The potential effects of in vitro digestion on the physicochemical and biological characteristics of polystyrene nanoplastics.

The presence of plastic waste in our environment has continued growing and become an important environmental concern. Because of its degradation into micro- and nanoplastics (MNPLs), MNPLs are becoming environmental pollutants of special environmental/health concern. Since ingestion is one of the main exposure routes to MNPLs, the potential effects of digestion on the physicochemical/biological characteristics of polystyrene nanoplastics (PSNPLs) were determined. The results indicated a high tendency of digested PSNPLs to agglomerate and a differential presence of proteins on their surface. Interestingly, digested PSNPLs showed greater cell uptake than undigested PSNPLs in all three tested cell lines (TK6, Raji-B, and THP-1). Despite these differences in cell uptake, no differences in toxicity were observed except for high and assumed unrealistic exposures. When oxidative stress and genotoxicity induction were determined, the low effects observed after exposure to undigested PDNPLs were not observed in the digested ones. This indicated that the greater ability of digested PSNPLs to internalize was not accompanied by a greater hazard. This type of analysis should be performed with other MNPLs of varying sizes and chemical compositions.

Nanometers-Thick Ferromagnetic Surface Produced by Laser Cutting of Diamond.

In this work, we demonstrate that cutting diamond crystals with a laser (532 nm wavelength, 0.5 mJ energy, 200 ns pulse duration at 15 kHz) produced a ≲20 nm thick surface layer with magnetic order at room temperature. We measured the magnetic moment of five natural and six CVD diamond crystals of different sizes, nitrogen contents and surface orientations with a SQUID magnetometer. A robust ferromagnetic response at 300 K was observed only for crystals that were cut with the laser along the (100) surface orientation. The magnetic signals were much weaker for the (110) and negligible for the (111) orientations. We attribute the magnetic order to the disordered graphite layer produced by the laser at the diamond surface. The ferromagnetic signal vanished after chemical etching or after moderate temperature annealing. The obtained results indicate that laser treatment of diamond may pave the way to create ferromagnetic spots at its surface.

Characterization of Total RNA, CD44, FASN, and PTEN mRNAs from Extracellular Vesicles as Biomarkers in Gastric Cancer Patients.

In-depth characterization has introduced new molecular subtypes of gastric cancer (GC). To identify these, new approaches and techniques are required. Liquid biopsies are trendsetting and provide an easy and feasible method to identify and to monitor GC patients. In a prospective cohort of 87 GC patients, extracellular vesicles (EVs) were isolated from 250 µL of plasma. The total RNA was isolated with TRIZOL. The total RNA amount and the relative mRNA levels of CD44, PTEN, and FASN were measured by qRT-PCR. The isolation of EVs and their contained mRNA was possible in all 87 samples investigated. The relative mRNA levels of PTEN were higher in patients already treated by chemotherapy than in chemo-naïve patients. In patients who had undergone neoadjuvant chemotherapy followed by gastrectomy, a decrease in the total RNA amount was observed after neoadjuvant chemotherapy and gastrectomy, while FASN and CD44 mRNA levels decreased only after gastrectomy. The amount of RNA and the relative mRNA levels of FASN and CD44 in EVs were affected more significantly by chemotherapy and gastrectomy than by chemotherapy alone. Therefore, they are a potential biomarker for monitoring treatment response. Future analyses are needed to identify GC-specific key RNAs in EVs, which could be used for the diagnosis of gastric cancer patients in order to determine their molecular subtype and to accompany the therapeutic response.

Enzymatic degradation of polyethylene terephthalate nanoplastics analyzed in real time by isothermal titration calorimetry.

Plastics are globally used for a variety of benefits. As a consequence of poor recycling or reuse, improperly disposed plastic waste accumulates in terrestrial and aquatic ecosystems to a considerable extent. Large plastic waste items become fragmented to small particles through mechanical and (photo)chemical processes. Particles with sizes ranging from millimeter (microplastics, <5 mm) to nanometer (nanoplastics, NP, <100 nm) are apparently persistent and have adverse effects on ecosystems and human health. Current research therefore focuses on whether and to what extent microorganisms or enzymes can degrade these NP. In this study, we addressed the question of what information isothermal titration calorimetry, which tracks the heat of reaction of the chain scission of a polyester, can provide about the kinetics and completeness of the degradation process. The majority of the heat represents the cleavage energy of the ester bonds in polymer backbones providing real-time kinetic information. Calorimetry operates even in complex matrices. Using the example of the cutinase-catalyzed degradation of polyethylene terephthalate (PET) nanoparticles, we found that calorimetry (isothermal titration calorimetry-ITC) in combination with thermokinetic models is excellently suited for an in-depth analysis of the degradation processes of NP. For instance, we can separately quantify i) the enthalpy of surface adsorption ∆AdsH = 129 ± 2 kJ mol-1, ii) the enthalpy of the cleavage of the ester bonds ∆EBH = -58 ± 1.9 kJ mol-1 and the apparent equilibrium constant of the enzyme substrate complex K = 0.046 ± 0.015 g L-1. It could be determined that the heat production of PET NP degradation depends to 95% on the reaction heat and only to 5% on the adsorption heat. The fact that the percentage of cleaved ester bonds (η = 12.9 ± 2.4%) is quantifiable with the new method is of particular practical importance. The new method promises a quantification of enzymatic and microbial adsorption to NP and their degradation in mimicked real-world aquatic conditions.

Calcium-sensing receptor-mediated NLRP3 inflammasome response to calciprotein particles drives inflammation in rheumatoid arthritis.

Increased extracellular Ca2+ concentrations ([Ca2+]ex) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR). To prevent extraosseous calcification in vivo, the serum protein fetuin-A stabilizes calcium and phosphate into 70-100 nm-sized colloidal calciprotein particles (CPPs). Here we show that monocytes engulf CPPs via macropinocytosis, and this process is strictly dependent on CaSR signaling triggered by increases in [Ca2+]ex. Enhanced macropinocytosis of CPPs results in increased lysosomal activity, NLRP3 inflammasome activation, and IL-1ß release. Monocytes in the context of rheumatoid arthritis (RA) exhibit increased CPP uptake and IL-1ß release in response to CaSR signaling. CaSR expression in these monocytes and local [Ca2+] in afflicted joints are increased, probably contributing to this enhanced response. We propose that CaSR-mediated NLRP3 inflammasome activation contributes to inflammatory arthritis and systemic inflammation not only in RA, but possibly also in other inflammatory conditions. Inhibition of CaSR-mediated CPP uptake might be a therapeutic approach to treating RA.

Picomolar glyphosate sensitivity of an optical particle-based sensor utilizing biomimetic interaction principles.

The continually growing use of glyphosate and its critically discussed health and biodiversity risks ask for fast, low cost, on-site sensing technologies for food and water. To address this problem, we designed a highly sensitive sensor built on the remarkably specific recognition of glyphosate by its physiological target enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs). This principle is implemented in an interferometric sensor by using the recently established soft colloidal probe (SCP) technique. EPSPs was site-specifically immobilized on a transparent surface utilizing the self-assembling properties of circadian clock gene 2 hydrophobin chimera and homogeneity of the layer was evidenced by atomic force microscopy. Exposure of the enzyme decorated biochip to glyphosate containing samples causes formation of enzyme-analyte complexes and a competitive loss of available binding sites for glyphosate-functionalized poly(ethylene glycol) SCPs. Functionalization of the SCPs with different types of linker molecules and glyphosate was assessed employing confocal laser scanning microscopy as well as confocal Raman microspectroscopy. Overall, reflection interference contrast microscopy analysis of SCP-biochip interactions revealed a strong influence of linker length and glyphosate coupling position on the sensitivity of the sensor. In employing a combination of pentaglycine linker and tethering glyphosate via its secondary amino group, concentrations in aqueous solutions down to 100 pM could be measured by the differential adhesion between SCP and biochip surface, supported by automated image analysis algorithms. This sensing concept could even prove its exceptional pM sensitivity in combination with a superior discrimination against structurally related compounds.

Docetaxel-loaded solid lipid nanoparticles prevent tumor growth and lung metastasis of 4T1 murine mammary carcinoma cells.

BACKGROUND: Metastasis causes the most breast cancer-related deaths in women. Here, we investigated the antitumor effect of solid lipid nanoparticles (SLN-DTX) when used in the treatment of metastatic breast tumors using 4T1-bearing BALB/c mice. RESULTS: Solid lipid nanoparticles (SLNs) were produced using the high-energy method. Compritol 888 ATO was selected as the lipid matrix, and Pluronic F127 and Span 80 as the surfactants to stabilize nanoparticle dispersion. The particles had high stability for at least 120 days. The SLNs' dispersion size was 128 nm, their polydispersity index (PDI) was 0.2, and they showed a negative zeta potential. SLNs had high docetaxel (DTX) entrapment efficiency (86%), 2% of drug loading and showed a controlled drug-release profile. The half-maximal inhibitory concentration (IC50) of SLN-DTX against 4T1 cells was more than 100 times lower than that of free DTX after 24 h treatment. In the cellular uptake test, SLN-DTX was taken into the cells significantly more than free DTX. The accumulation in the G2-M phase was significantly higher in cells treated with SLN-DTX (73.7%) than in cells treated with free DTX (23.0%), which induced subsequent apoptosis. TEM analysis revealed that SLN-DTX internalization is mediated by endocytosis, and fluorescence microscopy showed DTX induced microtubule damage. In vivo studies showed that SLN-DTX compared to free docetaxel exhibited higher antitumor efficacy by reducing tumor volume (p < 0.0001) and also prevented spontaneous lung metastasis in 4T1 tumor-bearing mice. Histological studies of lungs confirmed that treatment with SLN-DTX was able to prevent tumor. IL-6 serum levels, ki-67 and BCL-2 expression were analyzed and showed a remarkably strong reduction when used in a combined treatment. CONCLUSIONS: These results indicate that DTX-loaded SLNs may be a promising carrier to treat breast cancer and in metastasis prevention.

Photodynamic therapy mediated by aluminium-phthalocyanine nanoemulsion eliminates primary tumors and pulmonary metastases in a murine 4T1 breast adenocarcinoma model.

Photodynamic therapy (PDT) is effective in the treatment of different types of cancer, such as basal cell carcinoma and other superficial cancers. However, improvements in photosensitizer delivery are still needed, and the use of PDT against more deeply located tumors has been the subject of many studies. Thus, the goal of this study was to evaluate the efficacy of a nanoemulsion containing aluminium-phthalocyanine (AlPc-NE) as a mediator of photodynamic therapy (PDT-AlPc-NE) against grafted 4T1 breast adenocarcinoma tumors in mice (BALB/c). Short after the appearance of the tumor, the animals were divided into groups (n = 5) as follows: untreated; only AlPc-NE and treated with PDT-AlPc-NE. The tumor volume was measured with a digital calliper at specific times. The presence of metastasis in the lungs was evaluated by microtomography and histopathological analyses. The results show that the application of PDT-AlPc-NE eradicated the transplanted tumors in all the treated animals, while the animals from control groups presented a robust increase in the tumor volume. Still more significantly, microtomography showed the animals submitted the PDT-AlPc-NE to be free of detectable metastasis in the lungs. The histological analysis of the lungs further confirmed the results verified by the microtomography. Therefore, this study suggests that PDT-AlPc-NE is effective in the elimination of experimentally grafted breast tumors in mice and also in preventing the formation of metastasis in the lungs.

Single cell study of adipose tissue mediated lipid droplet formation and biochemical alterations in breast cancer cells.

Obesity is a known risk factor for breast cancer and a negative prognostic factor for cancer recurrence and survival. Several studies demonstrated that aggressive breast tumor cells contain higher numbers of intracellular lipid droplets (LDs). Here we applied simultaneous visualization, identification and quantification of the lipid accumulation in lipid droplets (LDs) of aggressive, human triple-negative MDA-MB-231 breast cancer cells treated with adipose tissue-conditioned medium (ACM) derived from overweight and obese patients. In addition to Oil Red O and AdipoRed fluorescent staining, label-free confocal Raman microspectroscopy (CRM) has been applied. CRM enables imaging of cell compartments as well as quantification and monitoring of specific biomolecules and metabolic processes on a single cell level. Interestingly, breast cancer cells incubated with ACM showed a significantly higher number of intracellular LDs. Cultivation of breast tumor cells with ACM of obese patients induced the formation of LDs with a 20-fold higher lipid concentration than cultivation with basal medium. This is in line with the significantly higher levels of NEFAs (non-esterified fatty acids) detected in the ACM obtained from obese patient compared to ACM obtained from overweight patients or basal medium. Further, by principal component analysis, we identified a significant increase in unsaturation, esterification and lipid to protein ratio in LDs in breast cancer cells incubated with ACM. CRM analyses might function as a valuable diagnostic tool to identify metabolic alterations in biological samples which in turn could provide more detailed insights in the pathogenesis of breast cancer in association with obesity.