Confocal Fluorescence Imaging Research Articles

Development of polysaccharide-complexed nano-sized rice protein dispersion.

The objective of this study was to improve water solubility of the rice protein (RP) by forming complexes with anionic polysaccharides, such as sodium alginate (SA) and xanthan gum (XG). In addition, utilization of the RP complexes as an emulsifier was evaluated. The prepared RP-SA or RP-XG complexes were analyzed by measuring their particle size, ζ-potential, and water solubility as well as by confocal laser scanning microscopy. The formation of a complex between RP-SA and RP-XG improved the water solubility and dispersibility of RP over a wide range of pH values (3, 5, 7, and 9). Confocal fluorescence images showed that the aggregation of RP molecules was prevented by the formation of complexes between RP and polysaccharides. When soybean oil-in-water emulsions were prepared with complexes, RP-SA (ratio 4:1) and RP-XG(ratio 4:1) complex-stabilized emulsions were stable for 4weeks of storage.

Characterization of a Novel Gja8 (Cx50) Mutation in a New Cataract Rat Model.

To describe a novel spontaneous cataract inbred strain isolated from large-scale breeding SD rats, identify the responsible gene mutation, and understand how this mutation affects lens function. Exome sequencing of 12 cataract-associated genes was performed in the affected and healthy relatives. Sequences of rat wild-type or mutant gap junction protein alpha 8 gene (Gja8) were transfected into cells. The expression level of protein was assayed by Western blot analysis. Subcellular localization of connexin 50 (Cx50) was analyzed in confocal fluorescent images. Wound-healing, 5-ethynyl-2'-deoxyuridine incorporation, and attachment assay were performed to characterize the cell migration, proliferation and adhesion. The abnormality was found to be inheritable in an autosomal semi-dominant pattern through different mating patterns. We found a G to T transversion at codon 655 in Gja8, leading to a substitution of valine by phenylalanine (p.V219F). Gja8V219F/+ heterozygotes expressed nuclear cataract while Gja8V219F/V219F homozygotes manifested microphthalmia in addition to cataract. Histology revealed fiber disorders and loss of organelle-free zone in the mutant lens. Cx50V219F altered its location in HeLa cells and inhibited the proliferation, migration and adhesion abilities of HLEB3 cells. The mutation also reduced the expression of focal adhesion kinase and its phosphorylation. The c.655G>T mutation (p.V219F) is a novel mutation in Gja8, inducing semi-dominant nuclear cataracts in a new spontaneous cataract rat model. The p.V219F mutation altered Cx50 distribution, inhibited lens epithelial cell proliferation, migration, and adhesion, and disrupted fiber cell differentiation. As a consequence, the nuclear cataract and small lens formed.

Coal humus acid functionalized high stability fluorescent copper nanoclusters for tumor identification by sequential off-on-off monitoring tryptophan and Hg2.

The abnormalities of Tryptophan (Trp) and mercury ions (Hg2+) not only easily activate diseases, including mental illness and cancer, but also seriously affect human wellbeing. Fluorescent sensors are profoundly attractive options for identifying amino acids and ions; however, most sensors remain challenging due to the multipliable cost and deviation from the asynchronous quenching detection. In particular, fluorescent copper nanoclusters with high stability that quantitatively monitoring Trp and Hg2+ successively have seldom been reported. Herein, we employ coal humus acid (CHA) as a protective ligand and successfully construct weak cyan fluorescent copper nanoclusters (CHA-CuNCs) by a rapid, environmentally benign and cost-effective method. Significantly, the fluorescence of CHA-CuNCs is obviously improved by introducing Trp, because the indole group of Trp enhances the radiative recombination and aggregation-induced emissions. Interestingly, CHA-CuNCs not only realizes the highly selective and specific detection of Trp with a linear range of 25-200μM and a detection limit of 0.043μM based on the turn-on fluorescence strategy, but also quickly achieves the consecutive turn-off detection of Hg2+ due to the chelation interaction between Hg2+ and pyrrole heterocycle in Trp. Moreover, this method is successfully applied in the analysis of Trp and Hg2+ in real samples. Furthermore, the confocal fluorescent imaging of tumor cells demonstrates that CHA-CuNCs can be used for bioimaging and cancer cell recognition with Trp and Hg2+ abnormalities. These findings provide new guidance for the eco-friendly synthesis of CuNCs with eminent sequential off-on-off optical sensing property, indicating good prospects in biosensing and clinical medicine applications.

A MEMS Mirror-Based Confocal Laser Endomicroscope with Image Distortion Correction

Scanning MEMS mirrors can extend confocal laser microscopy into endoscopic applications, but the practical use of MEMS mirror-based confocal endomicroscopy is hindered partially by various image distortions such as barrel, fan-shaped and nonlinear distortions. In this work, the nonlinear scanning behaviors of an electrothermal MEMS mirror are analyzed and incorporated into an optical scanning model that takes all these three types of distortions into account. The model generates a 2D spatial mapping that can be applied to correct all of the image distortions in one step without a calibration board. To experimentally validate this method, a confocal laser endomicroscope employing a two-axis scanning electrothermal MEMS micromirror is designed and constructed, and confocal fluorescence images of a patterned micro-structure are obtained with the MEMS endomicroscope. The results show that the overall image distortion is reduced by at least one order of magnitude in the length direction.

A triphenylamine-based fluorescence probe for detection of hypochlorite in mitochondria

The level of HClO/ClO− in mitochondria is essential to keep the normal function of mitochondria. Therefore, it is meaningful to accurately and quickly monitor ClO− in mitochondria. In this work, a new triphenylamine-based fluorescence probe PDTPA was designed and synthesized, in which pyridinium salt and dicyano-vinyl group were introduced as mitochondria targeting site and reaction site for ClO−. The probe showed high sensitivity and fast fluorescence response (<10 s) in the detection of ClO−. Moreover, the probe PDTPA had good linearity in a wide concentration range of ClO− and its detection limit was calculated as 10.5 μM. Confocal fluorescence images demonstrated that the probe could target mitochondria and track the fluctuations of endogenous/exogenous ClO− levels in the mitochondria of living cells.

Breast carcinoma detection in ex vivo fresh human breast surgical specimens using a fast slide-free confocal microscopy scanner: HIBISCUSS project.

New generation ultra-fast fluorescence confocal microscopy allows the ex vivo intraoperative analysis of fresh tissue. The High resolution Imaging for Breast carcInoma detection in ex vivo Specimens after breast Conserving sUrgery by hiStolog Scanner (HIBISCUSS) project aimed to develop an online learning program to recognize the main breast tissue features on ultra-fast fluorescence confocal microscopy images and to evaluate the performance of surgeons and pathologists in diagnosing cancerous and non-cancerous breast tissue in ultra-fast fluorescence confocal microscopy images. Patients who underwent conservative surgery or mastectomy for breast carcinoma (invasive or in situ lesions) were included. The fresh specimens were stained with a fluorescent dye and imaged using a large field-of-view (20 cm2) ultra-fast fluorescence confocal microscope. One hundred and eighty-one patients were included. The images from 55 patients were annotated to generate learning sheets and images from 126 patients were blindly interpreted by seven surgeons and two pathologists. The time for tissue processing and ultra-fast fluorescence confocal microscopy imaging was between 8 and 10 min. The training program was composed of 110 images divided into nine learning sessions. The final database for blind performance assessment comprised 300 images. The mean duration for one training session and one performance round was 17 and 27 min respectively. The performance of pathologists was almost perfect with 99.6 per cent (standard deviation (s.d.) 5.4 per cent) accuracy. Surgeons' accuracy significantly increased (P = 0.001) from 83 per cent (s.d. 8.4 per cent) in round 1 to 98 per cent (s.d. 4.1 per cent) in round 7 as well as the sensitivity (P = 0.004). Specificity increased without significance from 84 per cent (s.d. 16.7 per cent) in round 1 to 87 per cent (s.d. 16.4 per cent) in round 7 (P = 0.060). Pathologists and surgeons showed a short learning curve in differentiating breast cancer from non-cancerous tissue in ultra-fast fluorescence confocal microscopy images. Performance assessment for both specialties supports ultra-fast fluorescence confocal microscopy evaluation for intraoperative management. NCT04976556 (http://www.clinicaltrials.gov).

Anti‐proliferative potential of copper(I) acylthiourea complexes with triphenylphosphine against breast cancer cells

We report the synthesis of five new Cu(I) acylthiourea complexes (C1–C5) bearing the general formula [Cu(L‐R)Cl (PPh3)2] [L = monodentate acylthiourea ligand, R = C6H5 (L1), C6H4CH3(o) (L2), C6H4OCH2CH3(p) (L3), C10H7 (L4) or C6H4Cl(p) (L5)]. All the complexes were characterized by analytical and spectroscopic tools. The complexes (C1–C5) exhibited a distorted tetrahedral geometry as inferred from the single crystal X‐ray diffraction study. The complexes were subjected to interact with the biomolecules (calf thymus [CT] DNA/bovine serum albumin [BSA]); the one bearing naphthyl substituent (C4) exhibited the highest binding efficacy. Further, anticancer activity of the complexes was studied exclusively against breast cancer cell lines, namely, MCF7, T47D, and MDA MB 231. Complex C4 was found to be highly cytotoxic on the three cancer cell lines with the IC50 values of 0.75, 0.75, and 0.68 μM, respectively. Conveniently, the complexes displayed fourfold less toxicity against the normal MCF10a human breast cells. The ability of complex C4 to induce apoptosis was analyzed by acridine orange/ethidium bromide (AO/EB) and Hoechst 33258 staining assays. Furthermore, it was found that complex C4 induced apoptosis via reactive oxygen species (ROS)‐mediated mitochondrial signaling pathway. Confocal fluorescence images of the cells subjected to lyso and mitotracker staining assays revealed that complex C4 was primarily localized on the mitochondria, and finally, Western blot analysis also confirmed the apoptosis induced by complex C4 in the MDA MB 231 cancer cells.

Human intravital microscopy in the study of sarcomas: an early trial of feasibility.

ClinicalTrials.gov, identifier NCT03517852; ClinicalTrials.gov, identifier NCT03823144.

Proarrhythmic toxicity of low dose bisphenol A and its analogs in human iPSC-derived cardiomyocytes and human cardiac organoids through delay of cardiac repolarization

Bisphenol A (BPA) and its analogs are common environmental chemicals with many potential adverse health effects. The impact of environmentally relevant low dose BPA on human heart, including cardiac electrical properties, is not understood. Perturbation of cardiac electrical properties is a key arrhythmogenic mechanism. In particular, delay of cardiac repolarization can cause ectopic excitation of cardiomyocytes and malignant arrhythmia. This can occur as a result of genetic mutations (i.e., long QT (LQT) syndrome), or cardiotoxicity of drugs and environmental chemicals. To define the impact of low dose BPA on electrical properties of cardiomyocytes in a human-relevant model system, we examined the rapid effects of 1 nM BPA in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using patch-clamp and confocal fluorescence imaging. Acute exposure to BPA delayed repolarization and prolonged action potential duration (APD) in hiPSC-CMs through inhibition of the hERG K+ channel. In nodal-like hiPSC-CMs, BPA acutely increased pacing rate through stimulation of the If pacemaker channel. Existing arrhythmia susceptibility determines the response of hiPSC-CMs to BPA. BPA resulted in modest APD prolongation but no ectopic excitation in baseline condition, while rapidly promoted aberrant excitations and tachycardia-like events in myocytes that had drug-simulated LQT phenotype. In hiPSC-CM-based human cardiac organoids, the effects of BPA on APD and aberrant excitation were shared by its analog chemicals, which are often used in “BPA-free” products, with bisphenol AF having the largest effects. Our results reveal that BPA and its analogs have repolarization delay-associated pro-arrhythmic toxicity in human cardiomyocytes, particularly in myocytes that are prone to arrhythmias. The toxicity of these chemicals depends on existing pathophysiological conditions of the heart, and may be particularly pronounced in susceptible individuals. An individualized approach is needed in risk assessment and protection.

A naphthalimide functionalized fluoran with AIE effect for ratiometric sensing Hg2+ and cell imaging application

The pollution caused by mercury ions (Hg2+) poses a potential threat to public health. Therefore, monitoring Hg2+ concentration in the environment is necessary and significant. In this work, a naphthalimide functionalized fluoran dye NAF has been prepared, which shows a new red-shift in emission at 550 nm with the maximum intensity in a mixture of water-CH3CN (v/v = 7/3) due to aggregating induced emission (AIE) effect. Meanwhile, NAF can be employed as a Hg2+ ions sensor, which displays a selective and sensitive response to Hg2+ ions by the reduced fluorescence of naphthalimide fluorophore and increased fluorescence of fluoran group, respectively, showing ratiometric fluorescence signal changes with more than 65-fold emission intensity ratio increase and naked eyes visible color change. In addition, the response time is fast (within 1 min) and the sensing can be conducted in a wide pH range (4.0–9.0). Moreover, the detection limit has been evaluated to be 5.5 nM. The sensing mechanism may be attributed to the formation of a π-extended conjugated system due to the Hg2+ ions-induced conversion of spironolactone to the ring-opened form, partially accompanied by the fluorescence resonance energy transfer (FRET) process. Significantly, NAF exhibits suitable cytotoxicity to living HeLa cells, which allows it to be utilized for ratiometric imaging of Hg2+ ions assisted by confocal fluorescence imaging.

A near-infrared and lysosome-targeted coumarin-BODIPY photosensitizer for photodynamic therapy against HepG2 cells

Photodynamic therapy (PDT) is a promising modality to treat cancer therapy for its non-invasive advantage and good selectivity. Herein, an iodinated and coumarin fused BODIPY photosensitizer named as BDP-C with near-infrared (NIR) region absorption was designed and synthesized for PDT against cancer cells. The absorption and emission wavelength of BDP-C appears at 732 nm and 808 nm, respectively. Its molar absorption coefficient is up to 2.13 × 105 cm−1M−1. The photosensitizer BDP-C shows good ability to yield singlet oxygen (1O2) and its 1O2 yield was calculated to be 17% using methylene blue (MB) as the reference, and then the photodynamic properties of BDP-C were investigated through MTT assays and confocal fluorescence imaging. The results show that the photosensitizer BDP-C has good biocompatibility and photostability, good long-term cell imaging ability, and moderate 1O2 generation efficiency. In addition, BDP-C can also specifically target lysosomes and kill HepG2 cells through apoptosis pathway upon laser irradiation. It will be a promising candidate drug in the field of biologic imaging and PDT.

The Pd-catalyzed precise synthesis of self-assembled fluorescent nanoprobes with different molecular weights for enhancing the permeability of cell membrane and targeting lysosomes

As a common polymer, poly (dimethylaminoethyl methacrylate) (PDMAEMA) is mainly used to synthesize medical materials. However, there are few reports about the related polymer fluorescent probes. Moreover, as far as we know, no one has studied the effect of its molecular weight on the performance of polymer probes. In this work, polymer probes (NapNH2(1)-PDM(4), NapNH2(1)-PDM(9), NapNH2(1)-PDM(23), NapNH2(1)-PDM(34)) with different molecular weights were accurately constructed based on 1,8-naphthalimide fluorophore and poly(N,N-dimethylaminoethyl methacrylate) and their photophysical properties were systematically studied. The test results showed that they all showed good photophysical properties. Moreover, the polymer probe NapNH2(1)-PDM(23), which had the best self-assembly effect and good cell permeability, was successfully screened by confocal fluorescence imaging technology. Not only that, the probe NapNH2(1)-PDM(23) could also label lysosomes in cells quickly and accurately. Therefore, this work provided important reference to construct relevant polymer probes with good cell permeability in the future.

Comprehensive multiparameter evaluation of platelet function using a highly sensitive membrane capacitance sensor

An accurate and comprehensive assessment of platelet function is essential for managing patients who receive antiplatelet therapies or require platelet transfusion either for treating active bleeding or for prophylaxis. Platelets contribute to clotting by undergoing a series of highly regulated functional responses including adhesion, spreading, granular secretion, aggregation, and cytoskeletal contraction. However, current platelet function assays evaluate only partial aspects of this intricate process and often under non-physiological testing conditions. Herein, we describe the development of a new approach to measure multiple key platelet function-related parameters, in a more physiologically relevant ex vivo semi-rigid microenvironment using a membrane capacitance sensor (MCS). MCS response to clotting provided three sensing parameters with sensitivities towards platelet counts, stimulation strengths, and activation pathways. Live confocal fluorescent imaging of stimulated platelets on MCS suggests that the presented system can readily and accurately convert the dynamics of cytoskeletal reorganization into analyzable electrical signals. Together, this new completely electrical sensing platform can be a promising diagnostic venue to recognize the impairment of primary hemostatic functions, evaluate the efficacy of therapeutic interventions, and gain further insights into the mechanisms of platelets in hemostasis and thrombosis.

Novel graphene quantum dots modified NH2-MIL-125 photocatalytic composites for effective antibacterial property and mechanism insight

Bacterial infection threatens human health, so the design of efficient and stable photocatalytic antibacterial materials has attracted widespread attention. In this regard, a novel photocatalyst GQDs/NH2-MIL-125 (GQDs: graphene quantum dots) was prepared by the solvothermal method for photocatalytic disinfection. Since the addition of GQDs enhanced the absorption of visible light and also accelerated the transfer of photogenerated electrons, the photocatalytic performance of GQDs/NH2-MIL-125 was significantly improved. 5 wt% GQDs loading sample (2-GQDs/NH2-MIL-125) exhibited the best antibacterial properties. The killing efficiency by 2-GQDs/NH2-MIL-125 against E. coli and S. aureus (107 CFU·mL−1) increased from 40 % and 75 % by NH2-MIL-125 to 92 % and 93 % within 60 min, respectively. Moreover, through the single-wavelength antibacterial experiments, it was found that the introduction of GQDs led to a wide-range-responsive antibacterial effect. Through photocatalytic trapping experiments and ESR analysis, the dominant roles of superoxide radicals (•O2–) were confirmed in the antibacterial process. Finally, by studying the state of bacteria via TEM, confocal fluorescent images, and gel electrophoresis, it was found that the active substances not only destroyed the cell membranes but also further destroyed DNA to achieve the purpose of completely killing bacteria. This work provides a new method for the rapid disinfection of pathogenic microorganisms using GQDs-related photocatalysts.

Expansion microscopy reveals subdomains in C. elegans germ granules.

Light and electron microscopy techniques have been indispensable in the identification and characterization of liquid-liquid phase separation membraneless organelles. However, for complex membraneless organelles such as the perinuclear germ granule in C. elegans, our understanding of how the intact organelle is regulated is hampered by (1) technical limitations in confocal fluorescence imaging for the simultaneous examination of multiple granule protein markers and (2) inaccessibility of electron microscopy. We take advantage of the newly developed super resolution method of expansion microscopy (ExM) and in situ staining of the whole proteome to examine the C. elegans germ granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with WT animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, Mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. This study will serve as an important tool in our understanding of germ granule biology and the biological role of liquid-liquid phase separation.

Mitochondrial-Targeted Delivery of Polyphenol-Mediated Antioxidases Complexes against Pyroptosis and Inflammatory Diseases.

Excess accumulation of mitochondrial reactive oxygen species (mtROS) is a key target for inhibiting pyroptosis-induced inflammation and tissue damage. However, targeted delivery of drugs to mitochondria and efficient clearance of mtROS remain challenging. In current study, it is discovered that polyphenols such as tannic acid (TA) can mediate the targeting of polyphenol/antioxidases complexes to mitochondria. This affinity does not depend on mitochondrial membrane potential but stems from the strong binding of TA to mitochondrial outer membrane proteins. Taking advantage of the feasibility of self-assembly between TA and proteins, superoxide dismutase, catalase, and TA are assembled into complexes (referred to as TSC) for efficient enzymatic activity maintenance. In vitro fluorescence confocal imaging shows that TSC not only promoted the uptake of biological enzymes in hepatocytes but also highly overlapped with mitochondria after lysosomal escape. The results from an in vitro model of hepatocyte oxidative stress demonstrate that TSC efficiently scavenges excess mtROS and reverses mitochondrial depolarization, thereby inhibiting inflammasome-mediated pyroptosis. More interestingly, TSC maintain superior efficacy compared with the clinical gold standard drug N-acetylcysteine in both acetaminophen- and D-galactosamine/lipopolysaccharide-induced pyroptosis-related hepatitis mouse models. In conclusion, this study opens a new paradigm for targeting mitochondrial oxidative stress to inhibit pyroptosis and treat inflammatory diseases.

Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs.

Some membrane proteins cluster in nanoscale cholesterol and sphingomyelin rich membrane domains, called lipid rafts, that transform into ceramide-rich rafts and coalesce into large ceramide-rich platforms during cell stress. However, the clustering mechanisms and their impact on membrane protein stability and function remain uncertain. We reported previously that the cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in the hyperinflammatory pulmonary disease cystic fibrosis (CF), forms clusters that are cholesterol-dependent and become incorporated into large and long-lived ceramide-rich platforms during hormonal stimulation. Using a combination of optimized fluorescence confocal imaging and quantitative analyses such as image correlation spectroscopy (ICS) and the k-space ICS (kICS), we report here that CFTR clustering at the plasma membrane of primary human bronchial epithelial cells does not involve known tethering interactions of CFTR with PDZ domain-containing proteins, filamin A or the actin cytoskeleton. It also does not require CFTR palmitoylation but is critically dependent on membrane lipid order and is induced by detergents that increase the phase separation of membrane lipids. Clustering and integration of CFTR into ceramide-rich platforms are impaired by the CF disease-causing mutations F508del and S13F and rescued by the CFTR modulators elexacaftor plus tezacaftor. These results indicate that CF therapeutics that correct mutant protein folding restore both CFTR trafficking and normal lipid interactions in the plasma membrane which results in its stability at the plasma membrane and its functional restoration.

Early deficits in GABA inhibition parallels an increase in L-type Ca2+ currents in the jaw motor neurons of SOD1G93A mouse model for ALS

Amyotrophic Lateral Sclerosis (ALS) involves protracted pre-symptomatic periods of abnormal motor neuron (MN) excitability occurring in parallel with central and peripheral synaptic perturbations. Focusing on inhibitory control of MNs, we first compared longitudinal changes in pre-synaptic terminal proteins for GABA and glycine neurotransmitters around the soma of retrogradely identified trigeminal jaw closer (JC) MNs and ChAT-labeled midbrain extraocular (EO) MNs in the SOD1G93A mouse model for ALS. Fluorescence immunocytochemistry and confocal imaging were used to quantify GAD67 and GlyT2 synaptic bouton density (SBD) around MN soma at pre-symptomatic ages ∼P12 (postnatal), ∼P50 (adult) and near disease end-stage (∼P135) in SOD1G93A mice and age-matched wild-type (WT) controls. We noted reduced GAD67 innervation in the SOD1G93A trigeminal jaw closer MNs around P12, relative to age-matched WT and no significant difference around P50 and P135. In contrast, both GAD67 and GlyT2 innervation were elevated in the SOD1G93A EO MNs at the pre-symptomatic time points. Considering trigeminal MNs are vulnerable in ALS while EO MNs are spared, we suggest that upregulation of inhibition in the latter might be compensatory. Notable contrast also existed in the innate co-expression patterns of GAD67 and GlyT2 with higher mutual information (co-dependency) in EO MNs compared to JC in both SOD1G93A and WT mice, especially at adult stages (P50 and P135). Around P12 when GAD67 terminals expression was low in the mutant, we further tested for persistent GABA inhibition in those MNs using in vitro patch-clamp electrophysiology. Our results show that SOD1G93A JC MNs have reduced persistent GABA inhibition, relative to WT. Pharmacological blocking of an underlying tonically active GABA conductance using the GABA-α5 subunit inverse agonist, L-655-708, disinhibited WT JC MNs and lowered their recruitment threshold, suggesting its role in the control of intrinsic MN excitability. Quantitative RT-PCR in laser dissected JC MNs further supported a reduction in GABA-α5 subunit mRNA expression in the mutant. In light of our previous report that JC MNs forming putative fast motor units have lower input threshold in the SOD1G93A mice, we suggest that our present result on reduced GABA-α5 tonic inhibition provides for a mechanism contributing to such imbalance. In parallel with reduced GABA inhibition, we noted an increase in voltage-gated L-type Ca2+ currents in the mutant JC MNs around P12. Together these results support that, early modifications in intrinsic properties of vulnerable MNs could be an adaptive response to counter synaptic deficits.

Fluorescent Self-Reporting Lipid Nanoparticles for Nitric Oxide/Gene Co-Delivery and Combination Therapy.

The combination cancer therapy of nitric oxide (NO) with gene therapy is a promising method for tumor treatment. However, efficient co-delivery of gas and therapeutic genes to tumor cells remains a challenge. Herein, we designed a nano-sized ultraviolet (UV) light-responsive cationic lipid vector DPNO(Zn). Fluorescence spectroscopy and confocal imaging experiments revealed that DPNO(Zn) lipid nanoparticles (LNPs) could rapidly release NO under low-power UV light irradiation. Moreover, the fluorescence turn-on might take place along with the release of NO, indicating the self-reporting ability. Gene delivery experiments showed that DPNO(Zn) LNPs had good gene transfection ability, making such materials a good candidate for gas/gene combination therapy. In vitro antitumor assay demonstrated that the co-delivery system was more effective in inhibiting tumor cell proliferation than individual NO or pTrail treatment. Studies on the mechanism of tumor cell apoptosis induced by NO/pTrail co-delivery showed that NO could not only effectively increase the accumulation of p53 protein in tumor cells, thereby promoting the activation of caspase-3, but also induce mitochondrial damage. On the other hand, the Trail protein expressed by pTrail gene could enhance the degree of NO-induced caspase-3 activation, indicating the synergistic effect. These results proved that DPNO(Zn) LNP may serve as a multifunctional nanocarrier for potential tumor therapy.

Hispolon, A Bioactive Compound from Phellinus linteus, Induces Apoptosis of Human Breast Cancer Cells Through the Modulation of Oxidative Stress and Autophagy

Breast cancer (BC) is a major type of cancer found in women worldwide, accounting for more than 25% of all females diagnosed as BC patients. The BC treatment still needs to be highly effective with few adverse effects on the rest of the body. This study's objective was to investigate how hispolon enhanced anticancer activities and examine the underlying processes in human breast cancer cells. Hispolon was used to treat the HTB-26 cell lines. We investigated cell viability, intracellular reactive oxygen species (ROS) production, immunofluorescence straining, MDC assay, and RT-PCR. According to the findings, hispolon inhibited HTB-26 cells from proliferating and caused them to undergo apoptosis. When the mechanism of action of hispolon was investigated, it was discovered that BCL-2 expression was downregulated while pro-apoptotic genes including caspase-3, caspase-9, and BAX were upregulated. The level of intracellular ROS was also shown to be increased by hispolon, and the expression of antioxidant genes including SOD, GPX, and Catalase was found to be downregulated. The increase in the confocal fluorescence image of LC3I/II and the identification of upregulated autophagy-related genes, such as LC3I/II, ATG5, ATG10, and ATG12, served as indicators of the activation of autophagy. Therefore, our findings showed that hispolon had great potential for being an effective therapeutic agent and could cause cell death in breast cancer cells.