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Mateusz Wierzbicki,1 Ewa Sawosz,1 Marta Grodzik,1 Anna Hotowy,1 Marta Prasek,1 Slawomir Jaworski,1 Filip Sawosz,2 André Chwalibog2 1Division of Nanobiotechnology, Warsaw University of Life Sciences, Warsaw, Poland; 2Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Copenhagen, Denmark Abstract: Carbon nanoparticles, with their high biocompatibility and low toxicity, have recently been considered for biomedical applications, including antiangiogenic therapy. Critical to normal development and tumor formation, angiogenesis is the process of forming capillary blood vessels from preexisting vessels. In the present study, we evaluated the effects of diamond and graphite nanoparticles on the development of chicken embryos, as well as vascularization of the chorioallantoic membrane and heart at the morphological and molecular level. Nanoparticles did not affect either body/heart weight or serum indices of the embryos’ health. However, vascularization of the heart and the density of branched vessels were significantly reduced after treatment with diamond nanoparticles and, to a lesser extent, graphite nanoparticles. Application of nanoparticles significantly downregulated gene and protein expression of the proangiogenic basic fibroblast growth factor, indicating that both diamond and graphite nanoparticles inhibit angiogenesis. Keywords: diamond, graphite, nanoparticles, vasculogenesis, bFGF, VEGF

Slawomir Jaworski,1 Ewa Sawosz,1 Marta Grodzik,1 Anna Winnicka,2 Marta Prasek,1 Mateusz Wierzbicki,1 André Chwalibog31Division of Biotechnology and Biochemistry of Nutrition, Faculty of Animal Science, 2Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland; 3Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Copenhagen, DenmarkAbstract: Graphene is a single atom-thick material with exciting potential. It can be used in many fields, from electronics to biomedicine. However, little is known about its toxicity and biocompatibility. Herein, we report a study on the toxicity of graphene platelets (GPs) by examining the influence of GPs on the morphology, mortality, viability, membrane integrity, and type of cell death of U87 and U118 glioma cells. It was found that graphene is toxic to glioma cells, but it activated apoptosis only in the U118 cell line, without inducing necrosis, indicating the potential applicability of GP in anticancer therapy.Keywords: toxicity, glioma, apoptosis

Carbon nanoparticles, with their high biocompatibility and low toxicity, have recently been considered for biomedical applications, including antiangiogenic therapy. Critical to normal development and tumor formation, angiogenesis is the process of forming capillary blood vessels from preexisting vessels. In the present study, we evaluated the effects of diamond and graphite nanoparticles on the development of chicken embryos, as well as vascularization of the chorioallantoic membrane and heart at the morphological and molecular level. Nanoparticles did not affect either body/heart weight or serum indices of the embryos' health. However, vascularization of the heart and the density of branched vessels were significantly reduced after treatment with diamond nanoparticles and, to a lesser extent, graphite nanoparticles. Application of nanoparticles significantly downregulated gene and protein expression of the proangiogenic basic fibroblast growth factor, indicating that both diamond and graphite nanoparticles inhibit angiogenesis.

It has been demonstrated that the content of certain amino acids in eggs is not sufficient to fully support embryonic development. One possibility to supply the embryo with extra nutrients and energy is in ovo administration of nutrients. Nanoparticles of diamond are highly biocompatible non-toxic carbonic structures, and we hypothesized that bio-complexes of diamond nanoparticles with L-glutamine may affect molecular responses in breast muscle. The objective of the investigation was to evaluate the effect of diamond nanoparticle (ND) and L-glutamine (Gln) on expression of growth and differentiation factors of chicken embryo pectoral muscles. ND, Gln, and Gln/ND solutions (50 mg/L) were injected into fertilized broiler chicken eggs at the beginning of embryogenesis. Muscle tissue was dissected at day 20 of incubation and analysed for gene expression of FGF2, VEGF-A, and MyoD1. ND and especially Gln/ND up-regulated expression of genes related to muscle cell proliferation (FGF2) and differentiation (MyoD1). Furthermore, the ratio between FGF2 and MyoD1 was highest in the Gln/ND group. At the end of embryogenesis, Gln/ND enhanced both proliferation and differentiation of pectoral muscle cells and differentiation dominated over proliferation. These preliminary results suggest that the bio-complex of glutamine and diamond nanoparticles may accelerate growth and maturation of muscle cells.

Graphene is a single atom-thick material with exciting potential. It can be used in many fields, from electronics to biomedicine. However, little is known about its toxicity and biocompatibility. Herein, we report a study on the toxicity of graphene platelets (GPs) by examining the influence of GPs on the morphology, mortality, viability, membrane integrity, and type of cell death of U87 and U118 glioma cells. It was found that graphene is toxic to glioma cells, but it activated apoptosis only in the U118 cell line, without inducing necrosis, indicating the potential applicability of GP in anticancer therapy.

Nanoparticles of colloidal silver (AgNano) can influence gene expression. Concerning trials of AgNano application in poultry as antimicrobial and metabolic agents, it is useful to reveal whether they affect the expression of genes crucial for bird development. AgNano were administered to broiler chickens as a water solution in two concentrations (10 and 20 ppm). After dissection of the birds, breast muscles and hearts were collected. Gene expression of FGF2 and VEGF on the mRNA and protein levels were evaluated using qPCR and ELISA methods. The results for gene expression in breast muscle revealed significant changes on the mRNA level (FGF2 was up-regulated) but not on the protein level. In the heart, 20 ppm of AgNano in drinking water increased the expression of VEGFA, at the same time decreasing FGF2 expression both on the transcriptional and translational levels. Changes in the expression of these genes may lead to histological changes, but this needs to be proven using histological and immunohistochemical examination of tissues. In general, we showed that AgNano application in poultry feeding can influence the expression of FGF2 and VEGFA genes on the mRNA and protein levels in growing chicken.

The objective of the present investigation was to evaluate the effects of taurine and Au nanoparticles on the expression of genes related to embryonic muscle development and on the morphological characteristics of muscles. Fertilised chicken eggs (n = 160) were randomly divided into four groups: without injection (Control) and with injection of Au nanoparticles (NanoAu), taurine (Tau) or Au nanoparticles with taurine (NanoAu + Tau). The experimental solutions were given in ovo, on the third day of incubation, by injecting 0.3 ml of the experimental solution into the air sack. The embryos were evaluated on the 20th day of incubation. The methods included gene expression at the mRNA and protein levels, immunohistochemistry, histology and microscopy. In groups NanoAu, Tau and NanoAu + Tau, the muscle structure and the number of muscle cells were affected. Furthermore, taurine increased fibre diameter, the total number of nuclei, the proportion of proliferating cell nuclear antigen (PCNA)-positive cells and the total cell number. Also, gene expression of basic fibroblast growth factor-2 and PCNA was downregulated. There were no significant interactions between NanoAu and taurine, indicating that NanoAu did not enhance the effects of taurine. It may be concluded that 20 days after injection, NanoAu affected some parameters of muscle development, but the most profound effects were those of taurine.

Marlena Zielinska1,2, Ewa Sawosz1, Marta Grodzik1, Mateusz Wierzbicki1, Maria Gromadka1, Anna Hotowy3, Filip Sawosz3, Andrzej Lozicki1, Andrè Chwalibog31Division of Biotechnology and Biochemistry of Nutrition, Warsaw University of Life sciences, Warsaw, 2The Kielanowski Institute of Animal Physiology and Nutrition, Jablonna, Poland; 3Department of Basic Animal and Veterinary sciences, University of copenhagen, Frederiksberg, DenmarkPurpose: It was hypothesized that heparan sulfate (HS) as an essential compound for myogenesis and nanoparticles of gold (nano-Au) as highly reactive compounds can affect muscle development as a consequence of molecular regulation of muscle cell formation, and that these effects may be enhanced by a complex of HS conjugated with nano-Au. The objective of the present study was to determine the effect of administration of nano-Au, HS, and a nano-Au+HS complex on the morphological and molecular characteristics of breast muscle during embryogenesis.Methods: Chicken embryos were used as in vivo model. Fertilized chicken eggs (n = 350) were randomly divided into the control group and the groups treated with nano-Au, HS, and nano-Au+HS. The experimental solutions were given in ovo on the first day of incubation and the embryos were evaluated on day 20 of incubation. The methods included biochemical indi- ces in blood, immunohistochemistry, microscopy (transmission electron microscopy, scanning electron microscopy, confocal), and gene expression at the messenger ribonucleic acid and protein levels.Results: The treatments did not adversely affect mortality, organ weight, and homeostasis of the embryos. HS stimulated the development and maturation of breast muscle by increasing the number of nuclei, satellite cells, and muscle fibers and affected the expression of basic fibroblast growth factor-2 and paired-box transcription factor-7. Furthermore, the nano-Au+HS complex contributed to the increased number of myocytes and nuclei in chicke

Marta Grodzik1, Ewa Sawosz1, Mateusz Wierzbicki1, Piotr Orlowski1, Anna Hotowy2, Tomasz Niemiec1, Maciej Szmidt3, Katarzyna Mitura4, André Chwalibog21Division of Biotechnology and Biochemistry of Nutrition, Warsaw University of Life Sciences, Warsaw, Poland; 2Department of Basic Animal and Veterinary Science, University of Copenhagen, Copenhagen, Denmark; 3Division of Histology and Embryology, Warsaw University of Life Sciences, Warsaw, Poland; 4Department of Biomedical Engineering, Koszalin University of Technology, Koszalin, PolandAbstract: The objective of the study was to determine the effect of carbon nanoparticles produced by different methods on the growth of brain tumor and the development of blood vessels. Glioblastoma multiforme cells were cultured on the chorioallantoic membrane of chicken embryo and after 7 days of incubation, were treated with carbon nanoparticles administered in ovo to the tumor. Both types of nanoparticles significantly decreased tumor mass and volume, and vessel area. Quantitative real-time polymerase chain reaction analysis showed downregulated fibroblast growth factor-2 and vascular endothelial growth factor expression at the messenger ribonucleic acid level. The present results demonstrate antiangiogenic activity of carbon nanoparticles, making them potential factors for anticancer therapy.Keywords: cancer, nanoparticle, embryo, angiogenesis, FGF-2, VEGF

Purpose: It was hypothesized that heparan sulfate (HS) as an essential compound for myogenesis and nanoparticles of gold (nano-Au) ashighly reactive compounds can affect muscle development as a consequence of molecular regulation of muscle cell formation, and that these effects may be enhanced by a complex of HS conjugated with nano-Au. The objective of the present study was to determine the effect of administration of nano-Au, HS, and a nano-Au+HS complex on the morphological and molecular characteristics of breast muscle during embryogenesis. Methods: Chicken embryos were used as in vivo model. Fertilized chicken eggs (n = 350) were randomly divided into the control group and the groups treated with nano-Au, HS, and nano-Au+HS. The experimental solutions were given in ovo on the first day of incubation and the embryos were evaluated on day 20 of incubation. The methods included biochemical indices in blood, immunohistochemistry, microscopy (transmission electron microscopy, scanning electron microscopy, confocal), and gene expression at the messenger ribonucleic acid and protein levels. Results: The treatments did not adversely affect mortality, organ weight, and homeostasis of the embryos. HS stimulated the development and maturation of breast muscle by increasing the number of nuclei, satellite cells, and muscle fibers and affected the expression of basic fibroblast growth factor-2 and paired-box transcription factor-7. Furthermore, the nano-Au+HS complex contributed to the increased number of myocytes and nuclei in chicken embryo muscles. Conclusion: The results indicate that the administration of HS and nano-Au affects muscle development and that this effect is enhanced by conjugating HS with nano-Au.

The objective of the study was to determine the effect of carbon nanoparticles produced by different methods on the growth of brain tumor and the development of blood vessels. Glioblastoma multiforme cells were cultured on the chrioallantoic membrane of chicken embryo and after 7 days of incubation, were treated with carbon nanoparticles administered in ovo to the tumor. Both types of nanoparticles significantly decreased tumor mass and volume, fibroblast growth factor-2 and vascular endothelial growth factor expression at the messenger ribonucleic acid level. The present results demonstrate antiangiogenic activity of carbon nanoparticles, making them potential factors for anticancer therapy.

Currently, medicine intensively searches for methods to transport drugs to a target (sick) point within the body. The objective of the present investigation was to evaluate morphological characteristics of the assembles of silver or diamond nanoparticles with Salmonella Enteritidis (G-) or Listeria monocytogenes (G+), to reveal possibilities of constructing nanoparticle-bacteria vehicles. Diamond nanoparticles (nano-D) were produced by the detonation method. Hydrocolloids of silver nanoparticles (nano-Ag) were produced by electric non-explosive patented method. Hydrocolloids of nanoparticles (200 microl) were added to bacteria suspension (200 microl) in the following order: nano-D + Salmonella E.; nano-D + Listeria monocytogenes; nano-Ag + Salmonella E; nano-Ag + Listeria monocytogenes. Samples were inspected by transmission electron microscopy. Visualisation of nanoparticles and bacteria interaction showed harmful effects of both nanoparticles on bacteria morphology. The most spectacular effect of nano-D were strong links between nano-D packages and the flagella of Salmonella E. Nano-Ag were closely attached to Listeria monocytogenes but not to Salmonella E. There was no evidence of entering nano-Ag inside Listeria monocytogenes but smaller particles were placed inside Salmonella E. The ability of nano-D to attach to the flagella and the ability of nano-Ag to penetrate inside bacteria cells can be utilized to design nano-bacteria vehicles, being carriers for active substances attached to nanoparticles.

André Chwalibog1, Ewa Sawosz2, Anna Hotowy1, Jacek Szeliga2, Stanislaw Mitura3, Katarzyna Mitura3, Marta Grodzik2, Piotr Orlowski2, Aleksandra Sokolowska41University of Copenhagen, Copenhagen, Denmark; 2Warsaw University of Life Sciences, Warsaw, Poland; 3Technical University of Lódz, Lódz, Poland; 4Warsaw University of Technology, Warsaw, PolandPurpose: The objective of the present investigation was to evaluate the morphologic characteristics of self-assemblies of diamond (nano-D), silver (nano-Ag), gold (nano-Au), and platinum (nano-Pt) nanoparticles with Staphylococcus aureus (bacteria) and Candida albicans (fungi), to determine the possibility of constructing microorganism–nanoparticle vehicles.Methods: Hydrocolloids of individual nanoparticles were added to suspensions of S. aureus and C. albicans. Immediately after mixing, the samples were inspected by transmission electron microscopy.Results: Visualization of the morphologic interaction between the nanoparticles and microorganisms showed that nano-D, which are dielectrics and exhibit a positive zeta potential, were very different from the membrane potentials of microorganisms, and uniformly surrounded the microorganisms, without causing visible damage and destruction of cells. All metal nanoparticles with negative zeta potential had cell damaging properties. Nano-Ag showed the properties of self-organization with the cells, disintegrating the cell walls and cytoplasmic membranes, and releasing a substance (probably cytoplasm) outside the cell. Arrangement of nano-Au with microorganisms did not create a system of self-organization, but instead a "noncontact" interaction between the nanoparticles and microorganisms was observed to cause damage to fungal cells. Nano-Pt caused both microorganisms to release a substance outside the cell and disintegrated the cytoplasmic membrane and cell wall.Conclusion: Nano-Ag, nano-Au, and nano-Pt (all metal nanoparti

Ewa Sawosz1, André Chwalibog2, Jacek Szeliga3, Filip Sawosz2, Marta Grodzik1, Marlena Rupiewicz1, Tomasz Niemiec1, Katarzyna Kacprzyk11Division of Biotechnology and Biochemistry of Nutrition, Warsaw University of Life Sciences, Warsaw, Poland; 2Department of Basic Animal and Veterinary Sciences, University of Copenhagen, Copenhagen, Denmark; 3Division of Microbiology of Analytical Centre, Warsaw University of Life Sciences, Warsaw, PolandPurpose: Rapid development of nanotechnology has recently brought significant attention to the extraordinary biological features of nanomaterials. The objective of the present ­investigation was to evaluate morphological characteristics of the assembles of gold and platinum nanoparticles (nano-Au and nano-Pt respectively), with Salmonella Enteritidis (Gram-negative) and Listeria monocytogenes (Gram-positive), to reveal possibilities of constructing bacteria-nanoparticle vehicles.Methods: Hydrocolloids of nano-Au or nano-Pt were added to two bacteria suspensions in the following order: nano-Au + Salmonella Enteritidis; nano-Au + Listeria monocytogenes; nano-Pt + Salmonella Enteritidis; nano-Pt + Listeria monocytogenes. Samples were inspected by transmission electron microscope.Results: Visualization of morphological interaction between nano-Au and Salmonella Enteritidis and Listeria monocytogenes, showed that nano-Au were aggregated within flagella or biofilm network and did not penetrate the bacterial cell. The analysis of morphological effects of interaction of nano-Pt with bacteria revealed that nano-Pt entered cells of Listeria monocytogenes and were removed from the cells. In the case of Salmonella Enteritidis, nano-Pt were seen inside bacteria cells, probably bound to DNA and partly left bacterial cells. After washing and centrifugation, some of the nano-Pt-DNA complexes were observed within Salmonella Enteritidis.Conclusion: The results indicate that the bacteria could be used as a vehicle to deliver nano-Pt

Purpose: The objective of the present investigation was to evaluate the morphologic characteristics of self-assemblies of diamond (nano-D), silver (nano-Ag), gold (nano-Au), and platinum (nano-Pt) nanoparticles with Staphylococus aureus (bacteria) and Candida albicans (fungi), to determine the possiblility of constructing microorganism-nanoparticle vehicles. Methods: Hydrocolloids of individual nanoparticles were added to suspensions of S. aureus and C. albicans. Immediately after mixing, the samples were inspected by transmission electron microscopy. Results: Visualization of the morphologic interaction between the nanoparticles and microorganisms showed that nano-D, which are dielectrics and exhibit a positive zeta potential, were very different from the membrane potentials of microorganisms, and uniformly surrounded the microorganisms, without causing visible damage and destruction of cells. All metal nanoparticles with negative zeta potential had cell damaging properties. Nano-Ag showed the properties of self-organization with the cells, disintegrating the cell walls and cytoplasmic membranes, and releasing a substance (probably cytoplasm) outside the cell. Arrangement of nano-Au with microorganisms did not create a system of self-organization, but instead a "noncontact" interaction between the nanoparticles and microorganisms was observed to cause damage to fungal cells. Nano-Pt caused both microorganisms to release a substance outside the cell and disintegrated the cytoplasmic membrane and cell wall. Conclusion: Nano-Ag, nano-Au, and nano-Pt (all metal nanoparticles) are harmful to bacteria and fungi. In contrast, nano-D bind closely to the surface of microorganisms without causing visible damage to cells, and demonstrating good self-assembling ability. The results indicate that both microorganisms could be used as potential carriers for na

Purpose: Rapid development of nanotechnology has recently brought significant attention to the extraordinary biological features of nanomaterials. The objective of the present ­investigation was to evaluate morphological characteristics of the assembles of gold and platinum nanoparticles (nano-Au and nano-Pt respectively), with Salmonella Enteritidis (Gram-negative) and Listeria monocytogenes (Gram-positive), to reveal possibilities of constructing bacteria-nanoparticle vehicles. Methods: Hydrocolloids of nano-Au or nano-Pt were added to two bacteria suspensions in the following order: nano-Au + Salmonella Enteritidis; nano-Au + Listeria monocytogenes; nano-Pt + Salmonella Enteritidis; nano-Pt + Listeria monocytogenes. Samples were inspected by transmission electron microscope. Results: Visualization of morphological interaction between nano-Au and Salmonella Enteritidis and Listeria monocytogenes, showed that nano-Au were aggregated within flagella or biofilm network and did not penetrate the bacterial cell. The analysis of morphological effects of interaction of nano-Pt with bacteria revealed that nano-Pt entered cells of Listeria monocytogenes and were removed from the cells. In the case of Salmonella Enteritidis, nano-Pt were seen inside bacteria cells, probably bound to DNA and partly left bacterial cells. After washing and centrifugation, some of the nano-Pt-DNA complexes were observed within Salmonella Enteritidis. Conclusion: The results indicate that the bacteria could be used as a vehicle to deliver nano-Pt to specific points in the body.

The objective of this study was to evaluate the effects of two weeks' protein deprivation on the cellular parameters of non-specific immunity in rats. Wistar rats (200-250 g) were divided into two groups (2x12) and were fed two isoenergetic (control and protein-free) diets. The phagocytic activity of neutrophils and monocytes, and the oxidative-burst activity of neutrophils of peripheral blood, were determined by flow cytometry after stimulation with E. coli and phorbol 12-mirystate 13-acetate. Feeding the protein-free diet for two weeks did not influence the phagocytic activity of neutrophils, monocytes or blood morphology. However, the oxidative burst of stimulated neutrophils was increased indicating that two weeks' protein deprivation does not depress the oxygen-dependent killing mechanism in neutrophils, but may lead to the overproduction of reactive oxygen species.

Nitrogen balances (six days) were determined in male Wistar rats during feeding a diet with sufficient protein or a nearly protein-free diet (n = 2 x 24), and then during three days of starvation (n = 2 x 12). The objective was to evaluate the effect of protein withdrawal on minimum nitrogen excretion in urine (UN), corresponding to endogenous UN, during feeding and subsequent starvation periods. The rats fed the protein free-diet had almost the same excretion of urinary N during feeding and starvation (165 and 157 mg/kg W(0.75)), while it was 444 mg/kg W(0.75) in rats previously fed with protein, demonstrating a major influence of protein content in a diet on N excretion during starvation. Consequently, the impact of former protein supply on N losses during starvation ought to be considered when evaluating minimum N requirement necessary to sustain life.

The objective of the present study was to examine the effects of hydrocolloidal silver nanoparticles (Ag-nano) on microbial profile of caecum and morphology of enterocytes in duodenum of Japanese quail, as a model animal for poultry. Quails (Coturnix coturnix japonica) (10 d old) were randomly divided into four groups (15 quails each) and located into four cages for 12 days. Quails were fed with granulated diets given ad libitum and had free access to drinking water. Ag-nano were added to drinking water at concentrations of 0, 5, 15 and 25 mg/kg. At the end of the experiment, the animals were killed and samples of duodenum and caeca microflora were collected. This initial investigation demonstrated that silver nanoparticles did not influence emphatically microflora of quail caecum; however, water containing 25 mg/kg of Ag-nano significantly increased the population of lactic acid bacteria. Furthermore, Ag-nano did not show any damaging properties on enterocytes of duodenal villi.