Greater Cytotoxicity Research Articles

Protoporphyrin IX (PpIX) loaded PLGA nanoparticles for topical Photodynamic Therapy of melanoma cells

IntroductionNanoparticles (Np) can increase drug efficacy and overcome problems associated with solubility and aggregation in a solution of PpIX. PurposeEvaluate if Np interferes in the photophysical and photobiological capacity of the PpIX comparing with free PpIX intended for topical PDT of melanoma. MethodsIn vitro photophysical evaluation of Np-PpIX was carried out through singlet oxygen (1O2) quantum yield. In vitro cytotoxicity and phototoxicity assays have used murine melanoma cell culture. ResultsThe quantum yield of singlet oxygen has shown that Np did not influence the formation capacity of this reactive species. In the dark, all PpIX-Nps concentrations were less cytotoxic compared to free drugs. At a higher light dose (1500 mJ.cm2) 3.91 μg / mL PpIX had similar % viable cells for free and Np (∼34 %) meaning Nps did not interfere in the photodynamic effect of PpIX. However, at 7.91 μg / mL the phototoxicity increased for both (5.8 % viable cells for free versus 21.7 % for Nps). Despite the higher phototoxicity of free PpIX at this concentration, greater cytotoxicity in the dark was obtained (∼49 % viable cells for free versus ∼90.6 % Np) which means Nps protect the tumor tissue from the photodynamic action of PpIX. ConclusionsNp is a potential delivery system for melanoma skin cancer, since it maintained the photophysical properties of PpIX and excellent in vitro phototoxicity effect against melanoma cells, reducing cell viability ∼80 % (7.91 μg / mL PpIX in Nps) and provides safe PDT (due to lower cytotoxicity in the dark).

Catalase Modulates the Radio-Sensitization of Pancreatic Cancer Cells by Pharmacological Ascorbate.

Pancreatic cancer cells (PDACs) are more susceptible to an oxidative insult than normal cells, resulting in greater cytotoxicity upon exposure to agents that increase pro-oxidant levels. Pharmacological ascorbate (P-AscH−), i.e., large amounts given intravenously (IV), generates significant fluxes of hydrogen peroxide (H2O2), resulting in the killing of PDACs but not normal cells. Recent studies have demonstrated that P-AscH− radio-sensitizes PDAC but is a radioprotector to normal cells and tissues. Several mechanisms have been hypothesized to explain the dual roles of P-AscH− in radiation-induced toxicity including the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2), RelB, as well as changes in bioenergetic profiles. We have found that P-AscH− in conjunction with radiation increases Nrf2 in both cancer cells and normal cells. Although P-AscH− with radiation decreases RelB in cancer cells vs. normal cells, the knockout of RelB does not radio-sensitize PDACs. Cellular bioenergetic profiles demonstrate that P-AscH− with radiation increases the ATP demand/production rate (glycolytic and oxidative phosphorylation) in both PDACs and normal cells. Knocking out catalase results in P-AscH− radio-sensitization in PDACs. In a phase I trial where P-AscH− was included as an adjuvant to the standard of care, short-term survivors had higher catalase levels in tumor tissue, compared to long-term survivors. These data suggest that P-AscH− radio-sensitizes PDACs through increased peroxide flux. Catalase levels could be a possible indicator for how tumors will respond to P-AscH−.

Role of exopolysaccharides (EPSs) as anti-Mir-155 in cancer cells

Micro-RNAs (MiRNAs) are a class of small non-coding RNAs that regulate cellular gene expression. MiR-155 overexpression has been implicated in many types of cancer. Besides, miR-155 appears to help tumor invasion and migration and works as a moderator of epithelial-to-mesenchymal transition (EMT). Exopolysaccharides (EPSs) are a large group of natural heterogeneous polymers of sugars with a biologically antitumor effect. Herein, we test a hypothesis that EPS might promote its anti-tumorigenic effect via regulating miR-155 expression and its target pathways. Expression of miR-155 and a panel of targeted genes were investigated by real-time PCR. In our study, we have succeeded in the extraction, purification of exopolysaccharide with great cytotoxicity to different cancer cell lines, HepG II, Caco-2, and MCF-7. We reported that EPSs have a suppression effect on the oncogenic miR-155. In conclusion, this work clarifies a new possible mechanism for the anti-tumorigenic effect of EPSs in cancer cells and provides insights into the biological pathways through which EPSs act. Moreover, it paves the way for new prospective cancer therapeutics as anti-miRNA.

The Wonderful Activities of the Genus Mentha: Not Only Antioxidant Properties.

Medicinal plants and their derived compounds have drawn the attention of researchers due to their considerable impact on human health. Among medicinal plants, mint (Mentha species) exhibits multiple health beneficial properties, such as prevention from cancer development and anti-obesity, antimicrobial, anti-inflammatory, anti-diabetic, and cardioprotective effects, as a result of its antioxidant potential, combined with low toxicity and high efficacy. Mentha species are widely used in savory dishes, food, beverages, and confectionary products. Phytochemicals derived from mint also showed anticancer activity against different types of human cancers such as cervix, lung, breast and many others. Mint essential oils show a great cytotoxicity potential, by modulating MAPK and PI3k/Akt pathways; they also induce apoptosis, suppress invasion and migration potential of cancer cells lines along with cell cycle arrest, upregulation of Bax and p53 genes, modulation of TNF, IL-6, IFN-γ, IL-8, and induction of senescence phenotype. Essential oils from mint have also been found to exert antibacterial activities against Bacillus subtilis, Streptococcus aureus, Pseudomonas aeruginosa, and many others. The current review highlights the antimicrobial role of mint-derived compounds and essential oils with a special emphasis on anticancer activities, clinical data and adverse effects displayed by such versatile plants.

Pluronic-Coated Biogenic Gold Nanoparticles for Colon Delivery of 5-Fluorouracil: In vitro and Ex vivo Studies.

The aim of the study was to prepare 5-fluorouracil (5-FU)-loaded biogenic gold nanoparticles with pluronic-based coating (PFGNPs), their optimization (full factorial predicted OBPN-1) and in vitro-ex vivo evaluation. Several formulations were prepared, selected for optimization using Design Expert®, and compared for morphology, 5-FU release kinetics, compatibility, cell line toxicity, in vitro hemocompatibility, and ex vivo intestinal permeation across the rat duodenum, jejunum, and ileum. The pluronic-coated 5-FU-carrying GNPs were spherical, 29.11-178.21nm in diameter, with a polydispersity index (PDI) range of 0.191-292, and a zeta potential (ZP) range of 11.19-29.21 (-mV). The optimized OBPN-1 (desirability = 0.95) demonstrated optimum size (175.1nm), %DL as 73.8%, ZP as 21.7mV, % drug release (DR) as 75.7%, and greater cytotoxicity (viability ~ 8.9%) against the colon cancer cell lines than 5-FU solution (~ 24.91%), and less hemocompatibility. Moreover, OBPN-1 exhibited 4.5-fold permeation across the rat jejunum compared with 5-FU solution. Thus, the PFGNPs exhibit high DL capacity, sustained delivery, hemocompatibility, improved efficacy, and enhanced permeation profiles compared with 5-FU solution and several other NPs preparations suggesting it is a promising formulation for effective colon cancer control with reduced side effects.

Abstract PO075: Re-invigorating tumor infiltrating T lymphocytes against EBV positive nasopharyngeal cancer

Abstract Background/Aim: To determine if tumor-infiltrating T lymphocytes (TIL T) harvested from EBV-positive Nasopharyngeal cancer (NPC) is an effective source of cellular based immunotherapy. Methods: HLA-A02 Peripheral blood (PB; n=6; paired) and NPC tissue biopsies (n=10; undifferentiated, non-keratinizing and EBV positive) were collected from patients with written consent. Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat after density gradient centrifugation of PB. Tissue biopsies were minced, dissociated with human tumour dissociation kit (Miltenyi Biotec) and passed through nylon mesh to single cell suspension. Both PBMC and dissociated tumour were used for T cell expansion by incubation with anti-CD3/CD28 microbeads (Thermo Fisher Scientific) in RPMI-1640 (Hyclone) supplemented with 10% Fetal Bovine Serum (Hyclone) and 20IU/mL recombinant IL-2 (Miltenyi Biotec). TIL T cells were isolated with pan T cell isolation kit (Miltenyi Biotec) after expansion. Functionality of both PB T and TIL T cells were assessed using flow cytometry, trans-well assay and cytotoxicity assay against C666-A2 (NPC cell line expressing HLA-A02) thereafter. Results: Successful isolation and expansion of NPC TIL T cells was achieved using 1-2 fresh NPC biopsy samples (2-3 mm); with fold increase of 210 to 867 after 28 days (1.7-4.5 × 107 cells). Comparison of primary TIL and expanded TIL T demonstrated that the former has greater percentage of CD4+ (p<0.006) while the latter has more CD8+ cells (p<0.003) cells, suggesting preferential expansion of cytotoxic T cells. Expanded TIL T cells also shown greater cytotoxicity against NPC cell line (C666-A2; E:T =1:20) than paired, expanded PB T cells (19.5% vs 4.9%, p=0.02). Furthermore, the cytotoxicity against C666-A2 was even greater (p<0.015) after incubating the TIL T cells with EBV peptides (LMP-1, LMP-2, EBNA-1). Primed TIL T cells also demonstrated greater lytic cell degranulation (CD107a), higher T cell activation (CD137) and IFN-γ secretion than non-primed, expanded TIL T cells. Importantly, expanded TIL T cells also demonstrated increased migration towards C666-A2 than expanded PB T cells (p=0.002). Greater anti-tumour effector function was observed with expanded TIL T despite positive expression of T inhibitory molecules (PD-1, CTLA-4 and TIM-3). Conclusion: TIL T cells can be expanded reliably from NPC biopsies after 28-day culture. Expansion protocol demonstrated preferential expression of cytotoxic T cells over CD4 T cells. The greater anti-tumour effect of expanded TIL T over paired, expanded PB T suggested greater inherent anti-tumour T cells present in NPC TIL despite positive expression of T cell exhaustion markers. This warrant further investigations to explore therapeutic potential of TIL T in NPC. Citation Format: Yitian Png, Audrey Zhi-Yi Yang, Louise Soo-Yee Tan, See-Voon Seow, Swarnalatha Lucky Asidharan, Jamie Mong, Min-Han Tan, Han ChongohOH, Chwee Ming Lim. Re-invigorating tumor infiltrating T lymphocytes against EBV positive nasopharyngeal cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO075.

Synthesis of (Z)-3-((5-(benzylthio)-4H-1,2,4-triazol-3-yl)imino)-5-haloindolin-2-one derivatives: combined spectroscopic and computational investigations on the level and activity of matrix metalloproteinases 2 and 9 in cancer cell lines

Angiogenesis is an essential factor for cancer progression. Although more attention is paid in angiogenesis on its role in cancer biology, many other non-neoplastic diseases are also angiogenic-dependent. Recently, there is motivation to control cancer via inhibition of angiogenesis. Isatin-based scaffolds have been extensively used as anticancer agents in the recent years. Although some biological properties of isatin-based scaffolds are determined, their effects on angiogenesis are rare. So, we investigated the antiangiogenic effects of isatin-1,2,4-triazole conjugates. (Z)-3-((5-(benzylthio)-4H-1,2,4-triazol-3-yl)imino)-5-haloindolin-2-one macromolecules 1a–1l were synthesized and characterized, and the buffered solutions were used for evaluation of their cytotoxicity (cell viability) by MTT assay in vitro against U87MG (human glioblastoma astrocytoma) and A2780 (human ovarian carcinoma) cancer cell lines. Also, the effects of the compounds 1a–1l on supernatants activities and levels of matrix metalloproteinases (MMP-2 and MMP-9) were assayed using enzyme-linked immunosorbent assay (ELISA) and gelatin zymography. The compounds 1j–1l have the greatest cytotoxicity against studied cell lines. Moreover, our observations indicated that 1j–1l decreased the supernatants activity of MMP-2 and MMP-9 more than the others and all of the tested compounds considerably decreased the supernatant levels of MMP-9. The molecular mechanism of 1j binding to MMP-2 and MMP-9 was investigated by fluorescence quenching, absorption spectroscopy, FT-IR, molecular docking and molecular dynamics (MD) simulation procedures.

Eco-friendly graphene oxide-based magnesium oxide nanocomposite synthesis using fungal fermented by-products and gamma rays for outstanding antimicrobial, antioxidant, and anticancer activities

Green synthesis of nanoparticles is an eco-friendly new approach and an attractive field in biomedical applications. In the current investigation, a novel green synthesis of magnesium oxide nanoparticles (MgO NPs) and graphene oxide based magnesium oxide nanocomposite (GO/MgO nanocomposite) is accomplished using the fungal fermented agricultural by-products and gamma irradiation. MgO NPs are synthesized using wheat bran fermented by Erysiphe cichoracearum and gamma irradiation at 30.0 kGy. GO/MgO nanocomposite is produced efficiently at 20.0 kGy. The characterization of the synthesized nanoparticles is executed by FTIR, XRD, DLS, SEM, and TEM. The mean average diameter of MgO NPs is 14.5 nm and doped uniformly on the surface of the graphene oxide sheets. An excellent antioxidant activity has been shown by GO/MgO nanocomposite (500 μg/mL) of 56.71%. MgO NPs and GO/MgO nanocomposite prove a great antimicrobial effect against all the tested microbial human pathogens. Escherichia coli and Candida albicans are mostly inhibited by MgO NPs and GO/MgO nanocomposite. MgO NPs and GO/MgO nanocomposite show great cytotoxicity against Prostate cancer cell lines. IC50 of MgO NPs and GO/MgO nanocomposite against the Prostate cancer cell lines (PC3) are 86.7 μg/mL and 11.17 μg/mL, respectively. Owing to the strong attachment of MgO NPs and GO construction, the GO/MgO nanocomposite could be used as a promising candidate for new and potent antimicrobial and anticancer considered successful future applications.

Crosstalk of low density lipoprotein and liposome as a paradigm for targeting of 5-fluorouracil into hepatic cells: cytotoxicity and liver deposition

ABSTRACT This study aimed to utilize cholesterol conjugation of 5-fluorouracil (5-FUC) and liposomal formulas to enhance the partitioning of 5-FU into low density lipoprotein (LDL) to target hepatocellular carcinoma (HCC). Thus, 5-FU and 5-FUCwere loaded into liposomes. Later, the direct loading and transfer of 5-FU, and 5-FUC from liposomes into LDL were attained. The preparations were characterized in terms of particle size, zeta potential, morphology, entrapment efficiency, and cytotoxicity using the HepG2 cell line. Moreover, the drug deposition into the LDL and liver tissues was investigated. The present results revealed that liposomal preparations have a nanosize range (155 − 194 nm), negative zeta potential (- 0.82 to – 16 mV), entrapment efficiency of 69% for 5-FU, and 66% for 5-FUC. Moreover, LDL particles have a nanosize range (28–49 nm), negative zeta potential (- 17 to −27 mV), and the entrapment efficiency is 11% for 5-FU and 85% for 5-FUC. Furthermore, 5-FUC loaded liposomes displayed a sustained release profile (57%) at 24 h compared to fast release (92%) of 5-FU loaded liposomes. 5-FUC and liposomal formulas enhanced the transfer of 5-FUC into LDL compared to 5-FU. 5-FUC loaded liposomes and LDL have greater cytotoxicity against HepG2 cell lines compared to 5-FU and 5-FUC solutions. Moreover, the deposition of 5-FUC in LDL (26.87ng/mg) and liver tissues (534 ng/gm tissue) was significantly increased 5-FUC liposomes compared to 5-FU (11.7 ng/g tissue) liposomal formulation. In conclusion, 5-FUC is a promising strategy for hepatic targeting of 5-FU through LDL-mediated gateway.

In vivo trafficking of a tumor-targeting IgE antibody: molecular imaging demonstrates rapid hepatobiliary clearance compared to IgG counterpart

ABSTRACT IgE antibodies elicit powerful immune responses, recruiting effector cells to tumors more efficiently and with greater cytotoxicity than IgG antibodies. Consequently, IgE antibodies are a promising alternative to conventional IgG-based therapies in oncology (AllergoOncology). As the pharmacokinetics of IgE antibodies are less well understood, we used molecular imaging in mice to compare the distribution and elimination of IgE and IgG antibodies targeting the human tumor-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4). Anti-CSPG4 IgE and IgG1 antibodies with human Fc domains were radiolabeled with 111In. CSPG4-expressing A375 human melanoma xenografts implanted in NOD-scid IL2rg-/- mice were also engrafted with human immune cells by intravenous administration. 111In-anti-CSPG4 antibodies were administered intravenously. Their distribution was determined by single-photon emission computed tomography (SPECT) and ex vivo gamma-counting over 120 h. SPECT imaging was conducted from 0 to 60 min after antibody administration to precisely measure the early phase of IgE distribution. 111In-labeled anti-CSPG4 IgG and IgE showed serum stability in vitro of >92% after 5 days. In A375 xenograft-bearing mice, anti-CSPG4 IgE showed much faster blood clearance and higher accumulation in the liver compared to anti-CSPG4 IgG. However, tumor-to-blood and tumor-to-muscle ratios were similar between the antibody isotypes and higher compared with a non-tumor-targeting isotype control IgE. IgE excretion was much faster than IgG. In non-tumor-bearing animals, early SPECT imaging revealed a blood clearance half-life of 10 min for IgE. Using image-based quantification, we demonstrated that the blood clearance of IgE is much faster than that of IgG while the two isotypes showed comparable tumor-to-blood ratios.

Anti-EGFR liquid crystalline nanodispersions for docetaxel delivery: Formulation, characterization and cytotoxicity in cancer cells

Prostate cancer is one of the most frequent neoplasms, associated with high mortality. Some factors may intensify its aggressiveness, for example, the overexpression of the epidermal growth factor receptor (EGFR) in some subtypes of prostate tumors. In this context, the inhibition of EGFR helps to fight neoplasia, with the use of the chimeric anti-EGFR monoclonal antibody, cetuximab (CTX). For cancer treatment in the metastatic stage, chemotherapy using docetaxel (DTX) is widely employed, however the drug is very liposoluble, hampering its bioavailability. In this sense, liquid crystalline systems have great potential for the delivery of DTX. Particularly, liquid crystalline nanodispersions can be used in parenteral routes, with many advantages, including the possibility of prolonged drug release, improved pharmacokinetics, allowing passive tumor accumulation by the EPR effect. Thus, the objective of this work was to develop a liquid crystalline nanodispersion (LCN) based on ethoxylated cetyl alcohol 20 and propoxylated 5 as surfactant (Procetyl®), oleic acid, DSPE-PEG-MAL (anchor for CTX binding) and soy phosphatidylcholine as oily phase and 1.5 % poloxamer dispersion in PBS buffer as an aqueous phase. The formulation was loaded with DTX and covalently functionalized with CTX to evaluate its cytotoxic potential against prostate cancer cells. The results suggested that the obtained system has a predominantly hexagonal crystalline phase, was able to be nanodispersed with low polydispersity, and presented negative zeta potential. CTX did not have its structure compromised after thiolation and was successfully covalently linked to LCN. In a prostate cancer cells, PC3, LCNs functionalized with CTX underwent greater cell uptake, resulting in greater cytotoxicity, compared to free DTX and non-functionalized DTX-loaded LCNs. Therefore, the present study reports for the first time an EGFR-targeted liquid crystal nanodispersion for selective to deliver DTX to prostate cells

Anticancer activity of Momordica cochinchinensis (red gac) aril and the impact of varietal diversity

BackgroundMomordica cochinchinensis (Cucurbitaceae) is a nutritionally and medicinally important fruit restricted to South East Asia with diverse morphological and genetic variations but there is limited information on its medicinal potential.MethodsM. cochinchinensis aril from 44 different samples in Australia, Thailand and Vietnam were extracted using different solvents and tested for its anticancer potential. Anticancer activity of M. cochinchinensis aril on breast cancer (MCF7 and BT474) and melanoma (MM418C1 and D24) cells were compared to control fibroblasts (NHDF). The cytotoxicity of the cells following treatment with the aril extract was determined using CCK-8 assay. Biochemical and morphological changes were analysed using flow cytometry, confocal and transmission electron microscopy to determine the mechanism of cell death.ResultsThe water extract from the aril of M. cochinchinensis elicited significantly higher cytotoxicity towards breast cancer and melanoma cells than the HAE extract. The IC50 concentration for the crude water extract ranged from 0.49 to 0.73 mg/mL and induced both apoptotic and necrotic cell death in a dose- and time-dependant manner with typical biochemical and morphological characteristics. The greatest cytotoxicity was observed from Northern Vietnam samples which caused 70 and 50% melanoma and breast cancer cell death, respectively.ConclusionsThe water extract of M. cochinchinensis aril caused significant apoptosis and necrosis of breast cancer and melanoma cells, with varieties from Northern Vietnam possessing superior activity. This highlights the potential of this fruit in the development of novel anticancer agents against such tumours, with specific regions on where to collect the best variety and extraction solvent for optimum activity.

Characterization and cytotoxic activity on glial cells of alkaloid-enriched extracts from pods of the plants Prosopis flexuosa and Prosopis nigra (Fabaceae)

Introduction: Prosopis spp. pods have shown to be a potential source of protein and energy in livestock. However, prolonged ingestion of some of these species produces neurological symptoms in ruminants. Objective: In the present study, the alkaloid content and the in vitro neurotoxic activity of alkaloid enriched-extracts from P. flexuosa and P. nigra pods were determined in order to elucidate the mechanism of animal poisoning caused by these species. Methods: The main alkaloids present in both extracts were analyzed by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS). The cytotoxic activity of Prosopis alkaloid enriched-extracts in primary mixed glial cell culture was assessed by phase contrast microscopy and using neutral red, and lactate dehydrogenase (LDH) activity assays. Results: Juliprosine and juliprosopine were identified in P. flexuosa pods, while the absence of these alkaloids in P. nigra was confirmed. Both extracts (5-30 μg/mL) induced in a dose dependent manner, morphological alterations, such as swelling, enlargement and detachment from the culture surface. Consistent with this, decrease in cell viability and release of LDH 48 hours after exposure, revealed that P. flexuosa pods was significantly more cytotoxic than P. nigra. Conclusions: The present study suggest that the cytotoxic effect displayed by both extracts are due to the alkaloid content. However, the presence of the alkaloids juliprosine and juliprosopine in P. flexuosa could explain the greater cytotoxicity on glial cells with respect to P. nigra that was not shown to contain these alkaloids.

Glycyrrhetinic Acid-Functionalized Mesoporous Silica Nanoparticles for the Co-Delivery of DOX/CPT-PEG for Targeting HepG2 Cells.

A pH-triggered mesoporous silica nanoparticle (MSN)-based nano-vehicle for the dual delivery of doxorubicin (DOX)/camptothecin-PEG (CPT-PEG) has been prepared. To enhance its selectivity, the nanoparticles were decorated with glycyrrhetinic acid (GA) to target HepG2 cells. The highly insoluble CPT was derivatized with a reductive-cleavable PEG chain to improve its loading within the MSN. The preparation of these particles consisted of four steps. First, CPT-PEG was loaded within the pores of the MSN. Then, dihydrazide polyethylene glycol chains were introduced onto the surface of an aldehyde-functionalized MSN by means of a hydrazone bond. Afterwards, DOX was covalently attached to the other end of the dihydrazide polyethylene glycol chains. Finally, the resulting nanoparticles were decorated with GA by formation of an imine bond between the amino group of DOX and a benzaldehyde-GA derivative. The system was stable at physiological conditions and the release of both drugs was negligible. However, at acidic pH, a burst release of DOX and a gradual release of CPT-PEG takes place. GA-decorated drug delivery systems (DDS) selectively internalizes into HepG2. In vitro tests demonstrated that this system shows a great cytotoxicity towards HepG2 cells. Furthermore, glutathione cleavage of CPT prodrug assures the formation of free CPT leading to a synergistic effect in combination with DOX.

Benzoindole-based bifunctional ratiometric turn-on sensor with an ICT effect for trapping of H+ and Al3+ in dual-channel cell imaging and samples.

Abnormal changes in H+ and Al3+ concentrations in living cells can alter neurological diseases. A small-molecule sensor combined with a fluorescence imaging technique holds great promise for monitoring changes in proton and metal-ion concentration. In this work, a bifunctional ratiometric naked-eye fluorescence sensor (BIBC) was developed for turn-on detection of H+ and Al3+ in H2O/EtOH (v/v=1:1) mixtures. BIBC exhibits a pKa value of 4.58 within a linear pH variation from 4.1 to 4.7 (R2=0.9939). Moreover, the fluorescence intensity ratio (I566 nm/I524 nm) shows a good linear relationship (R2=0.9965) within an Al3+ concentration range of 7.0-10.0μM. The detection limit (DL) for the sensor was calculated to be 1.58μM. The practical application of BIBC for Al3+ detection in real samples was further discussed, and satisfactory results were obtained. Furthermore, the sensor was applied to real-time visualization of changes in H+ and Al3+ concentration in living cells, with great photostability and low cytotoxicity observed. Fluorescence images of H+ and Al3+ were collected by using a fluorescence microscope in a dual-channel configuration, wherein they were labeled green and yellow, respectively.

An in vitro study on the antitumor effect of sonodynamic therapy using sinoporphyrin sodium on human glioblastoma cells

Sonodynamic therapy (SDT) is a promising modality for cancer treatment. Sinoporphyrin sodium (DVDMS), purified from Photofrin II, shows great potential in SDT evidenced by growing studies. The purpose of the current study was to investigate the antitumor effect of SDT combined with DVDMS on human glioblastoma (U87 MG) cell line in vitro. The cellular uptake of DVDMS was investigated by confocal microscopy and IVIS spectrum imaging system. In addition, DVDMS toxicity and anti-tumor effect of SDT were assessed by flow cytometry. The generation of intracellular reactive oxygen species (ROS) was determined using DCFH-DA staining. Simultaneously, fluorescence microscopy was performed to access the destabilization of mitochondrial membrane potential (MMP). The results showed that DVDMS could easily enter the cells and accumulated in the cytoplasm, especially the mitochondria. And the intracellular DVDMS increased with incubation time or concentrations. The results also showed remarkable cytotoxicity of DVDMS-mediated SDT (center frequency: 0.970 MHz; peak-rarefactional pressure: 0.52-MPa; acoustic power: 0.32 W; pulse repetition frequency: 1 Hz; duty cycle: 1–30%; duration: 3 min) on U87 MG cells, while DVDMS alone was non-toxic to the cells. In comparison with the control group, the SDT-treated group showed significant generation of intracellular ROS and loss of MMP at 1 h post-treatment. These results indicated that DVDMS-mediated SDT could induce great cytotoxicity in U87 MG cells via the production of ROS and showed potentials in the treatment for glioblastoma.

The impact of pyrolysis temperature on physicochemical properties and pulmonary toxicity of tobacco stem micro-biochar

Biochars (BCs) are currently widely used, yet their impact on human health is mostly unknown. We generated micro-tobacco stem-pyrolysed BCs (mTBCs) at different pyrolysis temperatures and assessed pulmonary toxicity in normal human lung epithelial BEAS-2B cells. mTBCs generated at 350 °C (mTBC350) and 650 °C (mTBC650) were analysed and compared for physicochemical properties and adverse effects. Pyrolysis temperature had a significant influence on chemical composition, particle size, specific surface area and aromatic carbon structure. mTBC650 displayed a highly ordered aromatic carbon structure with smaller particle size, high surface area (20.09 m2/g) and high polycyclic aromatic hydrocarbon and metal content. This composition could promote reactive oxygen species accumulation accompanied by greater cytotoxicity, genotoxicity and epithelial barrier malfunction in cultured cells. Thus, the risk of pulmonary toxicity owing to micro-BCs (mBCs) is affected by pyrolysis temperature. Long-term exposure to mBCs produced at high temperatures may lead to or exacerbate pulmonary disease.

Manufacturing of natural killer cells for treating solid malignancies

ObjectivesNatural killer (NK) cells are an attractive and potent tool for cancer immunotherapy, however, ex vivo expansion of NK cells is required to achieve therapeutic cell dosages. As such, this review will discuss recent NK cell manufacturing methods applied in clinical trials for expanded NK cells for the treatment of solid tumors, as well as investigational NK cell manufacturing protocols. Given the unique challenges posed by the solid tumor microenvironment, the main objective of this review is to highlight key biological mechanisms associated with tumor homing and infiltration of NK cells and how manufacturing methods impact these functions. Key findingsFor efficient adoptive NK cell therapy for the treatment of solid malignancies, NK cells need to extravasate from the blood stream, migrate through the tumor extracellular matrix, lyse cancer cells, activate other immune cells, and persist in the blood stream. The NK cell manufacturing process is complex, with each parameter influencing the expansion rate, and final NK cell number, purity, phonotype, and cytotoxicity. Many investigational and clinical NK cell manufacturing protocols generate high numbers of NK cells with greater cytotoxicity than freshly isolated NK cells. The expression of factors related to homing and migration in NK cells after ex vivo expansion is largely overlooked, but the few studies which have explored this indicate manufacturing processes can affect these critical mechanisms. ConclusionThe current manufacturing protocols can generate high numbers of NK cells with increased cytotoxic functions, however understanding the effect of expansion on factors related to NK cell homing and migration is also important for treating solid malignancies. Furthermore, to progress the field of expanded NK cells for the treatment of solid tumors, improving “off-the-shelf” NK cell therapies and standardizing the manufacturing protocols and release criteria should be prioritized.

VAV1‐overexpressing YT cells display improved cytotoxicity against malignant cells

Immunotherapy based on adoptive transfer of genetically engineered T- and NK-cells is an area of active ongoing research and has proven highly efficacious for patients with certain B-cell malignancies. Use of NK cells and NK cell lines as carriers of chimeric antigen receptors (CARs) appears particularly promising, as this opens an opportunity for moving the therapy from autologous to the allogeneic (universal) format. This "off-the-shelf" approach is thought to significantly reduce the price of the treatment and make it available to many more patients in need. Yet, the efficacy of CAR-NK cells in vivo presently remains low, and boosting the activity of CAR NK cells via stronger tumor homing, resistance to tumor microenvironment, as well as greater cytotoxicity may translate into improved patient outcomes. Here, we established a derivative of a human NK cell line YT overexpressing a positive regulator of cytotoxicity, VAV1. Activity of YT-VAV1 cells obtained was assayed in vitro against several cancer cell lines and primary patient-derived cancer cells. YT-VAV1 cells outperform parental YT cells in terms of cytotoxicity.

Synthesis and Cytotoxicity Assessment of Novel 7-O- and 14-O-Derivatives of Glaucocalyxin A.

Rabdosia japonica has been historically used in China as a popular folk medicine for the treatment of cancer, hepatitis, and gastricism. Glaucocalyxin A (GLA), an ent-kaurene diterpene isolated from Rabdosia japonica, is one of the main active ingredients showing potent inhibitory effects against several types of tumor cells. To the best of our knowledge, studies regarding the structural modification and Structure- Activity Relations (SAR) of this compound have not yet been reported. The aim of this study was to discover more potent derivatives of GLA and investigate their SAR and cytotoxicity mechanisms. Novel 7-O- and 14-O-derivatives of GLA were synthesized by condensation of acids or acyl chloride. The anti-tumor activities of these derivatives against various human cancer cell lines were evaluated in vitro by MTT assays. Apoptosis assays of compound 17 (7,14-diacylation product) were performed on A549 and HL-60 cells by flow cytometry and TUNNEL. The acute toxicity of this compound was tested on mice, at the dose of 300mg per kg body weight. Seventeen novel 7-O- and 14-O-derivatives of GLA (1-17) were synthesized. These compounds showed potent cytotoxicity against the tested cancer cell lines, and almost all of them were found to be more cytotoxic than GLA and oridonin. Of the synthesized derivatives, compound 17 presented the greatest cytotoxicity, with IC50 values of 0.26μM and 1.10μM in HL-60 and CCRF-CEM cells, respectively. Furthermore, this compound induced weak apoptosis of A549 cells but showed great potential in stimulating the apoptosis of HL- 60 cells. Acute toxicity assays indicated that compound 17 is relatively safer. The results reported herein indicate that the synthesized GLA derivatives exhibited greater cytotoxicity against leukemia cells than against other types of tumors. In particular, 7,14-diacylation product of GLA was found to be an effective anti-tumor agent. However, the cytotoxicity mechanism of this product in A549 cells is expected to be different than that in other tumor cell lines. Further research is needed to confirm this hypothesis.