Accumulation and toxicity of biologically produced gold nanoparticles in different types of specialized mammalian cells.

The biologically produced gold nanoparticles (AuNPs) are novel carriers with promising use in targeted tumor therapy. Still, there are no studies regarding the efficacy of nanoparticle internalization by cancer and noncancer cells. In this study, AuNPs were produced by Fusarium oxysporum and analyzed by spectrophotometry, transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), and Zetasizer. Obtained AuNPs were about 15 nm in size with a zeta potential of -35.8 mV. The AuNPs were added to cancer cells (4T1), noncancer cells (NIH/3T3), and macrophages (RAW264.7). The viability decreased in 4T1 (77 ± 3.74%) in contrast to NIH/3T3 and RAW264.7 cells (89 ± 4.9% and 90 ± 3.5%, respectively). The 4T1 cancer cells also showed the highest uptake and accumulation of Au (∼80% of AuNPs was internalized) as determined by graphite furnace atomic absorption spectroscopy. The lowest amount of AuNPs was internalized by the NIH/3T3 cells (∼30%). The NIH/3T3 cells exhibited prominent reorganization of F-actin filaments as examined by confocal microscopy. In RAW264.7, we analyzed the release of proinflammatory cytokines by flow cytometry and we found the AuNP interaction triggered transient secretion of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In summary, we proved the biologically produced AuNPs entered all the tested cell types and triggered cell-specific responses. High AuNP uptake by tumor cells was related to decreased cell viability, while low nanoparticle uptake by fibroblasts triggered F-actin reorganization without remarkable toxicity. Thus, the biologically produced AuNPs hold promising potential as cancer drug carriers and likely require proper surface functionalization to shield phagocytizing cells.

Fate of the capping agent of biologically produced gold nanoparticles and adsorption of enzymes onto their surface.

Enzymotherapy based on DNase I or RNase A has often been suggested as an optional strategy for cancer treatment. The efficacy of such procedures is limited e.g. by a short half-time of the enzymes or a low rate of their internalization. The use of nanoparticles, such as gold nanoparticles (AuNPs), helps to overcome these limits. Specifically, biologically produced AuNPs represent an interesting variant here due to naturally occurring capping agents (CA) on their surface. The composition of the CA depends on the producing microorganism. CAs are responsible for the stabilization of the nanoparticles, and promote the direct linking of targeting and therapeutic molecules. This study provided proof of enzyme adsorption onto gold nanoparticles and digestion efficacy of AuNPs-adsorbed enzymes. We employed Fusarium oxysporum extract to produce AuNPs. These nanoparticles were round or polygonal with a size of about 5 nm, negative surface charge of about - 33 mV, and maximum absorption peak at 530 nm. After the adsorption of DNAse I, RNase A, or Proteinase K onto the AuNPs surface, the nanoparticles exhibited shifts in surface charge (values between - 22 and - 13 mV) and maximum absorption peak (values between 513 and 534 nm). The ability of AuNP-enzyme complexes to digest different targets was compared to enzymes alone. We found a remarkable degradation of ssDNA, and dsDNA by AuNP-DNAse I, and a modest degradation of ssRNA by AuNP-RNase A. The presence of particular enzymes on the AuNP surface was proved by liquid chromatography-mass spectrometry (LC-MS). Using SDS-PAGE electrophoresis, we detected a remarkable digestion of collagen type I and fibrinogen by AuNP-proteinase K complexes. We concluded that the biologically produced AuNPs directly bound DNase I, RNase A, and proteinase K while preserving their ability to digest specific targets. Therefore, according to our results, AuNPs can be used as effective enzyme carriers and the AuNP-enzyme conjugates can be effective tools for enzymotherapy.

Investigation of Protein Corona Formed around Biologically Produced Gold Nanoparticles.

Although there are several research articles on the detection and characterization of protein corona on the surface of various nanoparticles, there are no detailed studies on the formation, detection, and characterization of protein corona on the surface of biologically produced gold nanoparticles (AuNPs). AuNPs were prepared from Fusarium oxysporum at two different temperatures and characterized by spectrophotometry, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDS). The zeta potential of AuNPs was determined using a Zetasizer. AuNPs were incubated with 3 different concentrations of mouse plasma, and the hard protein corona was detected first by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then by electrospray liquid chromatography-mass spectrometry (LC-MS). The profiles were compared to AuNPs alone that served as control. The results showed that round and oval AuNPs with sizes below 50 nm were produced at both temperatures. The AuNPs were stable after the formation of the protein corona and had sizes larger than 86 nm, and their zeta potential remained negative. We found that capping agents in the control samples contained small peptides/amino acids but almost no protein(s). After hard protein corona formation, we identified plasma proteins present on the surface of AuNPs. The identified plasma proteins may contribute to the AuNPs being shielded from phagocytizing immune cells, which makes the AuNPs a promising candidate for in vivo drug delivery. The protein corona on the surface of biologically produced AuNPs differed depending on the capping agents of the individual AuNP samples and the plasma concentration.

Histopathological study of the maternal exposure to the biologically produced silver nanoparticles on different organs of the offspring.

This research for the first time presents the possibility of crossing the biologically produced SNPs through the placenta to different organs of rat offspring. SNPs were produced using Fusarium oxysporum. After adding 1 mmol final concentration of silver nitrate solution to the culture supernatant and 5 min heating, SNPs were produced, and their production was proved using visible spectrum, transmission electron microscope (TEM), and X-ray diffraction (XRD) analyses. SNPs were washed, and their concentration determined using inductively coupled plasma (ICP) instrument. SNPs were used for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and after determination of their half maximal inhibitory concentration (IC50) dose, their toxic and nontoxic doses were determined and used for in vivo studies. A total of 24 female rats, after detection of their vaginal plugs, were divided into 3 groups each having 8 members. A control group was treated with normal saline. The other two groups were treated by toxic and nontoxic doses of SNPs, respectively. After delivery and breastfeeding, the pups were scarified, and their organs were collected and analyzed using histological examinations. Results showed that SNPs had a maximum absorbance peak around 450 nm, with polygonal and round shapes. XRD results confirmed the presence of SNPs. The concentration of the SNPs after washing was 19 ppm/mL based on the ICP results. MTT assay results showed that SNPs had a dose-dependent toxic effect. Histopathological examination results showed that SNPs could pass through the placenta; both their nontoxic and toxic doses induced somehow mild alternations in the liver, kidney, testis, and ovary and had no effects on the brains of the rat offspring. In conclusions, the use of the biologically produced SNPs should be limited during pregnancy and breastfeeding.

One step conjugation of some chemotherapeutic drugs to the biologically produced gold nanoparticles and assessment of their anticancer effects.

Recent research tried to analyze the conjugation of some chemotherapeutic drugs to the biologically produced gold nanoparticles (GNPs) in one step, without the use of any additional linkers. GNPs was produced using Fusarium oxysporum and their presence was confirmed using spectrophotometer, transmission electron microscope (TEM), X-ray diffraction (XRD) and fourier transform infrared (FTIR) analyses. In order to carry out the conjugation study, capecitabine, tamoxifen, and paclitaxel were added dropwise to the GNPs solution under stirring condition and spectrophotometer, dynamic light scattering (DLS) and FTIR analyses were performed to prove the successful conjugation. Finally, AGS and MCF7 cell lines were used for methyl thiazol tetrazolium (MTT) assay to determine the toxicity of each drug and its conjugated form. Results showed that the spherical and hexagonal GNPs with maximum absorbance peak around 524 nm and average sizes less than 20 nm were produced. FTIR analysis clarified the presence of proteins on the surfaces of the GNPs. After the conjugation process although the FTIR analysis demonstrated that all the drugs were successfully conjugated to GNPs, MTT assay revealed that unlike the paclitaxel conjugated GNPs, capecitabine and tamoxifen conjugates displayed no toxic effects due to their deactivation and low half-lives. Moreover the average size and polydispersity index (PDI) of the GNPs after conjugation with all the three tested drugs increased. In conclusion different types of drugs could conjugate to the GNPs but it is important to employ high stable forms of the drugs in the conjugation procedure.

Effects of biologically produced gold nanoparticles: toxicity assessment in different rat organs after intraperitoneal injection.

Gold nanoparticles (GNPs) have different usage in the medical field. The plan of the present research was to evaluate the influence of the biologically produced GNPs on some rat organs. GNPs were produced using Fusarium oxysporum and their presence was confirmed using spectrophotometer, transmission electron microscope (TEM) and X-ray diffraction (XRD) analyses. The non-toxic and toxic doses of GNPs were determined using MTT assay and were injected intraperitoneally into rats in 3 continuous days and their effects on the kidney, liver and testis were analyzed using microscopic technique. Results revealed that GNPs that were produced had 525 nm absorbance peak and average sizes of about 50 nm, with round and hexagonal shapes. Results from the XRD analysis showed the presence of GNPs in the reaction mixture. MTT assay results revealed that GNPs had somehow toxic effects which depend on their doses. Histological examinations indicated that based on the tested organ, the distribution and effects of GNPs were different which in the testis, the non-toxic dose had no effects and in some parts of the liver and kidney, it induced mild changes. The toxic dose of the GNPs in all the three tested organs induced mild changes. In conclusion, the in vitro and in vivo behaviors of the produced GNPs were different and GNPs even in high concentration induced low changes in the rat organs. This may be due to the short exposure and the use of the biologically produced GNPs.

The healing property of a bioactive wound dressing prepared by the combination of bacterial cellulose (BC) and Zingiber officinale root aqueous extract in rats.

Gluconacetobacter xylinus was used for production of bacterial cellulose (BC). The obtained BC was washed and floated in the nontoxic dose of the herb aqueous extract that was obtained from the methyl thiazol tetrazolium (MTT) assay. Twenty-four Wistar rats were divided into four separated groups and after inducing the wounds (15 mm in diameter), each group was treated with honey, BC, herb aqueous extract, and the combination of BC-herb aqueous extract. Each day the contraction percentages of the wound sites were measured. On days 3, 7, and 14, two rats from each group were euthanized, the skin samples from the wound regions were achieved and their paraffin blocks were prepared. Finally after trichrome staining, the microscopic examinations were done. MTT assay results indicated that the herb aqueous extract had dose-dependent toxic effects and the nontoxic dose of the extract was prepared and utilized for in vivo assay. Although the macroscopic analysis revealed that the BC and the herb aqueous extract had better activity in the wound contraction percentages than their combination, microscopic analysis indicated that the combination of BC-herb aqueous extract revealed all the characteristics that each material induced in the wound site alone. In conclusion, the speed of the wound healing should not be solely considered and its quality should be considered as well.

Wound healing property of a gel prepared by the combination of Pseudomonas aeruginosa alginate and Alhagi maurorum aqueous extract in rats.

Although alginate has been known to be a good wound dressing, it does not have antimicrobial properties, has low availability, and is expensive. To overcome these problems, the present study was conducted, where the extraction of this material from an available small factory Pseudomonas aeruginosa and the improvement of its wound healing property by its combination with herb extract, Alhagi maurorum, done. Nineteen P. aeruginosa strains were isolated and identified from burned skin, and the one isolated strain with the highest ability of alginate production was selected. A. maurorum aqueous extract was prepared, and the toxicity of each material was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay. A mixture of nontoxic doses of each substance was then prepared. Thirty-two Wistar rats were divided into four groups (n = 8). The control group and the rest three groups, which were treated by alginate, A. maurorum extract, and alginate- A. maurorum extract. Throughout the 21 days of treatment, the open wound sites were checked. Finally, the rats were sacrificed and the effect of each substance on their skin tissue was evaluated. The results showed that the high alginate production without any toxic effect was obtained from the P. aeruginosa strain K1. A. maurorum aqueous extract had dose-dependent toxicity. The aqueous solution of alginate- A. maurorum extract complex group showed the best wound healing activity in both macroscopic and microscopic examinations. Recent research has introduced a new type of wound dressing with high wound healing properties. This could decrease the time for re-epithelialization and increase wound contraction percentage.

Assessment of the cutaneous wound healing efficiency of acidic, neutral and alkaline bacterial cellulose membrane in rat.

Recent research was conducted to evaluate the healing efficiency of bacterial cellulose (BC) as a wound dressing in different pHs and its possibility of being a smart wound dressing that can indicate pHs. BC was produced by environmentally isolated bacterial strains. After washing the best achieved BC, it was floated in normal saline with different pHs with phenol red used as a pH indicator. Finally the wound healing effects of the acidic, neutral and alkaline BC membranes were evaluated in rat cutaneous wounds. Results showed that one of the isolates which its partial 16srRNA genome had 95% similarity with Gluconacetobacter intermedius, had the thickest layer. The microscopic and macroscopic evaluations showed that the acidic BC had the best healing activity. Although the color of the films remained unchanged during the experiments because they were transparent and thin, these changes could not be easily seen. This suggests the use of thicker films such as the ones which are cross linked with some materials (e.g., sterile gauze). In conclusion the pH can affect the healing ability of natural BC and acidic pH had the best wound healing efficiency. In future it is better to use the acidic BC instead of natural one for different wound healing purposes.

Biosynthesis of gold nanoparticles by two bacterial and fungal strains, Bacillus cereus and Fusarium oxysporum, and assessment and comparison of their nanotoxicity in vitro by direct and indirect assays

Background: Although nanoparticles (NPs) have many advantages, it has been proved that they may be absorbed by and have toxic effects on the human body. Recent research has tried to evaluate and compare the nanotoxicity of gold nanoparticles (AuNPs) produced by two types of microorganisms in vitro by two different methods. AuNPs were produced by Bacillus cereus and Fusarium oxysporum, and their production was confirmed by visible spectral, transmission electron microscope, and X-ray diffraction (XRD) analyses. The human fibroblast cell line CIRC-HLF was treated with AuNPs, and the induced nanotoxicity was measured using direct microscopic and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Results: The results showed that the produced AuNPs had a maximum absorbance peak around 510­530 nanometer (nm), with spherical, hexagonal, and octagonal shapes and average sizes around 20­50 nm. The XRD results confirmed the presence of GNPs in the microbial culture supernatants. An MTT assay showed that GNPs had dose-dependent toxic effects, and microscopic analysis showed that GNPs induced cell abnormalities in doses lower than the determined half-maximal inhibitory concentrations (IC50s). Conclusions: In conclusion, the biologically produced AuNPs had toxic effects in the cell culture, and direct techniques such as microscopic evaluation instead of indirect methods such as MTT assay were more useful for assessing the nanotoxicity of the biologically produced AuNPs. Thus, the use of only MTT assay for nanotoxicity evaluation of AuNPs is not desirable.

Silver nanoparticles production by two soil isolated bacteria, Bacillus thuringiensis and Enterobacter cloacae, and assessment of their cytotoxicity and wound healing effect in rats.

Production of silver nanoparticles by Bacillus thuringiensis and Enterobacter cloacae was performed and confirmed through UV-visible spectrophotometry, transmission electron microscopy (TEM), and x-ray diffraction (XRD) analyses. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using mouse fibroblast cell line NIH-3T3 D4 was carried out and IC50s of AgNPs were obtained. The nontoxic dose of each AgNPs solution was selected for wound healing assay. Thirty-two rats were divided into four groups; two were used as the controls and two were treated by AgNPs that were produced by two bacterial strains. Results revealed that the produced AgNPs were amorphous, spherical in shapes, and had sizes under 100 nm. Histological analysis showed that AgNPs had better wound healing efficacy than the control groups. In conclusion, when the biologically produced AgNPs were used in vivo, they induced the epithelization, formation of the collagen bundles and fibroplasia and reduced the duration of completion of the epithelization and the angiogenesis.

Biological production of silver nanoparticles by soil isolated bacteria and preliminary study of their cytotoxicity and cutaneous wound healing efficiency in rat.

Biosynthesis of AgNPs by 37 different bacterial soil isolates was done and confirmed through visible spectrophotometry. Fifteen isolates were identified and two of them with the highest ability of AgNPs production were used for Transmission Electron Microscopy (TEM) and X-ray diffraction (XRD) tests. MTT assay for both of the obtained AgNPs was run and after determination their IC50s, two different toxic and nontoxic doses of each AgNPs solution were chosen for wound healing assay. Forty eight rats were divided into 6 groups; two were the controls, two were administrated by the toxic and two were administrated by the nontoxic doses of AgNPs produced by Bacillus cereus and Escherichia fergusonii. Administration of the nontoxic doses of AgNPs had better wound healing effect than both of the toxic ones. The control groups had less wound healing properties. In conclusion, biologically produced AgNPs in their nontoxic doses accelerated the collagen formation and the epithelization and decelerated the angiogenesis and duration of completion the epithelization.

Impregnation of the bacterial cellulose membrane with biologically produced silver nanoparticles.

Different wound dressings with antibacterial property have been surveyed and one among them is bacterial cellulose (BC). Since the BC does not have antibacterial property, the biologically produced silver nanoparticles (SNPs) were impregnated into the BC. For the BC production, Hestrin-Schramm broth was used. Formation of the BC was proven by enzymatic hydrolysis. For SNPs production, the bacterial supernatant was treated with AgNO3 and formation of SNPs was monitored through spectrophotometer, TEM and XRD. For impregnation of SNPs into the BC, the cleaned membrane was placed in the bacterial supernatant that contained 1 mmol of AgNO3. The antibacterial assay was done for the BC/SNPs. Enzymatic hydrolysis proved the presence of the BC. Spectrophotometer and XRD results showed the formation of SNPs. TEM analysis revealed the presence of SNPs with sizes around 5-100 nm. SEM micrographs showed the impregnation of SNPs into the BC. Antibacterial test exhibited the antibacterial activity of the BC/SNPs.

The effect of temperature on antibacterial activity of biosynthesized silver nanoparticles.

The purpose of this study was the evaluation of two different temperatures on antibacterial activity of the biosynthesized silver nanoparticles. 38 silver nanoparticles-producing bacteria were isolated from soil and identified. Biosynthesis of silver nanoparticles by these bacteria was verified through visible light spectrophotometry. Two strains were relatively active for production of silver nanoparticles. These strains were subjected for molecular identification and recognized as Bacillus sp. and Acinetobacter schindleri. In the present study, the effect of temperatures was evaluated on structure and antimicrobial properties of the silver nanoparrticles by transmission electron microscopy (TEM), X-ray diffraction (XRD) analysis and antimicrobial Agar well diffusion methods. The silver nanoparticles showed antibacterial activity against all the pathogenic bacteria; however, this property was lost after treatment of the silver nanoparticles by high temperatures (100 and 300 °C). TEM images showed that the average sizes of heated silver nanoparticles were >100 nm. However, these were <100 nm for non-heated silver nanoparticles. Although, XRD patterns showed the crystalline structure of heated silver nanoparticles, their antibacterial activities were less. This was possible because of the sizes and accordingly less penetration of the particles into the bacterial cells. In addition, elimination of the capping agents by heat might be considered another reason.