a transmission electron microscope .Hemocytes were pooled after being collected from 10 individuals per group as described above for TEM observation. Cells were fixed in an electron microscope fixation solution  for 4 h at 4°C. The suspension was centrifuged at 200 × TM first strand cDNA synthesis kit  and then subjected to quantification analysis by using 2 × RealStar Green Power mixture  in LightCycler® 480 II , according to the manufacturer’s instructions. Gene-specific primers were designed with Primer Premier v5.0, and their sequences are as listed in Total RNA of harvested hemocytes was isolated with TRIzol reagent  according to the manufacturer’s instructions. RNA quality and quantity were determined and assessed by using NanoDrop 2000C . Extracted RNA was reverse-transcribed into cDNA with the PrimerScript5 cells per well) for 30 min at 27°C. After harvest, three strains of live bacteria  were washed 3 times, resuspended in PBS, and diluted to a concentration of 8 × 107 CFU/mL. To initiate infection, 10 μL of bacterial suspension was added to each well to achieve an MOI (multiplicity of infection) = 1. Following incubation for 40 min at 27°C, extracellular bacteria were removed by washing 3 times with 0.02% trypsin-EDTA. Subsequently, hemocytes were lysed and homogenized in 1 mL cold PBS containing 0.05% Triton X-100 for 1 h. Ten microliters of lysates from each well were serially diluted in PBS, from which a 100-μL bacterial inoculum was spread on LB plates, followed by overnight culture to enumerate bacterial colony counts. Negative controls were set up with hemocyte lysates unexposed to bacteria . For superoxide detection, hemocytes were harvested after in vivo bacterial exposure to oysters. They were then resuspended in Ca2+ and Mg2+ supplemented Hank’s balanced salt solution  with 5 μM MitoSOX reagent  at 37°C for 10 min. Finally, hemocytes were washed and resuspended in fresh HBSS for detection of mitochondrial superoxide by flow cytometry. At least 10,000 gated cell events were included. Statistical analysis was performed with FlowJo (v10.0 software).Hemocytes were collected and resuspended in 100 μL binding buffer, followed by incubation (10 min in dark) with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) supplied by an apoptosis detection kit  at room temperature. Next, another 400 μL binding buffer was added to the suspension, before analysis by a Guava5 cells per well) for 30 min, then were washed by PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Subsequently, hemocytes were washed by PBS again and treated with 500 μL PBS containing 0.3% Triton X-100 for 5 min, then washed by PBS for three times and stained by using One-Step TUNEL Cell Apoptosis Detection Kit (Green Fluorescence)  according to manufacturer’s instructions. Samples were counterstaining with DAPI  and examined under fluorescence microscope .Hemocytes were collected and plated on 24-well plate . Quantitative measurements were obtained against a standard curve  and then normalized with respect to total proteins. For AMP assay, oyster hemocytes were seeded into a microplate after in vivo infection detected, followed by measurements at 450 nm according to the instructions for human AMP ELISA kit , which were then quantified with a standard curve  according to the manufacturer’s instructions. Briefly, hemocytes were collected and mixed isometrically in a working solution containing 10 U/mL horseradish peroxidase and 10 mM Amplex Red. The mix was incubated at room temperature for 30 min, shielded from light. Absorbance at 560 nm was determined by using an EnSightrd curve  of H2O2 rd curve . For antrd curve .t-test and those among more than two groups by one-way ANOVA, followed by Tukey’s or Dunn’s post hoc tests by SPSS (v22.0).Data processing and statistical analyses were performed by using GraphPad Prism (v8.0.1), and a heatmap was generated by R (v3.5.2). Comparisons between two groups of samples were performed by Student’s C. hongkongensis challenged with V. parahaemolyticus. Several genes expression exalted post-challenging, which reached peak levels at 6 h  of three strains including Escherichia coli, Vibrio alginolyticus, V. parahaemolyticus were dramatically increased approximately by 4 fold, 5 fold and 9 fold, respectively, with respect to the untreated control , indicating that inhibition of autophagy had no obvious effects on basal apoptosis (p < 0.05), which suggests stress being imposed within the infected hosts. Consistent with the observation on V.p. infection in V.p. in the presence of NH4Cl, more individual hemocytes emerged fragmented DNA (p < 0.05), suggesting that hemocytes indeed failed to contain the bacteria while prematurely entering apoptosis , swollen mitochondria and condensation of chromatins  and H2O2. Notably, mitochondrial superoxide levels increased significantly following bacterial challenge, culminated at 6 h post-infection, and declined moderately toward 12 h and 24 h post-infection  treatment. As anticipated, the combined treatment was much more effective than treatment with single simulants. This robust response in signal transduction seems to be mirrored in the expression patterns of 6 selected autophagy-related genes  and a seriously flawed immune armor reasoningly  + NH4Cl treatment, with features resembling those in the treatment used in this study (V.p. infection + NH4Cl). Based on our findings for V.p. infection as a more naturalistic stimulus of autophagy (Accumulating evidence has shown that oysters possess a highly intricate immune system molded through generations of evolutionary and competitive processes . Importapoptosis , whereasl groups . Of notequadrant  and matequadrant . This obytoplasm . As propytoplasm . Remarkautophagy , the molC. gigas extrinsically induced by OsHV-1 viral infection, Vibrio splendidus infection or heat stress was characterized by accelerated energy consumption and metabolic perturbations (V.p. infection could induce energy stress as reflected by AMP elevations and AMPK phosphorylation (C. hongkongensis and functionally involved in phagocytosis and cytoskeleton remodeling (2O2 paired with AMP to synergistically increase AMPK phosphorylation (C. hongkongensis’s ability to manage and survive bacterial assaults, thus in part accounting for the immunological resilience of mollusks. In order to illuminate the mechanistic roles and significance of energy and oxidative stress in the contexts of infection-induced autophagy, further investigation is warranted.Invasion by pathogens into host organisms generally evokes immune responses as well as inflammation, which could sap host energy and disrupt cellular metabolic pathways, giving rise to disordered conditions ultimately . Consistrbations . Nutritirylation . Increasmodeling . AMPK acmodeling , in agremodeling . In addimodeling . Infectimodeling  as a meamodeling . Our wormodeling , as H2O2rylation  and contrylation . CollectThe datasets generated for this study are available on request to the corresponding author.XD, N-KW, YZ, and ZY: conceptualization. XD, N-KW, and YZ: methodology. XD, N-KW, and YX: analysis and investigation. XD, YX, and VT: data curation. XD and N-KW: writing – original draft preparation. N-KW, FM, XZ, YL, and ZN: writing – review and editing. YX and YH: visualization. YZ and ZY: supervision. YZ, ZY, ZX, JL, and SX: project administration. YZ, ZY, ZX, and JL: funding acquisition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antimitotics are important anticancer agents and include the natural alkaloid prodrug colchicine (COL). However, a major challenge of using COL as an anticancer drug is its cytotoxicity. We developed a novel drug delivery system (DDS) for COL using mesoporous silica nanoparticles (MSNs). The MSNs were functionalized with phosphonate groups, loaded with COL, and coated with folic acid chitosan-glycine complex. The resulting nanoformulation, called MSNsPCOL/CG-FA, was tested for action against cancer and normal cell lines. The anticancer effect was highly enhanced for MSNsPCOL/CG-FA compared to COL. In the case of HCT116 cells, 100% inhibition was achieved. The efficiency of MSNsPCOL/CG-FA ranked in this order: HCT116 (colon cancer) > HepG2 (liver cancer) > PC3 (prostate cancer). MSNsPCOL/CG-FA exhibited low cytotoxicity (4%) compared to COL (~60%) in BJ1 normal cells. The mechanism of action was studied in detail for HCT116 cells and found to be primarily intrinsic apoptosis caused by an enhanced antimitotic effect. Furthermore, a contribution of genetic regulation (metastasis-associated lung adenocarcinoma transcript 1 (MALAT 1), and microRNA (mir-205)) and immunotherapy effects (angiopoietin-2 (Ang-2 protein) and programmed cell death protein 1 (PD-1) was found. Therefore, this study shows enhanced anticancer effects and reduced cytotoxicity of COL with targeted delivery compared to free COL and is a novel method of developing cancer immunotherapy using a low-cost small-molecule natural prodrug. Colchicine (COL) is a natural alkaloid compound derived mainly from the medicinal plant Colchicum automnale and has been used in the clinic for treating gout and familial Mediterranean fever. COL has also shown some benefit in primary biliary cirrhosis , amyloidDespite COL not yet being used clinically for cancer therapy because of its toxicity to normal cells, it is used as a lead compound in generating potent anticancer agents ,6,7,8,9.Recently, antimitotic agents such as COL were reported to have a regulatory effect on most immune cell types, leading to the development of effective cancer immunotherapies . With reTraditional treatments for cancer, including surgery, radiation, cryosurgery, and chemotherapy, can be used alone or in combination. These methods have several limitations, such as toxicity, side effects, and expense. When chemotherapy is considered, normal treatment protocols include anticancer drugs alone or in combination to inhibit/kill cancer cells by affecting cell division and proliferation through different mechanisms. Drugs can produce various side effects for patients  . TherefoDespite the promising anticancer effects of COL, a few COL drug delivery systems (DDSs) have been tested to improve its therapeutic efficiency through cancer-targeting ,29. We fThe targeted drug delivery system for COL, MSNsPCOL/CG-FA, comprised COL-loaded MSNsP subsequently coated with chitosan-glycine conjugated to folic acid. The schematic representation of the process is shown in To observe the structural changes between the prepared materials, we used TEM STEM and FE-SEM techniques. The FE-SEM images A show th2/g and 0.772 cm3/g of specific surface area and total pore volume, respectively). This observation indicates successful preparation, in agreement with previous results  × 100, where av(NC) is the average absorbance of the three negative control wells measured at 495 nm (reference 690 nm) and av(S) is the average absorbance of the three sample wells measured at 495 nm (reference 690 nm). IC50 and IC90 values were calculated using Probit analysis and SPSS for Windows statistical analysis software package, version 9 .For the estimation of in vitro cytotoxic potency, the 3-4,5-dimethylthiazol-Z-yl-2,5-diphenyltetrazolium bromide (MTT) assay was conducted according to Mosmann . Human p5 cells/per well in RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin and incubated at 37 °C in a 5% CO2 atmosphere. After 24 h, the medium was replaced with fresh, treated with different samples at 40 µg/mL (100 µL), and incubated for 24 h or 72 h. Control cells received no treatments. For cell cycle analysis, cells were trypsinized, washed with cold PBS, and fixed with 70% ethanol. The fixed cells were rinsed with PBS, and then labeled with propidium iodide (PI) following the manufacturer’s instructions . Finally, cells were analyzed by flow cytometry . Cell cycle analysis was performed with an FL2-A histogram of single cells. The Annexin V-FITC Apoptosis Detection Kit  was used to detect apoptosis according to the manufacturer’s instructions. After treating the cells as mentioned, the cells were trypsinized, fixed in 70% ethanol, washed with cold PBS, and suspended in binding buffer (500 µL). Next, 5 μL of Annexin V-FITC and 5 μL of PI were added and incubated for 10 min in the dark, and then immediately analyzed by flow cytometry .To analyze apoptosis and the cell cycle, we followed manufacture protocols and our pervious study ; HCT116 The caspase-3 activity was determined according to the manufacturer’s instructions using the human active caspase-3 content assay kit. HCT116 cells were cultured on 96-well plates  in 100 µL of RPMI 1640 containing 10% FBS, 1% penicillin/streptomycin at 37 °C. The cells were treated at 10 and 500 µg/mL (for MSNs and MSNsP) or 10 and 60 µg/mL  with 100 μL sample volume per well and incubated for 72 h prior to the assay. The procedures are reported in detail in our previous study . The absTo assess tubulin polymerization, the ELISA kit for TUBb  was used following the manufacturer’s instructions. HCT116 cells were seeded in 96-well plates at a cell density of 1.2–1.8 × 10,000 cells/well in 100 µL growth medium (DMEM); 100 µL of each sample was added per well. MSNs and MSNsP were tested at 125, 250, 500, and 1000 µg/mL, whereas MSNsPCOL, MSNsPCOL/CG-FA, and COL were used at 0.4, 2, 10, and 50 µg/mL. After 48 h of incubation, the solution was removed, cells were detached by trypsinization, washed with cold PBS buffer, suspended in PBS, and followed by three freeze/thaw cycles to lyse the cells. Cell lysates were centrifuged in a cooling centrifuge for 10 min, which permits the removal of cellular debris to detect β-tubulin in the supernatant. The assay was performed as instructed in the kit. Finally, ELISA reader ROBONIK P2000  was used to measure the color at 450 nm in triplicate. Data were calculated as percent inhibition.6 and incubated for 48 h at 10 and 500 µL (for MSNs and MSNsP) or 10 and 60 µg/mL . Cells were then collected for RNA extraction using the RNeasy extraction kit according to the manufacturer’s protocol . Cells were disrupted in buffer RLT, homogenized, and disrupted before adding ethanol to the lysates to create conditions that subsequently promoted the selective binding of RNA to the RNeasy membrane. A total of 100 μl of sample lysate was added to a RNeasy Mini spin column, with total RNA binding to the membrane. High-quality RNA was eluted using RNase-free water. Centrifugation in a micro-centrifuge was used during all steps .HCT116 cells were cultured at a density at 1 x 10TM One-Step RT-PCR Kit with SYBR® Green  according to the manufacturer’s instructions and as described by Labib et al. [Bax and Bcl-2 expression was investigated by real-time polymerase chain reaction (RT-PCR) using the BIORAD iScriptb et al. . The RT-b et al. . A react6 and incubated for 48 h at 125, 250, 500, and 1000 µl (for MSNs and MSNsP) or 0.4, 2, 10, and 50 µg/mL . The cells were then collected and pelleted by centrifugation to remove the supernatant before washing three times with PBS. Next, the cells were resuspended in PBS, lysed, and centrifuged at 1500× g for 10 min with cooling centrifugation to remove cellular debris. The supernatant was collected for assay, 100 µL added to the plate reader and incubated for 90 min at 37 °C, and the liquid removed. Subsequently, 100 μL of 1× Biotinylated Detection Antibody was added to each well and incubated for 1 h at 37 °C, followed by removal of the liquid and washing three times with wash buffer. Next, 100 μL of 1× HRP conjugate working solution was added to each well and incubated for 30 min at 37 °C, then replaced with 90 μL of TMB substrate solution and incubated for 15 min at 37 °C. Finally, 50 μL of stop solution was added and the absorbance measured at 450 nm using the microplate reader ROBONIK P2000 . Measurements were made in triplicate.The BRAF ELISA kit was used with cell lysates according to the manufacturer’s protocol . HCT116 cells were cultured at density of 1 × 10The cytochrome c was measured using the human cytochrome c ELISA kit in cell lysates according to the manufacturer’s protocol . HCT116 cells were cultured at density of 1.2–1.8 × 10,000 cells/well and incubated for 72 h at 500 µL (for MSNs and MSNsP) or 60 µg/mL . The cells were then collected and pelleted by centrifugation to remove the supernatant before washing with PBS. Next, several steps were procced as the kit protocol. Finally, absorbance was measured at 450 nm using the microplate reader . Measurements were made in triplicate.The mitochondrial membrane potential was done by means of flow cytometry with cell lysates according to the manufacturer’s protocol TMRE mitochondrial potential assay . HCT116 cells were cultured at density of 1.2–1.8 × 10,000 cells/well and incubated for 72 h at 500 µL (for MSNs and MSNsP) or 60 µg/mL  with volume of 100 µg/mL. The cells were then collected and pelleted by centrifugation to remove the supernatant before washing with PBS. Next, we resuspended it in 100 µL of assay buffer assay, followed by adding 100 µL of TMRE buffer, and incubated for 30 min. Then, it was centrifuged, and resuspended in assay buffer, and the data collected by flow cytometry . Finally, measurements were made in triplicate.® CCL-247), cultured in 96-well plates  in RPMI 1640 medium supplemented with 10% FBS, 10 μg/mL of insulin, and 1% penicillin-streptomycin, and allowed to attach and grow for 24 h. The cell culture was treated with different samples, and control cells were left untreated. To prepare cell culture supernatants, after incubation, cells were harvested after detaching with trypsin and lysates collected by centrifugation. The cells were lysed with cell lysis buffer and centrifuged at 1500g for 10 min at 2–8 °C to exclude cell debris. The expression levels of angiopoietin-2 (Ang-2), PD-1, CD44, metastasis-associated lung adenocarcinoma transcript 1 . The fold change was calculated using 2−Ct for relative quantitation [The supernatant was used to measure Ang-2 and PD-1 by ELISA using an ELISA plate reader  according to the manufacturer’s instructions. For quantitative detection of total soluble human CD44, normal and variant isoforms were measured by ELISA. For measurement of long non-coding RNAs (lncRNAs) for MALAT1 and mir-205, cells were collected, and RNA extracted using the miRNeasy extraction kit. Total RNA including non-coding RNAs was extracted from supernatants using the miRNeasy extraction kit  and QIAzollysis reagent according to the manufacturer’s instructions. The concentration of RNA was determined using NanoDrop2000, which is very accurate for measuring even the smallest quantities of RNA . Reverse transcription was carried out on extracted RNA in a final volume of 20 μL using the RT2 First Strand kit (Qiagen) according to the manufacturer’s instructions. The expression levels of the studied lncRNAs were evaluated using GAPDH, which is widely used as an internal control for serum lncRNAs in numerous studies ,99 accorp < 0.05. All statistical calculations were performed in triplicate using computer program IBM SPSS  release 22 for Microsoft Windows.Data for biological evaluations are expressed as mean ± SD. Significance differences were calculated using the Student t-test, Mann Whitney U test, and analysis of variance (ANOVA) analysis at We successfully designed a novel DDS for COL prodrug that efficiently targets cancer cells. The DDS was fabricated by loading COL into spherical mesoporous silica nanoparticles and their subsequent modification with phosphonate groups. They were subsequently coated with chitosan-glycine complex conjugated to folic acid, which acted as a targeting ligand for cancers. Full inhibition of HCT116 colon cancer cells was observed. A weaker effect was observed in HepG2 liver and PC3 prostate cancer cells. The most important characteristic of the DDS was its negligible cytotoxicity in normal cells. We observed, after 72 h of incubation with MSNsPCOL/CG-FA, low inhibition in normal BJ1 cells (4%) compared to free COL (~60%). Apoptosis (intrinsic) was found to be the main mechanism of action occurring as a consequence of the strong antimitotic effects. MSNsPCOL/CG-FA more strongly inhibited tubulin than free COL. It also increased the cell cycle at G2/M, caspase-3 activation, and Bax expression compared to COL. On the other hand, MSNsPCOL/CG-FA inhibited anti-apoptotic proteins  more strongly than clinically used COL and 5-FU anticancer drugs. New effects of the DDS on genetic regulation and cancer related immuno-effects were found. MSNsPCOL/CG-FA significantly inhibited MALAT1, mir-205 expression, Ang-2 protein, and PD-1 compared to COL and 5-FU. We expect that the tailored DDS for COL has the potential to become a nanomedical platform for cancer treatment.


Neuronal death was assessed upon PCN67 treatment using differentiated PC12 cells and primary hippocampal neurons. At 72 h postexposure, cell viability assays showed an IC50 value of 0.35 μg/ml and dose-dependent damage of neurites and concomitant downregulation of neurofilaments L and M. Moreover, we found that younger primary neurons (DIV4) were much more sensitive to PCN67 toxicity than mature cultures (DIV14). Our comprehensive analysis indicated that the application of PCN67 at the IC50 concentration caused necrosis, which was reflected by an increase in LDH release, HMGB1 protein export to the cytosol, nuclear swelling, and loss of homeostatic control of energy balance. The blockage of mitochondrial calcium uniporter partially rescued the cell viability, loss of mitochondrial membrane potential (ΔΨm), and the overproduction of reactive oxygen species, suggesting that the underlying mechanism of neurotoxicity involved mitochondrial calcium accumulation. Increased lipid peroxidation as a consequence of oxidative stress was additionally seen for 0.1 μg/ml of PCN67, while this concentration did not affect ΔΨm and plasma membrane permeability. Our results show for the first time that neuronal mitochondria act as a target for PCN67 and indicate that exposure to this drug may result in neuron loss via mitochondrial-dependent mechanisms.Hexachloronaphthalene (PCN67) is one of the most toxic among polychlorinated naphthalenes. Despite the known high bioaccumulation and persistence of PCN67 in the environment, it is still unclear to what extent exposure to these substances may interfere with normal neuronal physiology and lead to neurotoxicity. Therefore, the primary goal of this study was to assess the effect of PCN67 in neuronal  Polychlorinated naphthalenes (PCNs) were included in 2015 into the Stockholm Convention on Persistent Organic Pollutants to protect human health and the environment from highly dangerous, long-lasting chemicals by the elimination of their production and reduction of their unintentional release –10. The p-dioxins (PCDDs), polychlorinated dibenzo-p-furans (PCDFs), and polychlorinated biphenyls (PCBs) [in vivo drug administration [in vivo and in vitro suggest the action through GABAergic and/or glutamatergic systems [PCNs are often labelled as dioxin-like compounds (DLCs) not only because their mechanism of action (through the aryl hydrocarbon receptor (AhR)) is similar to polychlorinated dibenzo-s (PCBs) , 28 but s (PCBs) , 30. Pris (PCBs) , 32. In s (PCBs) , 34 and s (PCBs) , 36, thys (PCBs) , and pres (PCBs) –41. Somes (PCBs)  have alsstration –45. Whil systems , 46.It has been estimated that one of the most toxic among all 75 known PCNs are hexachloronaphthalenes, mainly congeners 1,2,3,4,6,7-hexachloronaphthalene (PCN66), 1,2,3,5,6,7-hexachloronaphthalene (PCN67), and 1,2,3,6,7,8-hexachloronaphthalene (PCN70) , 48. Besin vitro models of primary hippocampal neurons and differentiated PC12. Here, we demonstrate that PCN67 disrupts neuronal sprouting and the formation of neurites. Moreover, our studies show the induction of mitochondrial-related necrotic death in a dose- and time-dependent manner. Our report is the first study, showing that mitochondria may be a primary intracellular target for PCN67 in neuronal cells.Despite widely documented harmful and adverse effects of PCN67, it is still unknown whether it may disturb neuronal function and produce subsequent neurotoxicity. To investigate the role of this substance in neurological processes, we employed All reagents, if not separately mentioned, were purchased from Sigma-Aldrich. The PC12 rat pheochromocytoma cell line was obtained from the American Type Culture Collection (ATCC). Maxima SYBR Green Master Mix, M-MLV Reverse Transcriptase, Trizol®, Alexa Fluor 488, Lipofectamine LTX reagent, B27, and Neurobasal were from Thermo Fisher Scientific. Protein Assay Kit was from Bio-Rad. Anti-GAPDH (Cat. No. sc-32233) and anti-histone H3 (Cat. No. sc-517576) were from Santa Cruz Biotechnology. HMGB1 antibody (Cat. No. 3935) was from Cell Signaling Technology. LDH Cytotoxicity Assay Kit was from Cayman Chemical. GcAMP3 calcium sensor was a gift from Loren Looger (Addgene plasmid #22692). Primers were synthesized in the Institute of Biochemistry and Biophysics (Poland).The mixture of hexachloronaphthalene congeners (94.14% purity) used in this study contained 81.17% of PCN67 as a dominant congener and additionally congeners 1,2,3,4,6,7-; 2,3,4,5,6,7-; and 1,2,4,5,6,7-hexachloronaphthalene comprising together up to 12.98% and 5.85% of 1,2,3,4,5,6,7-heptachloronaphtalene. The synthesis, characteristic, and purity were described previously , 40, 45.2. Cells were plated at the density of 1‐2 × 105/ml and cultured for 2 days before PCN67 administration. PCN67 was dissolved in DMSO and was added together with 1 mM dibutyryl-cAMP (differentiating agent). Cells were cultured with both agents for up to 72 h. No more than 20 cell passages were used for all experiments. Cells simultaneously cultured in the presence of 0.1% DMSO were used as a control. The black/white pictures of cell morphology were taken using an Olympus CK-40 inverted microscope equipped with a CCD camera.PC12 cells derived from pheochromocytoma were routinely grown in collagen-coated (type I from rat tail) plastic dishes in RPMI 1640 medium supplemented with 10% horse serum, 5% fetal bovine serum, 25 mM HEPES, pH 7.4, 2 mM L-glutamine, 1 mM sodium pyruvate, and the mix of penicillin/streptomycin in a humidified incubator at 37°C with 5% COWe used two parameters to score the potency of PC12 cell to differentiate into neuronal phenotype: the length of the longest neurite (any protrusion longer than the diameter of the cell body) and the number of cells possessing at least one neurite. Cells were chosen randomly. The images were captured at 400x magnification, and the neurites were measured using Image J software .3 cells were plated in each well of a 96-well plate. For WST-1 assay, plates were incubated with WST-1 solution in a 1 : 10 ratio for 4 h at 37°C. The absorbance was measured at 450 nm using a Victor X3 multiwell plate reader (PerkinElmer). The IC50 value was determined by nonlinear regression analysis in Prism 8.0 software . When indicated, cells were pretreated for 4 h with 5 μM BAPTA A/M added 48 h following PCN67 administration and the viability was assessed on the next day. Membrane permeability was determined in the presence of 7.5 μM propidium iodide. Ru360 or cyclosporine A (CsA) was added 30 min before PCN67 treatment, and the viability was measured after 72 h. For experiments with 0 mM extracellular Ca2+, following 48 h of incubation with PCN67, the growing medium was changed to Hank's balanced salt solution with or without calcium for 4 h and cells were imaged 24 h after using a Leica DM4000 microscope. For some experiments, cells were cultures in the presence of galactose (10 mM) in RPMI medium for 5 days following treatment with PCN67 for another 24 h.5 × 10g for 2 min and then triturated in the presence of DNase (100 U/ml) with a fire-polished glass pipet in Hank's balanced salt solution (HBSS) containing calcium and magnesium. Dissociated neurons were plated on nitric acid-treated 25 mm cover glass coated with poly-L-lysine in a plating medium. Four hours after plating, the medium was replaced with Neurobasal medium supplemented with 2% B27, 1 mM sodium pyruvate, and 2 mM L-glutamine. After four days, 4 μM cytosine arabinoside was added to inhibit glial proliferation, and the neurons were transfected with the pEGFP-N3 vector using Lipofectamine LTX.Hippocampal cultures were prepared from Sprague Dawley rat embryonic day 18 embryos. Briefly, the rat hippocampal CA1-CA3 region was dissected in PBS medium with 10 mM D-glucose and digested with 0.05% trypsin-EDTA in PBS for 20 min at 37°C. The dissociated tissues were centrifuged at 200The primary hippocampal neurons were grown until they reached either DIV4 or DIV14, and they were next treated with indicated PCN67 concentration. These stages were chosen because they correspond to distinct neuronal developmental processes such as axon formation and dendrite outgrowth in immature neurons (DIV4) or synaptogenesis associated with maturation (DIV14) as described previously . 40 mM Kμg of RNA. The gene expression was quantified using Maxima SYBR Green Master Mix in the conditions: 15 min at 95°C followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s using the Abi Prism 7000 sequence detection system (Applied Biosciences). The specificity of primers was checked by running a melting curve. Each time, the expression level of the gene of interest was normalized to the endogenous expression of Gapdh and the relative fold change was calculated using the 2ΔΔCt- method [Total cellular RNA was isolated from PC12 cells using Trizol reagent based on the protocol provided by the manufacturer. Single-stranded cDNA was synthesized using M-MLV reverse transcriptase and oligo(dT) primers using 1 t method . Either 6 PC12 cells were double stained with Annexin V  and propidium iodide (sensitive to necrotic cells) for 15 min at 25°C in the dark using the Annexin V-FITC Apoptosis Detection Kit based on the information provided by the manufacturer and analyzed using the FACScan Becton Dickinson flow cytometer. The fluorescence was collected from 104 cells, and the data were plotted with CellQuest Becton Dickinson software.1 × 10Lactate dehydrogenase (LDH) release from PC12 cells after drug treatment was measured with the LDH Cytotoxicity Assay Kit per manufacturer's protocol.3VO4, and protein inhibitor cocktail for 30 min. The lysates were next centrifuged at 800 x g for 15 min at 4°C; the supernatant was boiled for 5 min in the Laemmli buffer and stored at -80°C for further use. Protein concentration was measured using Bradford reagent according to the manufacturer's instruction. Postnuclear fraction referred here as cytosolic was obtained based on the method of Blobel and Potter [μg of cytosolic proteins were separated using 10% SDS-PAGE and transferred to a nitrocellulose membrane with a semidry method. Membranes were blocked with 5% bovine serum albumin in TBS-T buffer  for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies: anti-HMGB1 (1 : 1000), anti-GAPDH (1 : 2500), or antihistone H3 (1 : 1000), followed by 4 h incubation with secondary antibodies (1 : 10000) coupled with alkaline phosphatase. BCIP/NBT, an artificial alkaline phosphatase substrate, was used to visualize immunoreactive bands. Membranes were densitometrically quantified using Image J software . The results are presented as arbitrary units obtained after normalization of the marker protein.PC12 cells were lysed on ice with RIPA buffer supplemented with 1 mM PMSF, 2 mM Nad Potter . The purATP concentration in PC12 cells was determined using adenosine 5′-triphosphate (ATP) Bioluminescent Assay Kit on GloMax 20/20 luminometer and normalized to the protein content. For each set of measurements, a second negative control (no cells) was included, and background fluorescence was further subtracted from all other values. The results are presented as nmoles ATP/mg protein.2+ and membrane permeability live cell images were acquired using an inverted Zeiss Axio Observer 7 Marianas™ Microscope equipped with a X-Cite 120LED Boost White Light LED System and a high-resolution Prime™ Scientific CMOS digital camera that is controlled by a workstation loaded with SlideBook imaging and microscope control software . Differentiated PC12 cells were transfected with a GcAMP3 calcium sensor using Lipofectamine LTX and treated with PCN67 as described in Drug Treatment subsection in the presence of 7.5 μM propidium iodide. The images were acquired at 63x with the excitation time of 100 ms and one-hour interval time using the following filter set: GFP Exciter FF01-474/27, GFP Emitter FF01-525/45, and for propidium iodide: Exciter FF01-578/21 and Emitter FF02-641/75. The environmental control system  was used to keep constant experimental conditions . The focus was automatically controlled by Definite Focus.2 . Regions of interest (ROIs) after background subtraction were normalized as ΔF/F0 and processed using Microsoft Excel software. ROIs were averaged over each condition. At least 10 cells were imaged per experimental condition, and all experiments were performed in triplicate.Simultaneous CaΔΨm) was measured with TMRE (tetra-methyl-rhodamine-ethyl ester) whereas plasma membrane potential (ΔΨp) was measured with DiSBAC2  trimethine oxonol). Following 72 h of incubation with PCN67, PC12 cells were loaded with 25 nM TMRE or 1 μM DiSBAC2 for 30 min at 25°C in the dark in a buffer containing 20 mM HEPES, pH 7.4, 2 mM CaCl2, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 10 mM glucose and the fluorescence of 104 cells was recorded by a FACScan Becton Dickinson flow cytometer. The accompanying software analyzed the data. Cells incubated with 0.1% DMSO, used as a solvent for TMRE and PCN67, were monitored to record background fluorescence, which was later subtracted from the recordings.Mitochondrial membrane potential  with 488 nm excitation wavelength and emission at 525 nm. During each experiment, 104 cells were recorded. The background fluorescence was obtained with 0.1% DMSO.The ROS level was measured with DCFH-DA  following 72 h treatment with PCN67. Briefly, at the end of the treatment, PC12 cells were washed with PBS and incubated with DCFH-DA at a final concentration of 10 The level of thiobarbituric acid reactive substances (TBARS) in PC12 cells was determined according to the method described previously . The resμl of culturing medium was transferred into a 96-well plate and mixed in a 1 : 1 ratio with Griess reagent (1% sulfanilamide/0.1% NED in 5% phosphoric acid) followed by 10 min incubation in the dark. The absorbance at 540 nm was measured using a Victor X3 multiwell plate reader (PerkinElmer). DMSO-treated cells were used as control.Nitric oxide generation in PC12 cells following 72 h of PCN67 treatment was determined in phenol red-free media according to the method described previously . BrieflyP values for experiments involving multiple comparisons were obtained by the Tukey post hoc testing, albeit P values for not all comparisons are indicated on the graphs. P < 0.05 was considered statistically significant. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.All data are expressed as mean ± S.E.M from at least 3 experiments unless otherwise stated. One-way or two-way ANOVA was performed with matching as appropriate. in vitro neurotoxicity of PCN67, differentiated PC12 cells were exposed to a concentration ranging from 0.001 μg/ml to 25 μg/ml , P < 0.05). Increasing PCN67 concentration only potentiated the prodeath effect. Based on the dose-response curve presented in 50 value was 0.35 μg/ml while the IC10 was 0.039 μg/ml. In this context, we decided to employ the IC50 concentration and the highest concentration that did not produce significant cell death (0.1 μg/ml) for the following experiments done 72 h after PCN67 treatment.To assess  Figures . A signiμg/ml of PCN67 for 72 h did not affect the general morphology, in contrast to the IC50 concentration, which led to some cell detachment, floating and losing the characteristic neuronal-like shape. Moreover, treatment with this concentration resulted in the formation of neurites shorter by 15% , P < 0.05) but did not affect the number of cells bearing neuronal protrusions , P < 0.001) after treatment with IC50 concentration of PCN67 when compared to the vehicle-treated control , P < 0.01). These results demonstrate that PCN67 in a dose-dependent manner may interfere with the gene expression of NF-L and NF-M associated with the differentiation of PC12 cells into neuronal phenotype.PC12 cells exposed to 1 mM dibutyryl-cAMP showed de novo outgrowth of bi- or tripolar neurites, which appeared mostly straight, occasionally forming branches in some areas. The body of the cell was mostly polygonal. We scored two parameters: the length of the neurites and the percentage of the cell bearing neurites, to evaluate the effect of PCN67 on the differentiation potential of PC12 cells. As shown in  Figures . We subs control . The exp control  was reduin vitro. To search for any developmental-dependent effect, we used neurons at DIV4 and DIV14 that were susceptible to chronic KCl stimulation. It has been shown that KCl protects neurons from death resulting from overinhibition and from neurotrophic factor deprivation [50 concentration , P < 0.01) and no prosurvival effect of KCl , P < 0.05) upon treatment with IC50 concentration of PCN67 was also demonstrated at DIV14; however, KCl was able to abolish the prodeath effect of PCN67 and rescue the axonal length  in the total cell population was increased to 45% , P < 0.001) only in PC12 cells treated with PCN67 at the concentration of IC50 (+/PI−) were detected and the population of late apoptotic cells (Ax-V+/PI+) was of minor importance (less than 3%). To study this further, we measured the necrosis-associated membrane changes by detecting the release of lactate dehydrogenase (LDH) [50 , P < 0.001). In parallel, we observed the accumulation of HMGB1 protein (high-mobility group protein B1) in the cytosol , P < 0.001). In contrast to apoptosis, which is an ATP-dependent process, necrosis is energy-independent but results in ATP depletion [F, P < 0.05) . Collectively, our data suggests that PCN67 dose equivalent to IC50 induces the necrotic death of differentiated PC12 cells.To investigate the nature of cell death, we next quantified Annexin V/propidium iodide (Ax-V/PI) staining . The per of IC50 . In addise (LDH) . After tLDH) [50 , LDH pre cytosol , which i cytosol  comparedepletion . In this < 0.05) . Interes50 but not 0.1 μg/ml, resulted in a gradual increase in calcium level starting within the first 24 h and reaching significance at approximately 48 h and onward. After 2 days of treatment, a significant increase in propidium iodide uptake was visible, indicating plasma membrane hyperpermeability , P < 0.001) indicating plasma membrane depolarization . Its uptake to the cell is driven by electrochemical gradient which results in increased fluorescence . The mearization . This corization , 72 h. LΔΨm) were monitored using TMRE (tetra-methyl-rhodamine-ethyl ester), which accumulates in the mitochondrial matrix according to the Nernst equation [50 concentration resulted in massive mitochondrial depolarization after 72 h , P < 0.05) which was reflected by a decrease in TMRE fluorescence , P < 0.05) when measured 72 h after exposure to PCN67 , P < 0.05). We next tested whether observed necrotic death involved mitochondrial permeability transition pore (mPTP) opening. Pretreatment with cyclosporin A, a potent mPTP inhibitor, failed to protect from PCN67-induced cell death , P < 0.001) was detected at IC50 concertation  to measure ROS. Significantly elevated ROS level  based on viability assay. The IC50 (0.35 μg/ml) was additionally used to evaluate the nature of cell death. We employed the lowest PCN67 concentration (0.1 μg/ml) that did not alter the viability of PC12 cells as compared to the control. Surprisingly, we found that already low PCN67 concentration of 0.1 μg/ml which did not alter the viability of PC12 cells as compared to control induced cellular stress. Previous studies have shown that higher chlorinated polychlorinated naphthalenes, including PCN67, did not affect the viability of MCF-7 cells, when treated with the concentration of 100-10.000 pg/ml for 72 h [C. elegans [Drosophila [in vitro differentiation observed in our study can be, at least in part, mediated by AhR signaling. We demonstrated here that PCN67 treatment at IC50 concentration significantly decreased the number of neurite-forming cells and the average length of neurite in PC12 cells. A similar effect on axon length was also observed in primary hippocampal neurons, although more advanced culture stages (DIV14) seemed to be more resistant to PCN67 toxicity than the younger one (DIV4). Although no in vitro studies explored the effect of PCN67 on primary neurons so far, the finding of developmental-dependent sensitivity appears to be in line with our earlier work, showing the fetotoxic and embryotoxic effect of PCN67 at the doses that did not cause maternal toxicity [p-dioxin (TCDD) on viability and neuronal outgrowth in different in vitro models [Polychlorinated naphthalenes, a group of substances widely used up to the 1980s, are still considered as a serious environmental threat as they have been found in practically all ecosystems, and their accumulation has also been confirmed in humans . Despitefor 72 h . However elegans , affect osophila , and dimosophila . Polychlosophila ; therefotoxicity . Moreoveo models –87. Howeo models . Thus, t50 concertation was mitochondrial membrane depolarization and significant depletion of ATP. In parallel to increased permeability of the plasma membrane to small molecules , that started to be detectable 2 days following the treatment, we observed a gradual increase in cytosolic calcium concertation. Calcium is immediately taken up by mitochondrial calcium uniporter (MCU) which has been linked to H2O2 production, electron transport chain alteration, and disruption of ΔΨm homeostasis [α pathway. AMP kinase-PGC1α-dependent control of mitochondrial metabolism is expected to limit ROS production [One of the main characteristics of PCNs is high lipophilicity which allows to anticipate high penetration through biological membranes and rapid modification of membrane homeostasis and permeability. In our study, the earliest molecular change observed following PCN67 treatment at the ICeostasis . A signieostasis , may actoduction , which iμg/ml, which did not affect ΔΨm and plasma membrane permeability. We hypothesize that the observed dose-dependent effect can be attributed to the potency of PCN67 to influence cellular antioxidant defense, in particular GSH/GSSG redox potential. Indeed, it has been demonstrated that prolonged administration of PCNs in vivo decreased GSH concentration [μg/ml may imply that cellular defense systems are still sufficiently enough, to counteract oxidative damage. Moreover, the lack of damage to neurites could suggest that neuronal viability and function are preserved during short time exposure (72 h in this study) to lower PCN67 concentrations. However, since PCNs are highly bioaccumulative, it is plausible that exposure to even lower doses during a long-time period and concomitant gradual accumulation may ultimately lead to neurophysiological dysfunction. To support that, a variety of neurobehavioral tests performed on rats exposed to PCN67 showed dose-dependent impaired long-term memory, reduced pain threshold, and stress-induced analgesia [Compounds that target mitochondrial metabolism usually induce cellular stress , 93. Sinntration . Therefonalgesia . HoweverNeuronal injury may activate other nonneuronal cells in the CNS-like astrocytes and microglia to trigger a neuroinflammatory response. Therefore, we investigated whether PCN67-induced damage and necrotic death could also involve excessive production of NO, one of the main mediators of inflammation. We found no significant changes between vehicle- and PCN67-treated groups. Although no studies demonstrated the effect of PCNs on NO production, data derived from persistent organic pollutants with a similar mechanism of action indicated no evidence of nitrative stress in cerebellar neurons and in rat hypothalamus , 97. SinIt has been documented that dysfunctional mitochondria or altered synaptic transmission due to changes in the neuronal network could be associated with several neurobehavioral disorders such as anorexia , depress