Abstract: The objectives of the first year of research activities were to develop the leather surfaces covered by Ag/TiO2NMs using two main techniques: the conventional one, which consists in spraying the new synthesized Ag-TiO2NMs embedded in film forming polymers (INCDTP-ICPI Division Romania) and an innovative method, by plasma and sputtering techniques (Universidade do Minho Portugal). Derivates objectives were: the selection of methods for cytotoxicological testing of Ag/TiO2NMs and leathers treated with NMs (Institute of Cellular Biology and Pathology "Nicolae Simionescu" Romania), selection of suitable conditions for tribological tests in view of wearing simulation (Montanuniversität Leoben Austria). The main results were: the syntheses of 3 kinds of Ag/TiO2NMs (D1.1), physical-chemical characterization of new NMs (D1.2), plasma technology for leather surface treatment (D2.1), sputtering technology parameter settling (D2.2), cytotoxicological evaluation of Ag/TiO2NMs and leather surface wearing simulation by tribological tests.
Abstract: The objectives of the second year of research activities were to develop leather surfaces with self protection properties by using Ag/TiO2 and Ag/N-TiO2 nanoparticles through spraying (INCDTP-ICPI Division Romania) or by sputtering (Universidade do Minho Portugal), the evaluation of NMs cytotoxicological impact on epithelial cells, the identification of cell death mechanism induced by NMs (Institute of Cellular Biology and Pathology "Nicolae Simionescu" Romania) and ontological model selection for cytotoxicological and inflammatory response of human cells (INCDTP-ICPI Division Romania). The main results were: Leathers for footwear with self protection properties (D1.3); Leather characterisation, Ag-TiO2NMs optimum content for surface functionalisation (D1.4.), Wearing characterisation and assessment of leachable Ag-TiO2NM (D1.5), Report on the in silico studies on cell morphology ( D5.1), Report on the cytotoxicity studies and intracellular signaling pathways activated by manufactured oxide nanoparticles in human skin cells and human epithelial alveolar cells (D3.1), Leathers with antibacterial, self cleaning and/or flame retardant properties for footwear manufacture (D6.1), Superhydrophobic leather surfaces (D2.3), Antimicrobial leather surfaces (D2.4); Technology for leather surface functionalization based on molecular layer deposition (D2.5).
The main achievements and outputs of NANO_SAFE_LEATHER project resulted in collaborative activities are listed as follows:
- New Ag/TiO2NMs with advanced multifunctional properties for leather surface finishing as alternative for volatile organic compounds with non durable properties, pollutant for environment (water, soil, air) and consumers;
- Cytotoxicological evaluation of Ag/TiO2NMs on human epitelial cells;
- Ontological model for cytotoxicological response of human cells to Ag/TiO2NMs exposure;
- Innovative techniques based on CD-sputtering for physical deposition of NMs on leather surface as an ecological alternative to spraying method;
- New method based on tribological tests, adapted to leather structure and simulating the wearing behavior of leather surface covered with NMs;
- A new transnational consortium dedicated to NMs study and evaluation risks, optimisation of industrial technologies for competitiveness increase based on safe use of NMs.
|Scheme of TiO2 and N-TiO2 nanoparticles doping with Ag|
|Ag/TiO2 and Ag/N-TiO2 NPs with 50-64 nm size (TEM images) and photocatalytic activity in visible light (DRS spectra)|
|Leathers covered with Ag/TiO2 and Ag/N-TiO2 NPs, with antimicrobial resistance, self-cleaning properties and resistant to heat/fire|
|HaCaT keratinocyte viability under NMs exposure: non toxic effect for 10 - 400µg/ml||A549 lung cell viability under NMs exposure: non toxic effect for 200-750 µg/ml|
|Cytotoxicological evaluation of Ag/TiO2NMs on human epitelial cells|
|Elaboration of ontological model for cytotoxicological effect on Ag/TiO2NMs on human epitelial cells|
Abstract: The objective of stage III of the project, "Research on the effects of exposure to Ag and TiO2 NMs on lung epithelial cells", was the evaluation of Ag/TiO2NP cytotoxicity through identification of intracellular signaling pathways for skin and lung epithelial cells and inflammatory effects at lung cells level, in view of estimation of risks in industrial environment (D3.1, M7, D4.1). Other objectives were related to data processing through in silico methods with the final aim of setting the alert limits for risk concentrations of NMs used in leather surface finishing (D5.1); leachability of NMs in simulated wearing conditions (D1.5., M3) leathers manufacturing with protection properties, dissemination of the project results and intellectual rights protection through patent demand application (D7.5).
CO-INCDTP - ICPI Division carried out the experiments on wearing simulation of finished leather with Ag/TiO2NMs. The leathers were finished by classical methods (by spraying) and by sputtering (leather samples covered by P1- Minho University Portugal). The investigations regarding the NPs leachebility were performed by SEM-EDX and showed that 33% of NPs deposited by spraying and 62% NPs deposited by sputtering are released after rubbing tests according to STAS 5049 method. The loss of Ti is also higher when the leathers were covered by sputtering (40-60%) and were not recorded for leather samples covered by spraying (Figure 1).
|Fig.1. Wearing simulation of leather surface covered by spraying with Ag/TiO2NMs|
The in silico study on alert limits and rapid evaluation of Ag/TiO2NP's cytotoxicity, performed by The National Institute of Research and Development for Biological Sciences (INCDSB), has used a mix of in silico techniques for ontological model implementation NIHCO (Nanoparticles Impact on the Human Cell Morphology Ontology). Thresholds values were introduced for the selected labels taken as markers for cytotoxicity. The risk values can be introduced as images or as absolute numbers. The images for risk values and the images to be analysed are processed by segmentation of numerical information (number of nuclei, nucleus surface, circularity etc). The results can be graphical or numerical displayed (Figure 2).
|Figure 2- Results of rapid evaluation of cytotoxicity according to the label "number of nuclei"|
P1 - Institute of Cellular Biology and Pathology "Nicolae Simionescu" has performed the investigations on skin and lung cellular response induced by Ag/TiO2 NMs, the production of cytokines and chemokines as inflammatory effect and ROS generation by lung cells. These studies are very important in setting the risks of NPs on consumer or worker health.
The main results of investigations revealed that the Ag/TiO2 NMs with 0.53% Ag-NP1, 1.14%Ag-NP2 and 1.63% Ag-NP2 do not have significant effect on the total level or phosphorylated p38 MAPK and JNK from epithelial lung cells. Only NP2 induces the increase of total level of ERK1/2 and of phosphorylated ERK1/2. NP3 is the formula that was selected for further experiments on leather finishing and shows that the major signaling pathways of skin and lung cell do not activate. ROS (Reactive Oxygen Species) were produced by lung cells exposed to more than 50 µg/ml AgTiO2 NMs (Figure 3).
The main results of this study will be the subject of future publications.
|Figure 3 - Intracellular ROS production by A549 epithelial lung cells under different concentrations of NP1, NP2 and NP3 NPs|
P3 - SC Taro Commimpex SRL has experimented leathers with self-protection properties by using nanopowders of Ag/NTiO2. (Figure 4). The leathers were tested for self-cleaning and antimicrobial properties and showed reproducible properties as compared to results recorded in laboratory trials performed in CO pilot plant. The original results were subject of patent applications in the frame of partner consortium.
The dissemination activity included 1 published paper, 3 oral presentations, 3 posters, the presentation of the project on Nanosafecluster Bulletin (www.nanosafetycluster.eu) and 2 patent applications (OSIM and EPO).
|Figure 4 - Leathers covered with Ag/NTiO2NPs|
The main achievements of Stage III were:
- Report on identification of cell response induced by NMs.
- Report on cytokine, chemokine and ROS production of lung cells under NMs exposure.
- Report on wearing simulation of leather surface treated with NMs.
- Leathers with self protection properties.
- Paper, communication, patent (see Dissemination).
Abstract. The objective of Stage IV of the project, “Elaboration of industrial application guide of NMs based on Ag and TiO2” was the elaboration of guide draft for safe use of NMs for leather finishing ( D4.2, D6.2). In view of reaching this objective we have experimented the concentration limits of NMs for risk minimization related to skin and lung contact (M8, M9 ), in silico method for risk evaluation (M10), manufacturing of leathers with protection functions induced by NMs (M11, D6.1 ). Results dissemination was accomplished by submitting papers (D3.2, D5.2) and communications (D7.4).
CO-INCDTP- ICPI Division has elaborated the Guide for NM industrial application in safe conditions according to the new knowledge accumulated in the frame of research activities for cytotoxicology evaluation on human cell lines and legislative rules released at UE and USA level (Fig.5).
The leachebility evaluation of NMs releasing from leather surface showed that the concentrations are under the cytotoxicological risk limits.
The in silico studies regarding the epithelial cell morphology under the influence of NMs was carried out in view of NMs risk evaluation as a fast tool for cytotoxicology evaluation (Fig.6) and were validated by experimental data.
|Fig.5 Safety sheet for powder NM||Fig.6- Cell nucleus number processing by in silico method|
P1- Institute of Cellular Biology and Pathology " Nicolae Simionescu has experimented the risks associated with NM inhaling and skin contact. The final aim was the setting of the risk limits of NMs’ concentrations in view of risk minimization. Cell viability was evaluated as a function of NM’s concentration (10-1000 μg/ml) by using MTT method on keratinocytes cells (HaCaT) and on lung cells (A549). The ROS (reactive oxygen species) analysis was performed by 2′, 7′-Dichlorofluorescin diacetate (DCFH-DA) method. The results showed that the handling of NM is more important as compared to the possibility to leach from the leather surface. The lung cells are more sensitive than skin cells, the concentrations of 0.5 mg/mL for Ag/NTiO2 powder and 0.3 mg/mL for Ag/TiO2 solution represent the risk threshold for NM’s handling and inhaling. ROS analyses showed that the skin cells are less sensitive to Ag/NTiO2 NPs and the possible mechanism for cell death can be connected to the reactive oxygen species generations under the NM’s contact.
P3- SC Taro Commimpex SRL has experimented leathers (Figure 7) with self-protection properties by using Ag/TiO2 in solution, a commercial product with particle average size of 8 nm (Figure 8). The leathers manufactured in industrial conditions on larger quantities of leathers were tested for self-cleaning and antimicrobial properties (Figure 9) and showed reproducible properties as compared to results recorded in previous stages.
Figure 7 –Leathers covered with Ag/TiO2NPs
Fig.8-TEM image of NPs
Figure 9- Inhibition zones of leathers covered with Ag/TiO2NPs against
Staficloccoccus aureus (left) and Escherichia coli (right)
Dissemination activities were completed with submitting of 2 papers to be published in ISI indexed journals (1 paper in evaluation and 1 accepted paper ), 5 international communications (9th International Conference on Nanotoxicology Neuss, Germany 2018; International Conference on Nanotechnology and Bioscience” Heraklion, Crete 2018; 11th Asia International Conference of Leather Science and Technology (AICLST), 2018, Xi’an, China; The 36 th Annual Scientific Session of the Romanian Society for Cell Biology and the 10th National Congress with International Participation”, 2018, Craiova, Romania and “Advances in proteomics and progress in precision medicine”, 2018, Bucuresti, Romania).