Correction: Salasinska et al. Burning Behaviour of Rigid Polyurethane Foams with Histidine and Modified Graphene Oxide. Materials 2021, 14, 1184.

In the original article [...].

Morphology and Chemical Purity of Water Suspension of Graphene Oxide FLAKES Aged for 14 Months in Ambient Conditions. A Preliminary Study.

Graphene and its derivatives have attracted scientists' interest due to their exceptional properties, making them alluring candidates for multiple applications. However, still little is known about the properties of as-obtained graphene derivatives during long-term storage. The aim of this study was to check whether or not 14 months of storage time impacts graphene oxide flakes' suspension purity. Complementary micro and nanoscale characterization techniques (SEM, AFM, EDS, FTIR, Raman spectroscopy, and elemental combustion analysis) were implemented for a detailed description of the topography and chemical properties of graphene oxide flakes. The final step was pH evaluation of as-obtained and aged samples. Our findings show that purified flakes sustained their purity over 14 months of storage.

Burning Behaviour of Rigid Polyurethane Foams with Histidine and Modified Graphene Oxide.

Since rigid polyurethane (PU) foams are one of the most effective thermal insulation materials with widespread application, it is an urgent requirement to improve its fire retardancy and reduce the smoke emission. The current work assessed the fire behavior of PU foam with non-halogen fire retardants system, containing histidine (H) and modified graphene oxide (GOA). For investigated system, three loadings (10, 20, and 30 wt.%) were used. The Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analysis, cone calorimetry (CC) and smoke density chamber tests as well as pre- and post-burning morphological evaluation using scanning electron microscope (SEM) were performed. Moreover, TGA combined with FT-IR was conducted to determine the substances, which could be evolved during the thermal decomposition of the PU with fire retardant system. The results indicated a reduction in heat release rate (HRR), maximum average rate of heat emission (MAHRE), the total heat release (THR) as well as the total smoke release (TSR), and maximum specific optical density (Dsmax) compared to the polyurethane with commercial fire retardant, namely ammonium polyphosphate (APP). A significantly improvement, especially in smoke suppression, suggested that HGOA system may be a candidate as a fire retardant to reduce the flammability of PU foams.

Investigation into morphological and electromechanical surface properties of reduced-graphene-oxide-loaded composite fibers for bone tissue engineering applications: A comprehensive nanoscale study using atomic force microscopy approach.

We decided to implement an extensive atomic force microscopy study in order to get deeper understanding of surface-related nanoscale properties of 3D printed pristine polycaprolactone and its reduced-graphene-oxide-loaded composites. The study included surface visualization and roughness quantification, elastic modulus and adhesion force assessment with force spectroscopy, along with kelvin probe force microscopy evaluation of local changes of surface potential. Atomic force microscopy examination was followed by scanning electron microscopy visualization and wettability assessment. Moreover, systematic examination of reduced graphene oxide flakes fabricated exclusively for this study was performed, including: scanning electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy and combustion elemental analysis. The addition of reduced graphene oxide resulted in thickening of the composite fibers and surface roughness enhancement. In addition, elastic modulus of composite fibers was higher and at the same time adhesion forces between scanning probe and tested surface was lower than for pristine polymeric ones. Lastly, we recorded local (nanoscale) alterations of surface potential of fibers with addition of graphene-derivative. The results clearly suggest graphene derivative's dose-dependent alteration of elastic modulus and adhesion force recorded with atomic force microscope. Moreover, changes of the material's surface properties were followed by changes of its electrical properties.

Graphene-based materials enhance cardiomyogenic and angiogenic differentiation capacity of human mesenchymal stem cells in vitro - Focus on cardiac tissue regeneration.

Cell-based therapies have recently emerged as promising strategies for the treatment of cardiovascular disease. Mesenchymal stem cells (MSCs) are a promising cell type that represent a class of adult stem cells characterized by multipotency, high proliferative capacity, paracrine activity, and low immunogenicity. To improve the functional and therapeutic efficacy of MSCs, novel biomaterials are considered as scaffolds/surfaces that promote MSCs growth and differentiation. One of them are graphene-based materials, including graphene oxide (GO) and reduced graphene oxide (rGO). Due to the unique physical, chemical, and biological properties of graphene, scaffolds comprising GO/rGO have been examined as novel platforms to improve the differentiation potential of human MSCs in vitro. We verified different i) size of GO flakes, ii) reduction level, and iii) layer thickness to select the most suitable artificial niche for MSCs culture. The results revealed that graphene-based substrates constitute non-toxic substrates for MSCs. Surfaces with large flakes of GO as well as low reduced rGO are the most biocompatible for MSCs propagation and do not affect their proliferation and survival. Interestingly, small GO flakes and highly reduced rGO decreased MSCs proliferation and induced their apoptosis. We also found that GO and rGO substrates did not alter the MSCs phenotype, cell cycle progression and might modulate the adhesive capabilities of these cells. Importantly, we demonstrated that both materials promoted the cardiomyogenic and angiogenic differentiation capacity of MSCs in vitro. Thus, our data indicates that graphene-based surfaces represent promising materials that may influence the therapeutic application of MSCs via supporting their pro-regenerative potential.

Synthesis and Characterization of Graphene Oxide and Reduced Graphene Oxide Composites with Inorganic Nanoparticles for Biomedical Applications.

Graphene oxide (GO) and reduced graphene oxide (RGO), due to their large active surface areas, can serve as a platform for biological molecule adhesion (both organic and inorganic). In this work we described methods of preparing composites consisting of GO and RGO and inorganic nanoparticles of specified biological properties: nanoAg, nanoAu, nanoTiO2 and nanoAg2O. The idea of this work was to introduce effective methods of production of these composites that could be used for future biomedical applications such as antibiotics, tissue regeneration, anticancer therapy, or bioimaging. In order to characterize the pristine graphene materials and resulting composites, we used spectroscopic techniques: XPS and Raman, microscopic techniques: SEM with and AFM, followed by X-Ray diffraction. We obtained volumetric composites of flake graphene and Ag, Au, Ag2O, and TiO2 nanoparticles; moreover, Ag nanoparticles were obtained using three different approaches.

Gradient Chitosan Hydrogels Modified with Graphene Derivatives and Hydroxyapatite: Physiochemical Properties and Initial Cytocompatibility Evaluation.

In this study, we investigated preparation of gradient chitosan-matrix hydrogels through a novel freezing-gelling-thawing method. The influence of three types of graphene family materials (GFM), i.e., graphene oxide (GO), reduced graphene oxide (rGO), and poly(ethylene glycol) grafted graphene oxide (GO-PEG), as well as hydroxyapatite (HAp) on the physicochemical and biological properties of the composite hydrogels was examined in view of their potential applicability as tissue engineering scaffolds. The substrates and the hydrogel samples were thoroughly characterized by X-ray photoelectron spectroscopy, X-ray diffractometry, infrared spectroscopy, digital and scanning electron microscopy, rheological and mechanical analysis, in vitro chemical stability and bioactivity assays, as well as initial cytocompatibility evaluation with human umbilical cord Wharton's jelly mesenchymal stem cells (hUC-MSCs). We followed the green-chemistry approach and avoided toxic cross-linking agents, using instead specific interactions of our polymer matrix with tannic acid, non-toxic physical cross-linker, and graphene derivatives. It was shown that the most promising are the gradient hydrogels modified with GO-PEG and HAp.

The Influence of Laser Ablation Parameters on the Holes Structure of Laser Manufactured Graphene Paper Microsieves.

The graphene paper microsieves can be applied in the filtration of biological fluids or separation of solid particles from exploitation fluids. To produce graphene paper microsieves for specific applications, good control over fabrication should be achieved. In this study, a laser ablation method using a picosecond laser was applied to fabricate graphene paper microsieves. Holes in the microsieves were drilled using pulsed laser radiation with a pulse energy from 5 to 100 µJ, a duration of 60 ps, a wavelength of 355 nm, and a repetition rate of 1 kHz. The impact method was applied using 10 to 100 pulses to drill one hole. To produce holes of a proper diameter which could separate biological particles of a certain size (≥10 µm), optimum parameters of graphene paper laser ablation were defined using the MATLAB software taking into account laser pulse energy, repetition rate, and a desired hole diameter. A series of structural tests were carried out to determine the quality of an edge and a hole shape. Experimental results and Laguerre-Gauss calculations in MATLAB were then compared to perform the analysis of the distribution of diffraction fringes. Optimum experimental parameters were determined for which good susceptibility of the graphene paper to laser processing was observed.

Functional Properties of Poly(Trimethylene Terephthalate)-Block-Poly(Caprolactone) Based Nanocomposites Containing Graphene Oxide (GO) and Reduced Graphene Oxide (rGO).

This work reports a study on the influence of graphene oxide (GO) and reduced graphene oxide (rGO) on the functional properties of poly(trimethylene terephthalate)-block-poly(caprolactone) (PTT-block-PCL-T) (75/25 wt.%/wt.%) copolymer, obtained from dimethyl terephthalate (DMT), 1,3-biopropanediol and polycaprolactone diol (PCL) via in situ polymerization. The article presents, if and how the reduction of graphene oxide, in comparison to the non-reduced one, can affect morphological, thermal, electrical and mechanical properties. SEM examination confirms/reveals the homogeneous distribution of GO/rGO nanoplatelets in the PTT-block-PCL-T copolymer matrix. More than threefold increase in the value of the tensile modulus is achieved by the addition of 1.0 wt.% of GO and rGO. Moreover, the thermal conductivity and thermal stability of the GO and rGO-based nanocomposites are also improved. The differential scanning calorimetry (DSC) measurement indicates that the incorporation of GO and rGO has a remarkable impact on the crystallinity of the nanocomposites (an increase of crystallization temperature up to 58 °C for nanocomposite containing 1.0 wt.% of GO is observed). Therefore, the high performances of the PTT-block-PCL-T-based nanocomposites are mainly attributed to the uniform dispersion of nanoplatelets in the polymer matrix and strong interfacial interactions between components.

Impact of Graphene-Based Surfaces on the Basic Biological Properties of Human Umbilical Cord Mesenchymal Stem Cells: Implications for Ex Vivo Cell Expansion Aimed at Tissue Repair.

The potential therapeutic applications of mesenchymal stem/stromal cells (MSCs) and biomaterials have attracted a great amount of interest in the field of biomedical engineering. MSCs are multipotent adult stem cells characterized as cells with specific features, e.g., high differentiation potential, low immunogenicity, immunomodulatory properties, and efficient in vitro expansion ability. Human umbilical cord Wharton's jelly-derived MSCs (hUC-MSCs) are a new, important cell type that may be used for therapeutic purposes, i.e., for autologous and allogeneic transplantations. To improve the therapeutic efficiency of hUC-MSCs, novel biomaterials have been considered for use as scaffolds dedicated to the propagation and differentiation of these cells. Nowadays, some of the most promising materials for tissue engineering include graphene and its derivatives such as graphene oxide (GO) and reduced graphene oxide (rGO). Due to their physicochemical properties, they can be easily modified with biomolecules, which enable their interaction with different types of cells, including MSCs. In this study, we demonstrate the impact of graphene-based substrates (GO, rGO) on the biological properties of hUC-MSCs. The size of the GO flakes and the reduction level of GO have been considered as important factors determining the most favorable surface for hUC-MSCs growth. The obtained results revealed that GO and rGO are suitable scaffolds for hUC-MSCs. hUC-MSCs cultured on: (i) a thin layer of GO and (ii) an rGO surface with a low reduction level demonstrated a viability and proliferation rate comparable to those estimated under standard culture conditions. Interestingly, cell culture on a highly reduced GO substrate resulted in a decreased hUC-MSCs proliferation rate and induced cell apoptosis. Moreover, our analysis demonstrated that hUC-MSCs cultured on all the tested GO and rGO scaffolds showed no alterations of their typical mesenchymal phenotype, regardless of the reduction level and size of the GO flakes. Thus, GO scaffolds and rGO scaffolds with a low reduction level exhibit potential applicability as novel, safe, and biocompatible materials for utilization in regenerative medicine.

Effects of Reduced Graphene Oxides on Apoptosis and Cell Cycle of Glioblastoma Multiforme.

Graphene (GN) and its derivatives (rGOs) show anticancer properties in glioblastoma multiforme (GBM) cells in vitro and in tumors in vivo. We compared the anti-tumor effects of rGOs with different oxygen contents with those of GN, and determined the characteristics of rGOs useful in anti-glioblastoma therapy using the U87 glioblastoma line. GN/ExF, rGO/Term, rGO/ATS, and rGO/TUD were structurally analysed via transmission electron microscopy, Raman spectroscopy, FTIR, and AFM. Zeta potential, oxygen content, and electrical resistance were determined. We analyzed the viability, metabolic activity, apoptosis, mitochondrial membrane potential, and cell cycle. Caspase- and mitochondrial-dependent apoptotic pathways were investigated by analyzing gene expression. rGO/TUD induced the greatest decrease in the metabolic activity of U87 cells. rGO/Term induced the highest level of apoptosis compared with that induced by GN/ExF. rGO/ATS induced a greater decrease in mitochondrial membrane potential than GN/ExF. No significant changes were observed in the cytometric study of the cell cycle. The effectiveness of these graphene derivatives was related to the presence of oxygen-containing functional groups and electron clouds. Their cytotoxicity mechanism may involve electron clouds, which are smaller in rGOs, decreasing their cytotoxic effect. Overall, cytotoxic activity involved depolarization of the mitochondrial membrane potential and the induction of apoptosis in U87 glioblastoma cells.

Interaction of different forms of graphene with chicken embryo red blood cells.

With the rapid development of graphene synthesis and functionalization approaches, graphene and its related derivatives have shown great potential in many applications in material science, including biomedical applications. Several in vitro and in vivo studies clearly showed no definitive risks, while others have indicated that graphene might become health hazards. In this study, we explore the biocompatibility of graphene-related materials with chicken embryo red blood cells (RBC). The hemolysis assay was employed to evaluate the in vitro blood compatibility of reduced graphene, graphene oxide, and reduced graphene oxide, because these materials have recently been used for biomedical applications, including injectable graphene-related particles. This study investigated structural damage, ROS production and hemolysis of chicken embryo red blood cells. Different forms of graphene, when incubated with chicken embryo RBC, were harmful to cell structure and induced hemolysis.

Toxicity of different forms of graphene in a chicken embryo model.

In the present work, the toxicity of three forms of graphene: pristine graphene (pG), graphene oxide (GO), and reduced graphene oxide (rGO) was investigated using a chicken embryo model. Fertilized chicken eggs were divided into the control group and groups administered with pG, GO, and rGO, in concentrations of 50, 500, and 5000 µg/ml. The experimental solutions were injected in ovo into the eggs, and at day 18 of incubation, the embryo survival, body and organ weights, the ultrastructure of liver samples, and the concentration of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the livers were measured. Survival of embryos decreased significantly after treatment with all types of graphene, but not in a dose-dependent manner. The body weights were only slightly affected by the highest doses of graphene, while the organ weights were not different among treatment groups. In all experimental groups, atypical hepatocyte ultrastructure and mitochondrial damage were observed. The concentration of the marker of DNA damage 8-OHdG in the liver significantly decreased after pG and rGO treatments. Further in vivo studies with different animal models are necessary to clarify the level of toxicity of different types of graphene and to estimate the concentrations appropriate to evaluate their biomedical applications and environmental hazard.

Long Term Influence of Carbon Nanoparticles on Health and Liver Status in Rats.

Due to their excellent biocompatibility, carbon nanoparticles have been widely investigated for prospective biomedical applications. However, their impact on an organism with prolonged exposure is still not well understood. Here, we performed an experiment investigating diamond, graphene oxide and graphite nanoparticles, which were repeatedly administrated intraperitoneally into Wistar rats for four weeks. Some of the animals was sacrificed after the last injection, whereas the rest were sacrificed twelve weeks after the last exposure. We evaluated blood morphology and biochemistry, as well as the redox and inflammatory state of the liver. The results show the retention of nanoparticles within the peritoneal cavity in the form of prominent aggregates in proximity to the injection site, as well as the presence of some nanoparticles in the mesentery. Small aggregates were also visible in the liver serosa, suggesting possible transportation to the liver. However, none of the tested nanoparticles affected the health of animals. This lack of toxic effect may suggest the potential applicability of nanoparticles as drug carriers for local therapies, ensuring accumulation and slow release of drugs into a targeted tissue without harmful systemic side effects.

Graphene Functionalized with Arginine Decreases the Development of Glioblastoma Multiforme Tumor in a Gene-Dependent Manner.

Our previous studies revealed that graphene had anticancer properties in experiments in vitro with glioblastoma multiforme (GBM) cells and in tumors cultured in vivo. We hypothesized that the addition of arginine or proline to graphene solutions might counteract graphene agglomeration and increase the activity of graphene. Experiments were performed in vitro with GBM U87 cells and in vivo with GBM tumors cultured on chicken embryo chorioallantoic membranes. The measurements included cell morphology, mortality, viability, tumor morphology, histology, and gene expression. The cells and tumors were treated with reduced graphene oxide (rGO) and rGO functionalized with arginine (rGO + Arg) or proline (rGO + Pro). The results confirmed the anticancer effect of graphene on GBM cells and tumor tissue. After functionalization with amino acids, nanoparticles were distributed more specifically, and the flakes of graphene were less agglomerated. The molecule of rGO + Arg did not increase the expression of TP53 in comparison to rGO, but did not increase the expression of MDM2 or the MDM2/TP53 ratio in the tumor, suggesting that arginine may block MDM2 expression. The expression of NQO1, known to be a strong protector of p53 protein in tumor tissue, was greatly increased. The results indicate that the complex of rGO + Arg has potential in GBM therapy.

Biodistribution of a High Dose of Diamond, Graphite, and Graphene Oxide Nanoparticles After Multiple Intraperitoneal Injections in Rats.

Carbon nanoparticles have recently drawn intense attention in biomedical applications. Hence, there is a need for further in vivo investigations of their biocompatibility and biodistribution via various exposure routes. We hypothesized that intraperitoneally injected diamond, graphite, and graphene oxide nanoparticles may have different biodistribution and exert different effects on the intact organism. Forty Wistar rats were divided into four groups: the control and treated with nanoparticles by intraperitoneal injection (4 mg of nanoparticles/kg body weight) eight times during the 4-week period. Blood was collected for evaluation of blood morphology and biochemistry parameters. Photographs of the general appearance of each rat's interior were taken immediately after sacrifice. The organs were excised and their macroscopic structure was visualized using a stereomicroscope. The nanoparticles were retained in the body, mostly as agglomerates. The largest agglomerates (up to 10 mm in diameter) were seen in the proximity of the injection place in the stomach serous membrane, between the connective tissues of the abdominal skin, muscles, and peritoneum. Numerous smaller, spherical-shaped aggregates (diameter around 2 mm) were lodged among the mesentery. Moreover, in the connective and lipid tissue in the proximity of the liver and spleen serosa, small aggregates of graphite and graphene oxide nanoparticles were observed. However, all tested nanoparticles did not affect health and growth of rats. The nanoparticles had no toxic effects on blood parameters and growth of rats, suggesting their potential applicability as remedies or in drug delivery systems.

Thrombogenicity and biocompatibility studies of reduced graphene oxide modified acellular pulmonary valve tissue.

Current strategies in tissue engineering seek to obtain a functional tissue analogue by either seeding acellular scaffolds with cells ex vivo or repopulating them with cells in vivo, after implantation in patients. To function properly, the scaffold should be non-thrombogenic and biocompatible. Especially for the case of in vivo cell repopulation, the scaffold should be prepared in a manner that protects the tissue against platelet activation and adhesion. Anti-thrombogenicity can be achieved by chemical or physical surface modification. The aim of our study was to evaluate the platelet activation and thrombogenic properties of an acellular tissue scaffold that was surface modified with reduced graphene oxide (rGO). Graphene oxide was prepared by a modified Hummers method. For the study, an acellular pulmonary valve conduit modified with rGO was used. The rGO modified tissue samples were subjected to in vitro testing through interaction with whole blood under simulated laminar flow conditions. The following cellular receptors were then analysed: CD42a, CD42b, CD41a, CD40, CD65P and PAC-1. In parallel, the adhesion of platelets (CD62P positive), leukocytes (CD45 positive) and platelet-leukocyte aggregates (CD62P/CD45 positive) on the modified surface was evaluated. As a reference, non-coated acellular tissue, Poly-l lysine and fibronectin coated tissue were also tested. The rGO surface was also analysed for biocompatibility by performing a cytotoxicity test, TUNEL assay and Cell Cycle analysis. There was no significant difference in platelet activation and adhesion between the study groups. The only significant difference was observed for the PAC-1 receptor between Poly-l lysine group and rGO and the percentage of PAC-1 positive cells was 6% and 18% respectively. The average number of activated platelets (CD62P) in the field of view was 1, while the average number of leukocytes in the field of view was 3. No adherent platelet-leukocyte aggregates were observed. There were no significant differences in the DNA fragmentation. No significant effect of rGO on the amount of cells in different phases of the cell cycle was observed. Cytotoxicity indicates that the rGO can damage cells in direct contact but have no effect on the viability of fibroblasts in indirect contact.

In vitro and in vivo effects of graphene oxide and reduced graphene oxide on glioblastoma.

Graphene and its related counterparts are considered the future of advanced nanomaterials owing to their exemplary properties. However, information about their toxicity and biocompatibility is limited. The objective of this study is to evaluate the toxicity of graphene oxide (GO) and reduced graphene oxide (rGO) platelets, using U87 and U118 glioma cell lines for an in vitro model and U87 tumors cultured on chicken embryo chorioallantoic membrane for an in vivo model. The in vitro investigation consisted of structural analysis of GO and rGO platelets using transmission electron microscopy, evaluation of cell morphology and ultrastructure, assessment of cell viability by XTT assay, and investigation of cell proliferation by BrdU assay. Toxicity in U87 glioma tumors was evaluated by calculation of weight and volume of tumors and analyses of ultrastructure, histology, and protein expression. The in vitro results indicate that GO and rGO enter glioma cells and have different cytotoxicity. Both types of platelets reduced cell viability and proliferation with increasing doses, but rGO was more toxic than GO. The mass and volume of tumors were reduced in vivo after injection of GO and rGO. Moreover, the level of apoptotic markers increased in rGO-treated tumors. We show that rGO induces cell death mostly through apoptosis, indicating the potential applicability of graphene in cancer therapy.

Interaction of graphene family materials with Listeria monocytogenes and Salmonella enterica.

Graphene family materials have unique properties, which make them valuable for a range of applications. The antibacterial properties of graphene have been reported; however, findings have been contradictory. This study reports on the antimicrobial proprieties of three different graphene materials (pristine graphene (pG), graphene oxide (GO), and reduced graphene oxide (rGO)) against the food-borne bacterial pathogens Listeria monocytogenes and Salmonella enterica. A high concentration (250 µg/mL) of all the analyzed graphenes completely inhibited the growth of both pathogens, despite their difference in bacterial cell wall structure. At a lower concentration (25 µg/mL), similar effects were only observed with GO, as growth inhibition decreased with pG and rGO at the lower concentration. Interaction of the nanoparticles with the pathogenic bacteria was found to differ depending on the form of graphene. Microscopic imaging demonstrated that bacteria were arranged at the edges of pG and rGO, while with GO, they adhered to the nanoparticle surface. GO was found to have the highest antibacterial activity.

Toxicity of pristine graphene in experiments in a chicken embryo model.

Evaluation of the potential cytotoxicity of graphene is a key factor for medical applications, where flakes or a surface of graphene may be used as bioactive molecules, drug carriers, or biosensors. In the present work, effects of pristine graphene (pG) on the development of a living organism, with an emphasis on morphological and molecular states of the brain, were investigated using a chicken embryo model. Fertilized chicken eggs were divided into the control group and groups administered with pG suspended in milli-Q water at concentrations of 50 µg/L, 100 µg/L, 500 µg/L, 1,000 µg/L, 5,000 µg/L, and 10,000 µg/L (n=30 per group). The experimental solutions were injected in ovo into the albumin and then the eggs were incubated. After 19 days of incubation, the survival, weight of the body and organs, and blood serum biochemical indices were measured. The brain samples were collected for microscopic examination of brain ultrastructure and measurements of gene and protein expression. Survival of embryos was significantly decreased after treatment with pG, but the body and organ weights as well as biochemical indices were not affected. In all treatment groups, some atypical ultrastructures of the brain were observed, but they were not enhanced by the increasing concentrations of pG. Expression of proliferating cell nuclear antigen at the messenger ribonucleic acid level was downregulated, and the number of proliferating cell nuclear antigen-positive nuclei was significantly reduced in the 500-10,000 µg/L groups compared with the control group, indicating a decreased rate of deoxyribonucleic acid synthesis in the brain. The present results demonstrate some harmful effects of the applied pG flakes on the developing organism, including brain tissue, which ought to be considered prior to any medical applications.