Alginate-gelatine hydrogel microspheres protect NK cell proliferation and cytotoxicity under hypoxic conditions.

AIMS: This study aimed to encapsulate natural killer (NK) cells in a hydrogel to sustain their function within the hypoxic tumour microenvironments. METHODS: An alginate-gelatine hydrogel was generated via electrospray technology. Hydrogel biocompatibility was assessed through cell counting kit-8 and Live/Dead assays to ascertain cell. Moreover, we analysed lactate dehydrogenase assays to evaluate the cytotoxicity against tumours and utilised RT-qPCR to analyse cytokine gene level. RESULTS: Alginate and gelatine formed hydrogels with diameters ranging from 489.2 ± 23.0 µm, and the encapsulation efficiency was 34.07 ± 1.76%. Encapsulated NK cells exhibited robust proliferation and tumour-killing capabilities under normoxia and hypoxia. Furthermore, encapsulation provided a protective shield against cell viability under hypoxia. Importantly, tumour-killing cytotoxicity through cytokines upregulation such as granzyme B and interferon-gamma was preserved under hypoxia. CONCLUSION: The encapsulation of NK cells not only safeguards their viability but also reinforces anticancer capacity, countering the inhibition of activation induced by hypoxia.

Enhanced cytotoxic activity of natural killer cells from increased calcium influx induced by electrical stimulation.

Natural killer (NK) cells play a crucial role in immunosurveillance independent of antigen presentation, which is regulated by signal balance via activating and inhibitory receptors. The anti-tumor activity of NK cells is largely dependent on signaling from target recognition to cytolytic degranulation; however, the underlying mechanism remains unclear, and NK cell cytotoxicity is readily impaired by tumor cells. Understanding the activation mechanism is necessary to overcome the immune evasion mechanism, which remains an obstacle in immunotherapy. Because calcium ions are important activators of NK cells, we hypothesized that electrical stimulation could induce changes in intracellular Ca2+ levels, thereby improving the functional potential of NK cells. In this study, we designed an electrical stimulation system and observed a correlation between elevated Ca2+ flux induced by electrical stimulation and NK cell activation. Breast cancer MCF-7 cells co-cultured with electrically stimulated KHYG-1 cells showed a 1.27-fold (0.5 V/cm) and 1.55-fold (1.0 V/cm) higher cytotoxicity, respectively. Electrically stimulated KHYG-1 cells exhibited a minor increase in Ca2+ level (1.31-fold (0.5 V/cm) and 1.11-fold (1.0 V/cm) higher), which also led to increased gene expression of granzyme B (GZMB) by 1.36-fold (0.5 V/cm) and 1.58-fold (1.0 V/cm) by activating Ca2+-dependent nuclear factor of activated T cell 1 (NFAT1). In addition, chelating Ca2+ influx with 5 µM BAPTA-AM suppressed the gene expression of Ca2+ signaling and lytic granule (granzyme B) proteins by neutralizing the effects of electrical stimulation. This study suggests a promising immunotherapeutic approach without genetic modifications and elucidates the correlation between cytolytic effector function and intracellular Ca2+ levels in electrically stimulated NK cells.

Dual effects of hypoxia on proliferation and osteogenic differentiation of mouse clonal mesenchymal stem cells.

Mouse clonal mesenchymal stem cells (mc-MSCs) were cultured on a Cytodex 3 microcarrier in a spinner flask for a suspension culture under hypoxia condition to increase mass productivity. The hypoxia environment was established using 4.0 mM Na2SO3 with 10 µM or 100 µM CoCl2 for 24 h in a low glucose DMEM medium. As a result, the proliferation of mc-MSCs under hypoxic conditions was 1.56 times faster than the control group over 7 days. The gene expression of HIF-1a and VEGFA increased 4.62 fold and 2.07 fold, respectively. Furthermore, the gene expression of ALP, RUNX2, COL1A, and osteocalcin increased significantly by 9.55, 1.55, 2.29, and 2.53 times, respectively. In contrast, the expression of adipogenic differentiation markers, such as PPAR-γ and FABP4, decreased. These results show that the hypoxia environment produced by these chemicals in a suspension culture increases the proliferation of mc-MSCs and promotes the osteogenic differentiation of mc-MSCs.

Hypergravity-induced changes in actin response of breast cancer cells to natural killer cells.

Although immunotherapy holds promising cytotoxic activity against lymphoma or leukemia, the immunosuppressive mechanisms of solid tumors remain challenging. In this study, we developed and applied a hypergravity exposure system as a novel strategy to improve the responsiveness of breast cancer cells to natural killer (NK) cells for efficient immunotherapy. Following exposure to hypergravity, either in the presence or absence of NK cells, we investigated for changes in the cell cytoskeletal structure, which is related to the F-actin mediated immune evasion mechanism (referred to as "actin response") of cancer cells. Breast cancer cell line MDA-MB-231 cells were exposed thrice to a 20 min hypergravitational condition (10 × g), with a 20 min rest period between each exposure. The applied hypergravity induces changes in the intracellular cytoskeleton structure without decreasing the cell viability but increasing the cytotoxicity of MDA-MB-231 from 4 to 18% (4.5-fold) at a 3:1 ratio (NK-to-target). Analyses related to F-actin further demonstrate that the applied hypergravity results in rearrangement of the cytoskeleton, leading to inhibition of the actin response of MDA-MB-231. Taken together, our results suggest that the mechanical load increases through application of hypergravity, which potentially improves efficiency of cell-based immunotherapies by sensitizing tumors to immune cell-mediated cytotoxicity.

Vibrational stress affects extracellular signal-regulated kinases activation and cytoskeleton structure in human keratinocytes.

As the outermost organ, the skin can be damaged following injuries such as wounds and bacterial or viral infections, and such damage should be rapidly restored to defend the body against physical, chemical, and microbial assaults. However, the wound healing process can be delayed or prolonged by health conditions, including diabetes mellitus, venous stasis disease, ischemia, and even stress. In this study, we developed a vibrational cell culture model and investigated the effects of mechanical vibrations on human keratinocytes. The HaCaT cells were exposed to vibrations at a frequency of 45 Hz with accelerations of 0.8g for 2 h per day. The applied mechanical vibration did not affect cell viability or cell proliferation. Cell migratory activity did increase following exposure to vibration, but the change was not statistically significant. The results of immunostaining (F-actin), western blot (ERK1/2), and RT-qPCR (FGF-2, PDGF-B, HB-EGF, TGF-ß1, EGFR, and KGFR) analyses demonstrated that the applied vibration resulted in rearrangement of the cytoskeleton, leading to activation of ERK1/2, one of the MAPK signaling pathways, and upregulation of the gene expression levels of HB-EGF and EGFR. The results suggest that mechanical vibration may have wound healing potential and could be used as a mechanical energy-based treatment for enhancing wound healing efficiency.

Effects of collected road dusts on cell viability, inflammatory response, and oxidative stress in cultured human corneal epithelial cells.

Most studies on the adverse effects of air pollution have focused on respiratory and cardiovascular diseases, and there are relatively few studies on eye diseases following exposure of ambient particulate matter (PM). Epidemiological and clinical researches correlating the eye and PMs have recently received attention. PMs are complex mixture of particles that vary in chemical composition and size. This study investigated the influence of collected road dust on cell viability, inflammatory responses, and oxidative stress in human corneal epithelial cells. The collected road dust was classified with respect to aerodynamic diameter and solubility. Exposure concentration was calculated based on the particle deposition rate. We observed a dose-dependent decrease in cell viability at total PM2.5 and PM10. The pellet fractions of total PM2.5 and PM10 mainly contributed to the mitochondrial activity. Although both total PM2.5 and PM10 did not change the membrane integrity, the supernatant fractions significantly affected cell membrane integrity. Both total and fractions induced nitric oxide production and interleukin 8 expression. In addition, total PM2.5 and PM10 increased the oxidative stress; the pellet fractions of total PM2.5 and PM10 also induced higher oxidative stress. However, there was no significant difference between the cellular responses of total PM2.5 and PM10. We observed that the effects of collected road dust on cellular responses were strongly dependent on their concentration and solubility.

Porous NH2-MIL-125 as an efficient nano-platform for drug delivery, imaging, and ROS therapy utilized Low-Intensity Visible light exposure system.

Metal-organic frameworks are a novel class of organic-inorganic hybrid polymer with potential applications in bioimaging, drug delivery, and ROS therapy. NH2-MIL-125, which is a titanium-based metal organic framework with a large surface area of 1540m2/g, was synthesized using a hydrothermal method. The material was characterized by powder X-ray diffreaction (PXRD), thermogravimetric analysis (TGA), and transmission electron microscopy (TEM), and N2 isotherm analyses. The size of the polymer was reduced to the nanoscale using a high-frequency sonication process. PEGylation was carried out to improve the stability and bioavailability of the NMOF. The as-synthesized nano-NH2-MIL-125/PEG (NMOF/PEG) exhibited good biocompatibility over the (Cancer) MCF-7 and (Normal) COS-7 cell line. The interaction of NMOF/PEG with the breast cancer cell line (MCF-7) was examined by BIO-TEM analysis and laser confocal imaging. 2',7'-dichlorofluorescin diacetate (DCFDA) analysis confirmed that NMOF/PEG produced free radicals inside the cancer cell line (MCF-7) upon visible light irradiation. NMOF/PEG absorbed a large amount of DOX (20wt.% of DOX) and showed pH, and photosensitive release. This controlled drug delivery was attributed to the presence of NH2, Ti group in MOF and a hydroxyl group in PEG. This combination of chemo- and ROS-therapy showed excellent efficiency in killing cancer MCF-7 cells.

Enhanced production of recombinant proteins by a small molecule protein synthesis enhancer in combination with an antioxidant in recombinant Chinese hamster ovary cells.

The improvement in the production of recombinant proteins has been linked in a number of small molecules such as carboxylic acids to the inhibition of histone deacetylase, leading to increased transcription of genes. However, carboxylic acids such as pentanoic acid and butanoic acid have been shown to promote an apoptotic response in Chinese hamster ovary (CHO) cell culture. Supplementation of cultures with antioxidants has shown the ability to reduce the apoptotic response of carboxylic acid supplementation, leading to increased therapeutic protein production. In this study, we showed that pentanoic acid reduced the number of cells entering early apoptosis relative to butanoic acid by 15.4%. Additionally, supplementation of butanoic acid- and pentanoic acid-treated cultures with N-acetyl cysteine (NAC) reduced the population of cells entering early apoptosis by 5.3 and 10.0%, respectively, while increasing productivity by 19.5% in the presence of pentanoic acid and NAC. Conversely, a decrease of 5.7% in production was observed in response to combined butanoic acid and N-acetyl cysteine treatment. The results presented herein provide evidence that a culture supplementation method is critical for optimization of biopharmaceutical manufacturing processes.

Evaluation of Cytotoxicity and Hypoxic Effect of Nitroimidazole Embedded Nanoparticles.

Adenylate cyclase is a key intracellular enzyme involved in energy imbalance leading to tumor hypoxia and cytotoxicity. In this study, adenylate cyclase activities in isolated hepatocytes and Kupffer cells were compared in the presence of several metabolic stimulators. In cultured hepatocyte cells, adenylate cyclase was stimulated by guanylyl imidotriphosphate (GITP), guanosine triphosphate (GTP), progesterone and nitroimidazole embedded nanoparticle (NNP) effectors, while prostaglandin E2 and F2α were used as effectors in cultured Kupffer cells. The results showed that NNPs decreased adenylate cyclase specific activity in a dose-dependent manner after preincubation of hepatocytes with NNPs. The NNPs stimulated adenylate cyclase activities in hepatocytes were evaluated based on measurement of cyclic adenosine monophosphate (cAMP). The stimulatory effects of NNPs on adenylate cyclase were independent of the presence of GTP and may have been due to a direct effect on the catalytic subunit of adenylate cyclase. In addition, basal cAMP generation in hepatocyte cells was efficiently suppressed by the NNPs. In conclusion, NNPs exerted direct effects on the catalytic subunit of the adenylate cyclase system, and adenylate cyclase was hormone sensitive in liver cells.

Porous Covalent Triazine Polymer as a Potential Nanocargo for Cancer Therapy and Imaging.

A microporous covalent triazine polymer (CTP) network with a high surface area was synthesized via the Friedel-Crafts reaction and employed as a potential transport system for drug delivery and controlled release. The CTP was transformed to the nanoscale region by intense ultrasonication followed by filtration to yield nanoscale CTP (NCTP). This product showed excellent dispersibility in physiological solution while maintaining its chemical structure and porosity. An anticancer drug, doxorubicin (DOX), was loaded onto the NCTP through hydrophobic and π-π interactions, and its release was controlled at pH 4.8 and 7.4. The NCTP showed no toxicity toward cancer or normal cells, but the NCTP-DOX complex showed high efficacy against both types of cells in vitro. In-vitro cell imaging revealed that NCTP is a potential material for bioimaging. The potency of NCTP on cellular senescence was confirmed by the expression of senescence associated marker proteins p53 and p21. These results suggest that NCTP can be used as a new platform for drug delivery and imaging with potential applications in diagnosis and therapy.

Length-dependent effect of single-walled carbon nanotube exposure in a dynamic cell growth environment of human alveolar epithelial cells.

Despite the great use of nanomaterials for engineering and medical applications, nanomaterials may have adverse consequences by accidental exposure, because of their nanoscale size, composition and shape. Like many nanomaterials, carbon nanotubes (CNTs) have been used for many proven applications, but the size of the CNTs makes them more readily become airborne and can therefore create the risk of being inhaled by a worker. In this study, we evaluated single-walled CNT (SWCNT)-induced effects on cellular responses such as cell proliferation, inflammatory response and oxidative stress in dynamic cell growth condition. A dynamic cell growth environment was established to mimic the dynamic changes in the amount of circumferential and longitudinal expansion and contraction occurred during normal breathing movement in the lung. Two different length (short: outer diameter (OD) 1-2 nm, length 0.5-2 µm; long: OD 1-2 nm, length 5-30 µm) of SWCNTs were used at different exposure concentrations (5, 10 and 20 µg/ml) during the different exposure duration (24, 48 and 72 h). Dynamic environment facilitated altered interaction between SWCNTs and A549 monolayer. Cellular responses in dynamic condition were significantly different from those in static condition. Moreover, cellular responses were dependent on the length of SWCNTs both in static and dynamic cell growth conditions.

Effect of exposure conditions on SWCNT-induced inflammatory response in human alveolar epithelial cells.

There has been an increasing interest in the development and applications of carbon nanotubes (CNTs) due to their huge potential in industrial and medical applications, but the toxicological properties of these materials have not been well characterized, especially the effects of nanoparticle exposure under different conditions on cellular responses. Nano-structured particles are potentially hazardous when they deposit in the respiratory system. In this study, we characterized the effects of single walled CNT (SWCNT) exposure on interleukin-8 (IL-8) expression in human alveolar epithelial cells (A549) under various exposure conditions. We measured the level of IL-8 expression in the presence and absence of serum following exposure of SWCNTs. The results demonstrated that IL-8 expression was enhanced in the presence of serum. When A549 cells were exposed to low concentrations of SWCNTs, IL-8 expression kept increasing, even after removal of SWCNTs from the media. In addition, SWCNT exposure under dynamic cell growth condition induced changes in IL-8 expression.

Ultrasonic differentiation of normal versus malignant breast epithelial cells in monolayer cultures.

Normal and malignant mammary epithelial cells were studied using laboratory measurements, wavelet analysis, and numerical simulations of monolayer cell cultures to determine whether microscopic breast cancer can be detected in vitro with high-frequency ultrasound. Pulse-echo waveforms were acquired by immersing a broadband, unfocused 50-MHz transducer in the growth media of cell culture well plates and collecting the first reflection from the well bottoms. The simulations included a multilayer pulse-reflection model and a model of two-dimensional arrays of spherical cells and nuclei. The results show that normal and malignant cells produce time-domain signals and spectral features that are significantly different.