Intracellular Fluorescence Intensity Research Articles

Photodynamic effect of vascular-targeted polyphenol nanoparticles on Endothelial cells

BackgroundPort wine stains (PWS) are vascular malformations, and photodynamic therapy (PDT) is a promising treatment. Emerging drug delivery methods employ nanoparticles (NPs) to enhance drug permeability and retention in diseased blood vessels and improve drug bioavailability. (-) -epigallocatechin-3-gallate glycine (EGCG) has anti-angiogenetic effects and boosts photodynamic therapy. Chlorin e6 (Ce6) is capable of efficiently producing singlet oxygen, rendering it a very promising photosensitizer for utilization in nanomedicine. Material and MethodsEGCG-Ce6-NPs were synthesized and characterized using various techniques. The photodynamic effects of EGCG-Ce6-NPs on endothelial cells were evaluated. The compatibility and toxicity of the nanoparticle was tested using the CCK-8 assay. The intracellular uptake of the nanoparticle was observed using an inverted fluorescence microscope, and the intracellular fluorescence intensity was detected using flow cytometry. The ROS generation and apoptosis induced by EGCG-Ce6-NPs was observed using confocal laser scanning microscopy and flow cytometry respectively. ResultsEGCG-Ce6-NPs exhibited stability, spherical shape of uniform size while reducing the particle diameter, low polydisperse profile and retaining the ability to effectively generate singlet oxygen. These characteristics suggest promising potential for enhancing drug permeability and retention. Additionally, EGCG-Ce6-NPs demonstrated good compatibility with endothelial cells and enhanced intracellular uptake of Ce6. Furthermore, EGCG-Ce6-NPs increased activation efficiency, induced significant toxicity, more reactive oxygen species, and a higher rate of late apoptosis after laser irradiation. ConclusionThis in vitro study showed the potentials EGCG-Ce6-NPs for the destruction of endothelial cells in vasculature.

Enhancing Glioma Treatment by Using Novel Cryptotanshinone-Loaded Nano-Liposomes

Despite the efficacy of cryptotanshinone (CPT) against glioma, it has issues such as poor solubility and limited tumor penetration. To address these challenges, the development of a nano drug delivery system with high penetration and precise targeting is crucial. In this study, we utilized the emulsification-evaporation technique to create tLyp-1 modified liposomes that contain CPT. The tLyp-1 peptide, which is a membrane-penetrating agent, allows for precise targeting of brain glioma and drug release. We characterized the TLCP using particle size, polydispersity index (PDI), intracellular fluorescence, and transmission electron microscopy and found that it had a mean particle size of (169.1±22.0) nm and a PDI of 0.34±0.093. High performance liquid chromatography was used to quantify the encapsulation efficiency (74.33±8.9%). Our results showed that tLipo, the targeting liposome modified with tLyp-1, was taken up more by GL261 cells than regular liposomes. The intracellular fluorescence intensity of the tLipo group also increased. Fluorescence was observed in the mouse brain 0.5 h after tail vein injection of DiR-labeled tLipo, confirming its ability to penetrate the blood–brain barrier (BBB). The fluorescence was still present in the brain 24 h later. Our results further confirmed the BBB-penetration and anti-glioma efficacy of the nanodrug in reducing glioma cell growth.

Near-infrared absorption and emission probes with optimal connection bridges for live monitoring of NAD(P)H dynamics in living systems

Two NAD(P)H-biosensing probes consisting of 1,3,3-trimethyl-3H-indolium and 3-quinolinium acceptors, linked by thiophene, A, and 3,4-ethylenedioxythiophene, B, bridges are detailed. We synthesized probes C and D, replacing the thiophene connection in probe A with phenyl and 2,1,3-benzothiadiazole units, respectively. Probe E was prepared by substituting probe A's 3-quinolinium unit with a 1-methylquinoxalin-1-ium unit. Probe solutions are non-fluorescent but in the presence of NADH, exhibit near-infrared fluorescence at 742.1 nm and 727.2 nm for probes A and B, respectively, and generate absorbance signals at 690.6 nm and 685.9 nm. In contrast, probes C and D displayed pronounced interference from NADH fluorescence at 450 nm, whereas probe E exhibited minimal fluorescence alterations in response to NAD(P)H. Pre-treatment of A549 cells with glucose in the presence of probe A led to a significant increase in fluorescence intensity. Additionally, subjecting probe A to lactate and pyruvate molecules resulted in opposite changes in NAD(P)H levels, with lactate causing a substantial increase in fluorescence intensity, conversely, pyruvate resulted in a sharp decrease. Treatment of A549 cells with varying concentrations of the drugs cisplatin, gemcitabine, and camptothecin (5, 10, and 20 µM) led to a concentration-dependent increase in intracellular fluorescence intensity, signifying a rise in NAD(P)H levels. Finally, fruit fly larvae were treated with different concentrations of NADH and cisplatin illustrating applicability to live organisms. The results demonstrated a direct correlation between fluorescence intensity and the concentration of NADH and cisplatin, respectively, further confirming the efficacy of probe A in sensing changes in NAD(P)H levels within a whole organism.

Development of light and pH-dual responsive self-quenching theranostic SPION to make EGFR overexpressing micro tumors glow and destroy.

Drug resistant and undetectable tumors easily escape treatment leading metastases and/or recurrence of the lethal disease. Therefore, it is vital to diagnose and destroy micro tumors using simple yet novel approaches. Here, we present fluorescence-based detection and light-based destruction of cancer cells that are known to be resistant to standard therapies. We developed a superparamagnetic iron oxide nanoparticle (SPION)-based theranostic agent that is composed of self-quenching light activated photosensitizer (BPD) and EGFR targeting ligand (Anti-EGFR ScFv or GE11 peptide). Photosensitizer (BPD) was immobilized to PEG-PEI modified SPION with acid-labile linker. Prior to stimulation of the theranostic system by light its accumulation within cancer cells is vital since BPD phototoxicity and fluorescence is activated by lysosomal proteolysis. As BPD is cleaved, the system switches from off to on position which triggers imaging and therapy. Targeting, therapeutic and diagnostic features of the theranostic system were evaluated in high and moderate level EGFR expressing pancreatic cancer cell lines. Our results indicate that the system distinguishes high and moderate EGFR expression levels and yields up to 4.3-fold increase in intracellular fluorescence intensity. Amplification of fluorescence signal was as low as 1.3-fold in the moderate or no EGFR expressing cell lines. Anti-EGFR ScFv targeted SPION caused nearly 2-fold higher cell death via apoptosis in high EGFR expressing Panc-1 cell line. The developed system, possessing advanced targeting, enhanced imaging and effective therapeutic features, is a promising candidate for multi-mode detection and destruction of residual drug-resistant cancer cells.

Cellular Efficacy of Fattigated Nanoparticles and Real-Time ROS Occurrence Using Microfluidic Hepatocarcinoma Chip System: Effect of Anticancer Drug Solubility and Shear Stress.

The objective of this study was to evaluate the effectiveness of organ-on-chip system investigating simultaneous cellular efficacy and real-time reactive oxygen species (ROS) occurrence of anticancer drug-loaded nanoparticles (NPs) using hepatocarcinoma cells (HepG2) chip system under static and hepatomimicking shear stress conditions (5 dyne/cm2). Then, the role of hepatomimetic shear stress exposed to HepG2 and drug solubility were compared. The highly soluble doxorubicin (DOX) and poorly soluble paclitaxel (PTX) were chosen. Fattigated NPs (AONs) were formed via self-assembly of amphiphilic albumin (HSA)-oleic acid conjugate (AOC). Then, drug-loaded AONs (DOX-AON or PTX-AON) were exposed to a serum-free HepG2 medium at 37 °C and 5% carbon dioxide for 24 h using a real-time ROS sensor chip-based microfluidic system. The cellular efficacy and simultaneous ROS occurrence of free drugs and drug-loaded AONs were compared. The cellular efficacy of drug-loaded AONs varied in a dose-dependent manner and were consistently correlated with real-time of ROS occurrence. Drug-loaded AONs increased the intracellular fluorescence intensity and decreased the cellular efficacy compared to free drugs under dynamic conditions. The half-maximal inhibitory concentration (IC50) values of free DOX (13.4 μg/mL) and PTX (54.44 μg/mL) under static conditions decreased to 11.79 and 38.43 μg/mL, respectively, under dynamic conditions. Furthermore, DOX- and PTX-AONs showed highly decreased IC50 values of 5.613 and 21.86 μg/mL, respectively, as compared to free drugs under dynamic conditions. It was evident that cellular efficacy and real-time ROS occurrence were well-correlated and highly dependent on the drug-loaded nanostructure, drug solubility and physiological shear stress.

DNA Nanoribbon-Assisted Intracellular Biosynthesis of Fluorescent Gold Nanoclusters for Cancer Cell Imaging.

Metal nanoclusters (NCs) have emerged as a promising class of fluorescent probes for cellular imaging due to their high resistance to photobleaching and low toxicity. Nevertheless, their widespread use in clinical diagnosis is limited by their unstable intracellular fluorescence. In this study, we develop an intracellularly biosynthesized fluorescent probe, DNA nanoribbon-gold NCs (DNR/AuNCs), for long-term cellular tracking. Our results show that DNR/AuNCs exhibit a 4-fold enhancement of intracellular fluorescence intensity compared to free AuNCs. We also investigated the mechanism underlying the fluorescence enhancement of AuNCs by DNRs. Our findings suggest that the higher synthesis efficiency and stability of AuNCs in the lysosome may contribute to their fluorescence enhancement, which enables long-term (up to 15 days) fluorescence imaging of cancer cells (enhancement of ∼60 times compared to free AuNCs). Furthermore, we observe similar results with other metal NCs, confirming the generality of the DNR-assisted biosynthesis approach for preparing highly bright and stable fluorescent metal NCs for cancer cell imaging.

Protective effect of curcumin on hepatolenticular degeneration through copper excretion and inhibition of ferroptosis

BackgroundHepatolenticular degeneration (HLD) is an autosomal recessive disorder concerning copper metabolism. Copper overload is also accompanied by iron overload in HLD patients, which can lead to ferroptosis. Curcumin, the active component in turmeric, has the potential to inhibit ferroptosis. PurposeThe current study proposed a systematic investigation of the protective effects of curcumin against HLD and the underlying mechanisms. MethodsThe protective effect of curcumin on toxic milk (TX) mice was studied. Liver tissue was observed via hematoxylin-eosin (H&E) staining and the ultrastructure of the liver tissue was observed through transmission electron microscopy. Copper levels in the tissues, serum, and metabolites were measured by atomic absorption spectrometry (AAS). In addition, serum and liver indicators were evaluated. In cellular experiments, the effect of curcumin on the viability of rat normal liver cells (BRL-3A) was determined via the 3-[4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) assay. Cell and mitochondrial morphology were observed in curcumin-mediated HLD model cells. The intracellular copper ion fluorescence intensity was observed via fluorescence microscopy, and intracellular copper iron content was detected using AAS. Further, oxidative stress indicators were evaluated. Cellular reactive oxygen species (ROS) and cellular mitochondrial membrane potential were examined via flow cytometry. Furthermore, the expression levels of nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutathione peroxidase 4 (GPX4) were determined via western blotting (WB). ResultsThe histopathology of the liver confirmed the hepatoprotective effects of curcumin. Curcumin improved copper metabolism in TX mice. Both serum liver enzyme markers and antioxidant enzyme levels indicated the protective effect of curcumin against HLD-related liver injury. The MTT assay results showed that curcumin was protective against excess copper-induced injury. Curcumin improved the morphology of HLD model cells and their mitochondrial morphology. The Cu2+ fluorescent probe and the AAS results indicated that curcumin reduced Cu2+ content in HLD hepatocytes. In addition, curcumin improved oxidative stress levels and prevented the decline of mitochondrial membrane potential in HLD model cells. The ferroptosis inducer Erastin reversed these effects of curcumin. WB revealed that curcumin promoted Nrf2, HO-1, and GPX4 protein expression in HLD model cells, and the Nrf2 inhibitor ML385 reversed the effects of curcumin. ConclusionCurcumin demonstrates a protective role by expelling copper and inhibiting ferroptosis, activating the Nrf2/HO-1/GPX4 signaling pathway in HLD.

Exogenous melatonin stimulated isoflavone biosynthesis in NaCl-stressed germinating soybean (Glycine max L.)

Melatonin (MT) has gained increasing attention due to its pleiotropic effects. In this study, the function of exogenous MT on the response to NaCl stress and isoflavone biosynthesis in germinating soybeans was investigated. Results showed the exogenous MT (100 μM) application neutralised the negative effects of NaCl stress (60 mM), induced sprout growth, biomass and fluorescence intensity of intracellular free calcium, decreased malondialdehyde, H2O2 content and fluorescence intensity of O2•-, and enhanced superoxide dismutase, catalase and peroxidas activities of germinating soybeans. Meanwhile, total flavonoids and different forms of isoflavone content were enhanced by MT application, not only companied by the up-regulated relative gene expression of cinnamic acid 4-hydroxylase chalcone reductase, chalcone isomerase 1A, isoflavone reductase and isoflavone synthase 1 that involved in isoflavone biosynthesis, but also increased activities of phenylalanine ammonia lyase and 4-coumarate coenzyme A ligase. Given the evidence from the present study, it's proposed that the exogenous MT could relieve NaCl stress and stimulate isoflavone biosynthesis in germinating soybeans.

Dual Cathepsin B and Glutathione-Activated Dimeric and Trimeric Phthalocyanine-Based Photodynamic Molecular Beacons for Targeted Photodynamic Therapy.

Two dual stimuli-activated photosensitizers were developed, in which two or three glutathione (GSH)-responsive 2,4-dinitrobenzenesulfonate (DNBS)-substituted zinc(II) phthalocyanine units were connected via one or two cathepsin B-cleavable Gly-Phe-Leu-Gly peptide linker(s). These dimeric and trimeric phthalocyanines were fully quenched in the native form due to the photoinduced electron transfer to the DNBS substituents and the self-quenching of the phthalocyanine units. In the presence of GSH and cathepsin B, or upon internalization into A549 and HepG2 cancer cells, these probes were activated through the release of free phthalocyanine units. The intracellular fluorescence intensity was increased upon post-incubation with GSH ester or reduced upon pre-treatment with a cathepsin B inhibitor. Upon light irradiation, these photosensitizers became highly cytotoxic with IC50 values of 0.21-0.39 μM. The photocytotoxicity was also dependent on the intracellular GSH and cathepsin B levels. The results showed that these conjugates could serve as smart photosensitizers for targeted photodynamic therapy.

Protective effect of 18β-glycyrrhetinic acid against H2O2-induced injury in Schwann cells based on network pharmacology and experimental validation.

The aim of the present study was to assess the protective effects of 18β-GA against hydrogen peroxide (H2O2)-induced injury. First, the SMILES annotation for 18β-GA was used to search PubChem and for reverse molecular docking in Swiss Target Prediction, the Similarity Ensemble Approach Search Server and the TargetNet database to obtain potential targets. Injury-related molecules were obtained from the GeneCards database and the predicted targets of 18β-GA for injury treatment were selected by Wayne diagram analysis. Subsequently, Kyoto Encyclopedia of Genes and Genomes analysis was performed by WebGestalt. The experimental cells were assorted into control, model, 10 µM SB203580-treated, 5 µM 18β-GA-treated and 10 µM 18β-GA-treated groups. Hoechst 33258 staining was performed and intracellular reactive oxygen species (ROS) levels, cell apoptosis, Bcl-xl, Bcl-2, Bad, Bax, cleaved-caspase 3, cleaved-caspase 7, transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) levels, as well as p38 MAPK phosphorylation were measured. The ‘Inflammatory mediator regulation of TRP channels’ pathway was selected for experimental verification. The results indicated that 10 µM 18β-GA significantly increased cell viability as compared with the H2O2-treated model group. As suggested by the difference in intracellular ROS fluorescence intensity, 18β-GA inhibited H2O2-induced ROS production in Schwann cells. Hoechst 33258 staining indicated that 18β-GA reversed chromatin condensation and the increase in apoptotic nuclei following H2O2 treatment. Furthermore, flow cytometry suggested that 18β-GA substantially inhibited H2O2-induced apoptosis. Pre-treatment with 18β-GA obviously reduced Bad, Bax, cleaved-caspase3, cleaved-caspase 7, TRPA1 and TRPV1 levels and p38 MAPK phosphorylation after H2O2 treatment and increased Bcl-2 and Bcl-xl levels. In conclusion, 18β-GA inhibited Schwann cell injury and apoptosis induced by H2O2 and may be a potential drug to prevent peripheral nerve injury.

Study on Cancer Cell Invasiveness via Application of Mechanical Force to Induce Chloride Ion Efflux.

Chloride channels regulate cell volume by an efflux of chloride ions in response to osmotic stresses. These have been shown to play a role in cancer invasion. However, their function in cancer metastasis remains unclear. As the internal environment of the human body is rarely exposed to osmotic stress, we presumed that Cl- efflux in cancer cells is induced by mechanical stress caused by their crowded environment and invasion of their narrow interstitial spaces. In this study, we recruited atomic force microscopy to apply mechanical stress to mouse or human breast cancer cells with varying degrees of malignancy and examined their Cl- efflux by N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE), which is quenched via collision with Cl- ions. We found that intracellular MQAE fluorescence intensity increased immediately after cell compression, demonstrating induction of Cl- efflux by mechanical force. Furthermore, Cl- efflux ability showed correlation with the cancer metastatic potential. These results suggested that mechanical stress induced Cl- efflux may serve as a potential reporter for estimating the invasion ability of cancer cells.

5‑Nitro‑2‑(3‑phenylpropylamino) benzoic acid induces apoptosis of human lens epithelial cells via reactive oxygen species and endoplasmic reticulum stress through the mitochondrial apoptosis pathway.

Cataracts have a high incidence and prevalence rate worldwide, and they are the leading cause of blindness. Lens epithelial cell (LEC) apoptosis is often analysed in cataract research since it is the pathological basis of cataracts, except for congenital cataract. Chloride channels are present in ocular tissues, such as in trabecular cells, LECs and other cells. They serve an important role in apoptosis and participate in endoplasmic reticulum (ER) stress and oxidative stress. However, their role in the apoptosis of LECs has not been discussed. The present study examined the effects of the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) in human LECs (HLECs) to elucidate the role of NPPB in HLECs and investigate the role and mechanism of chloride channels in cataract formation. HLECs were exposed to NPPB. Cell survival rate was evaluated using Cell Counting Kit-8 assays. Oxidative stress was detected as reactive oxygen species (ROS) in cells by using a ROS assay kit. Apoptosis was examined by assessing mitochondrial membrane potential and using a JC-1 assay kit, and western blot analysis was performed to measure the expression levels of mitochondrial-dependent apoptosis pathway-associated proteins. ER stress was evaluated by determining the intracellular calcium ion fluorescence intensity, and western blot analysis was performed to measure ER stress-associated protein expression. The results revealed that NPPB treatment decreased the viability of HLECs and increased apoptosis. Additionally, NPPB increased intracellular ROS levels, as well as the number of JC-1 monomers and the protein expression levels of B-cell lymphoma-2 (Bcl-2)-associated X and cleaved caspase-3, and decreased Bcl-2 protein expression. NPPB increased intracellular calcium ions, the protein expression levels of activating transcription factor 6, JNK, C/EBP homologous protein and caspase-12, and the phosphorylation of protein kinase R-like endoplasmic reticulum kinase. N-acetylcysteine and 4-phenylbutyric acid inhibited NPPB-induced oxidative stress, ER stress and apoptosis. Therefore, NPPB treatment decreased cell viability and promoted apoptosis of HLECs via the promotion of oxidative and ER stress.

Protective effect of paeoniflorin on H2O2 induced Schwann cells injury based on network pharmacology and experimental validation.

This study was to investigate the protective effect of paeoniflorin (PF) on hydrogen peroxide-induced injury. Firstly, "SMILES" of PF was searched in Pubchem and further was used for reverse molecular docking in Swiss Target Prediction database to obtain potential targets. Injury-related molecules were obtained from GeenCards database, and the predicted targets of PF for injury treatment were selected by Wayne diagram. For mechanism analysis, the protein-protein interactions were constructed by String, and the KEGG analysis was conducted in Webgestalt. Then, cell viability and cytotoxicity assay were established by CCK8 assay. Also, the experimental cells were allocated to control, model (200 μmol·L-1 H2O2), SB203580 10 μmol·L-1 (200 μmol·L-1 H2O2+ SB203580 10 μmol·L-1), PF 50 μmol·L-1 (200 μmol·L-1 H2O2+ PF 50 μmol·L-1), and PF 100 μmol·L-1 (200 μmol·L-1 H2O2+ PF 100 μmol·L-1) groups. We measured the intracellular ROS, Hoechst 33258 staining, cell apoptosis, the levels of Bcl-xl, Bcl-2, Caspase-3, Cleaved-caspase3, Cleaved-caspase7, TRPA1, TRPV1, and the phosphorylation expression of p38MAPK. There are 96 potential targets that may be associated with PF for injury treatment. Then, we chose the "Inflammatory mediator regulation of TRP channels" pathway for the experimental verification from the first 10 KEGG pathway. In experimental verification, H2O2 decreased the cell viability moderately (P < 0.05), and 100 μmol·L -1 PF increased the cell viability significantly (P < 0.05). Depending on the difference of intracellular ROS fluorescence intensity, PF inhibited H 2O2-induced reactive oxygen species production in Schwann cells. In Hoechst 33258 staining, PF reversed the condensed chromatin and apoptotic nuclei following H2O2 treatment. Moreover, Flow cytometry results showed that PF could substantially inhibit H2O2 induced apoptosis (P < 0.05). Pretreatment with PF obviously reduced the levels of Caspase3, Cleaved-caspase3, Cleaved-caspase7, TRPA1, TRPV1, and the phosphorylation expression of p38MAPK after H 2O2 treatment (P < 0.05), increased the levels of Bcl-2, and Bcl-xl ( P < 0.05). PF inhibited Schwann cell injury and apoptosis induced by hydrogen peroxide, which mechanism was linked to the inhibition of phosphorylation of p38MAPK.

Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells

Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells

Effects of BαP and TBBPA on multixenobiotic resistance (MXR) related efflux transporter activity and gene expressions in gill cells of scallop Chlamys farreri.

The multixenobiotic resistance mechanism (MXR) provides aquatic organisms with the capacity to adapt to polluted environments, which can be inhibited by chemosensitizers. In the present study, the effect of two typical marine persistent organic pollutants, benzo(a)pyrene (BaP) and tetrabromobisphenol A (TBBPA), on the most relevant ABC transporters, ABCB1, ABCC1, and ABCG2 of scallop Chlamys farreri was tested. MXR transporter efflux activity of cultured gill cells of the scallops was evaluated by measuring the intracellular fluorescent intensity of Calcein-AM and rhodamine 123 with flow cytometry. The results showed that ABCB1 and ABCC1 transporters demonstrated increased activity compared with ABCG2 in mediating MXR efflux activity. BaP and TBBPA were able to suppress the efflux transporter activity of ABC transporters significantly, of which BaP revealed block effects by acting on the ABCB1 transporter. Additionally, exposure of BaP and TBBPA only significantly upregulated the expression level of ABCC1 gene.This study demonstrated the promising utility of efflux transporter activity in conjunction with biomarkers such as mRNA levels in identification of chemosensitizer.

SURG-10. SPECTROSCOPIC MEASUREMENT OF 5-ALA-INDUCED INTRACELLULAR PROTOPORPHYRIN IX IN PEDIATRIC BRAIN TUMORS

OBJECTIVE5-ALA guided resection of glioma in adults enables better delineation between tumor and normal brain, allowing improved resection and improved patients’ outcome. Recently, several reports were published regarding 5-ALA for resection of pediatric brain tumors. The aim of the study was to determine the intracellular fluorescence of PPIX in pediatric brain tumors by hyperspectral imaging and to compare it with visually observed intraoperative fluorescence.METHODS5-ALA was administered orally four hours prior to surgery. During tumor resection the surgeon assessed the fluorescence signal to be strong, weak or absent. Subsequently, fluorescence intensity of samples was measured via spectroscopy. In addition, clinical data, imaging and laboratory data were analyzed.RESULTSEleven children (1–16 years) were operated. Tumor entities included: three medulloblastomas, two pilocytic astrocytomas (PA), two anaplastic ependymomas and one diffuse astrocytoma, anaplastic astrocytoma, pilomyxoid astrocytoma and anaplastic pleomorphic xanthoastrocytoma. Strong fluorescence was visible in all anaplastic tumors and one PA; one PA demonstrated weak fluorescence. Visible fluorescence was strongly associated with intracellular fluorescence intensity and PPIX concentration (P<0.05). Within all tumors with visible fluorescence the intracellular PPIX concentration was greater than 4 µg/ml. Except for moderate and transient elevation of liver enzymes, no 5-ALA related adverse events were reported.CONCLUSIONWe demonstrate a strong association between intraoperative observations and spectrometric measurements of PPIX fluorescence in tumor tissue. As in former studies, fluorescence signal was more commonly observed in malignant glial tumors. Further prospective controlled trials should be conducted to investigate the feasibility of 5-ALA guided resection of pediatric brain tumors.

Mechanisms of the mechanically activated ion channel Piezo1 protein in mediating osteogenic differentiation of periodontal ligament stem cells via the Notch signaling pathway

To explore the mechanism of Piezo1 protein in mediating the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) via the Notch signaling pathway. In this study, young permanent teeth extracted from impacted teeth of 8-14-year- old children from January 1, 2016 to January 1, 2018 in the Department of Orthodontic, Beijing Children's Hospital were selected as cell sources. hPDLSCs were extracted by enzymatic digestion. Immunohistochemical staining was used to detect the expression of keratin and vimentin, and flow cytometry was used to identify the markers (CD146 and STRO-1) of hPDLSCs. The construction and screening of Piezo1 siRNA gene interference vector and Piezo1 gene overexpression plasmid were completed. Flexcell 4000T mechanical distraction stress instrument was used to construct hPDLSC cell model in vitro. According to the preliminary results, the experiment was divided into five groups: siRNA interference group, overexpression group, blank control group, stretch stress group, and negative control group. Real time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of Piezo1, Notch1, alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and bone sialoprotein (BSP). Western blot was used to detect the expression of ALP and Runx2. Fluo-3 AM probe was used to detect intracellular calcium content. Vimentin staining of hPDLSCs was positive, and keratin staining was negative. Flow cytometry was used to detect the expression of STRO-1 and CD146, markers of hPDLSC. Empty viral vectors, siRNA-Piezo1 interference sequence, and Piezo1 overexpression vector sequence could be transfected into hPDLSC by lentivirus, and the transfection efficiency was high (approximately 90%). The reverse transcription-polymerase chain reaction (RT-PCR) results showed that there were significant differences in Piezo1 gene levels among the siRNA interference group, overexpression group, blank control group, stretch stress group, and negative control group (F=9.573, P<0.05). The level of Piezo1 in the overexpression group was significantly higher than that in the siRNA interference group (q=3.893, P<0.05). The level of Piezo1 in the stretch stress group was significantly higher than that in the blank control group (q=2.006, P<0.05). The expression of Notch1 and osteogenic genes ALP, Runx2, OCN, and BSP had the same trend. Western blot results showed that there were significant differences in the expression of ALP in the siRNA interference group, overexpression group, blank control group, stretch stress group, and negative control group (F=11.207, P<0.001). The expression level of ALP in the overexpression group was significantly higher than that in the siRNA interference group (q=2.991, P<0.05). The expression of ALP in the stretch stress group was significantly higher than that in the blank control group (q=3.007, P<0.05). The expression of Runx2 protein showed the same trend. The intracellular calcium fluorescence intensity of the overexpression group was significantly higher than that of the siRNA interference group, and the intracellular calcium fluorescence intensity of the stretch stress group was significantly higher than that of the siRNA interference group. Mechanical stretch stress can promote the expression of Piezo1 protein. Ca2+ is the second messenger, activates the Notch1 signaling pathway and the expression of ALP, Runx2, OCN, and BSP; and promotes the osteogenic differentiation of hPDLSC. The siRNA-Piezo1 interfering plasmid can block this process. On the contrary, the overexpression plasmid of Piezo1 can promote the osteogenic differentiation of PDLSCs.

Valproic Acid Increased Autophagic Flux in human Multiple Myeloma Cells in Vitro

BackgroundTo investigate the effects of valproic acid (VPA) on autophagic flux in multiple myeloma (MM) cells. Methods and ResultsCell proliferation was assayed by the Cell Counting Kit-8 assay. The qRT-PCR was used to measure the expressions of LC3-II at mRNA level. Autophagic flux was measured by LC3-II turnover using western blot analysis and flow cytometry using the fluorescent dye Cyto-ID. An assay using the RFP-GFP-LC3 tandem construct was performed to monitor autophagic flux. Cell proliferation assay showed that VPA could inhibit the proliferation of MM cells and the inhibitory effects were enhanced with the extension of time. The qRT-PCR and western blot showed that the expression level of LC3-II in the VPA plus CQ group was significantly higher than that in CQ group. Cyto-ID autophagy test showed that the intracellular average fluorescence intensity in VPA plus CQ group was significantly higher than that in control and VPA group (all p < 0.001). The results of RFP-GFP-LC3 tandem construct showed that the numbers of yellow puncta and red puncta in VPA group was higher than that in control group. ConclusionsVPA could inhibit the proliferation of MM cells and the inhibitory effects were enhanced with the extension of time. VPA could enhance autophagic flux in MM cells, and the increase of autophagosomes was caused by autophagy enhancement rather than inhibition. These findings provided rationale for the treatment of MM with VPA.

Preferential drug delivery to tumor cells than normal cells using a tunable niosome\u2013chitosan double package nanodelivery system: a novel in vitro model

BackgroundWe previously described the properties of a targeted drug delivery system (DDS) in a cell-free system. Here, in this comparative cell-based study (normal and tumor cells), we provide a quantitative analysis of the extracellular diffusion and intracellular localization of this DDS. This DDS consists of fluorescence-labeled paclitaxel encapsulated in non-ionic surfactant vesicles/niosomes embedded in a thermo-sensitive cross-linked chitosan hydrogel with an affinity for the MUC1 mucin surface antigen overexpressed on tumor cells, and designed for a sustained and controlled, localized delivery of embedded drugs. We evaluated DDS in our novel in vitro model using MatTek’ glass-bottom culture plates and compared human cancer cell lines (OV2008 epithelial origin carcinoma and U373 glioma, both overexpressing MUC1) with human normal epithelial control cell lines (IMMC3 and IOSE-121 using differential contrast and confocal microscopy.ResultsTumor cells incubated in the presence of chitosan alone or DDS-containing chitosan–niosome–paclitaxel–BODIPY 564/570, showed a prominent granular accumulation on their surface when compared to the normal cells. Quantitation of gray value light intensity of the extracellular region of chitosan alone treated OV2008 and IOSE-121 controls done by analysis of multiple radial line segments, 4 µm each, using ImageJ software showed 2 times higher intensity around the OV2008 than around normal IOSE-121 controls (p < 0.05). In the DDS-treated OV2008 cells, extracellular fluorescence intensity measured at different diffusion distances outside of the cells, in three different zones showed the difference in means of fluorescence intensity in these zones (p < 0.05) with the highest level of fluorescence near the cell surface indicating a concentration gradient, most likely driven by the high affinity of chitosan to the MUC1 receptor. Also, as chitosan alone accumulated two times more along the edge of tumor cells compared to normal cells, we found intracellular fluorescence intensity quantified at time intervals to be also 2 times higher in OV2008 than in normal IMCC3 cells (p < 0.05).ConclusionBased on the observation of the DDS preferentially targeting tumor cells, there is a potential implication for the localized delivery of therapeutic drug doses to solid tumors or post-surgical solid tumors cavities containing residual tumor cells.

Spectroscopic measurement of 5-ALA-induced intracellular protoporphyrin IX in pediatric brain tumors.

5-Aminolevulinic acid (5-ALA)-guided resection of gliomas in adults enables better delineation between tumor and normal brain, allowing improved resection and improved patients' outcome. Recently, several reports were published regarding 5-ALA for resection of pediatric brain tumors. The aim of the study was to determine the intracellular fluorescence of protoporphyrin IX (PPIX) in pediatric brain tumors by hyperspectral imaging and to compare it with visually observed intraoperative fluorescence. 5-ALA was administered orally 4h prior to surgery. During tumor resection, the surgeon assessed the fluorescence signal to be strong, weak, or absent. Subsequently, fluorescence intensity of tumor samples was measured via spectroscopy. In addition, clinical data, imaging, and laboratory data were analyzed. Eleven children (1-16years) were operated. Tumor entities included three (n = 3) medulloblastomas, two (n = 2) pilocytic astrocytomas (PA), two (n = 2) anaplastic ependymomas and one (n = 1) diffuse astrocytoma, anaplastic astrocytoma (n = 1), pilomyxoid astrocytoma (n = 1) and anaplastic pleomorphic xanthoastrocytoma (n = 1). Strong fluorescence was visible in all anaplastic tumors and one PA; one PA demonstrated weak fluorescence. Visible fluorescence was strongly associated with intracellular fluorescence intensity and PPIX concentration (P < 0.05). Within all tumors with visible fluorescence, the intracellular PPIX concentration was greater than 4μg/ml. Except for moderate and transient elevation of liver enzymes, no 5-ALA related adverse events were reported. We demonstrate a strong association between intraoperative observations and spectrometric measurements of PPIX fluorescence in tumor tissue. As in former studies, fluorescence signal was more commonly observed in malignant glial tumors. Further prospective controlled trials should be conducted to investigate the feasibility of 5-ALA-guided resection of pediatric brain tumors.