Porphysome Engineered With Specific Protein Binding Sites as a Multimodal Theranostic Nanocarrier for Targeted Protein Delivery.

The immobilization of proteins on the surface of carriers is challenging due to the loss of protein structure and function in this process. Here, we report the development of the protein immobilization on the surface of the metallated-porphyrin complex in the porphysome nanocarrier. The conjugated Ni-porphyrin to fatty acid (as a tail) has been synthesized and independently placed at the depth of the bilayer center of Dipalmitoylphosphatidylcholine (DPPC) in which the Ni-porphyrin was at the polar region of the membrane and is thus superficial. This porphysome (DPPC: Ni-porphyrin, 4 : 1 mole ratio) was formed by supramolecular self-assembly with a diameter of 173±7 nm and zeta potential -8.5±3.4 mv, which exhibited no significant toxicity at the experimental concentrations and acceptable cellular uptake on MCF-7 cells. The physicochemical properties and specific protein binding sites of the firefly luciferase as a model protein into the porphysome (1 : 2 mole ratio) show the conjugation efficiency about 80 % and the conformation of protein was completely maintained. Furthermore, bioluminescence assay and SDS-PAGE confirmed the preservation of protein function. The stabilized platform of porphyrin-lipid structure can potentially improve the efficacy of protein functionality for a particular display, shifting porphysomes from a simple carrier to a therapeutic agent.

Chitosan nanoparticle and its effect on Neoscytalidium novaehollandiae, the causal agent of mulberry canker in Tehran.

Fungal pathogen "Neoscytalidium novaehollandiae" is the causal agent of trunk canker in mulberry trees. Mulberry is considered as most valuable tree for landscaping in Tehran. Here in, for the first time, chitosan nanoparticles (CSNPs) were used to inhibit canker disease causal agent of mulberry. For this purpose, CSNPs were synthesized with a yield of 86%, and after characterization of the synthesized nanoparticles, the growth inhibition rate of fungus (GI%) was evaluated. The results of in vitro assays showed that the concentration of 1500 ppm significantly (P ≤ 0.05) decreased the radial growth of the fungus in comparison with control. For in vivo experiments, 2-year-old branches from healthy randomly selected mulberry trees in the landscape, were inoculated artificially in the laboratory with mycelial plugs from a 7-day-old culture of fungus. The infected branches were then treated with 500, 1000, and 1500 ppm of CSNPs. The results indicated that the disease severity (DS%) in all the treatments and the control plants increased over time. However, the slope of the changes in DS was less in CSNPs treated compared to control. This effect was concentration dependent so that no disease progress was observed at 1500 ppm of CSNPs. The findings indicate the effectiveness of CSNPs in control of canker disease of mulberry caused by N. novaehollandiae.

A review of recent developments in the application of nanostructures for sperm cryopreservation.

In the 1970s, sperm cryopreservation was presented as a unique route to fertility preservation. The ability to cryopreserve sperm from all species is challenging. The sperm cryopreservation process encompasses various cellular stresses such as increased osmotic pressure, ice crystal formation, and thermal shock, therefore decreasing the quality of sperm. The nanostructures due to their inherent features such as reactivity, high uptake, active surface area, and antioxidant activity, have contributed to modifying freezing protocols. In this review, the current state of the art with regards to emerging applications of nanotechnology in sperm cryopreservation are reviewed, some of the most promising advances are summarized, and the limitations and advantages are comprehensively discussed.

A comparative study of structural and catalytic activity alterations in firefly luciferase induced by carbon quantum dots containing amine and carboxyl functional groups.

Despite of growing interest in use of carbon-based nanomaterials as carriers of functional proteins, less attention has been paid to the effects of these nanomaterials on the structure and function of the proteins. In this study, with the aim of shedding light on the mechanisms of interaction between carbon-based nanomaterials and proteins, the interactions of carbon quantum dots (CQDs) containing amine (CQD-NH2) or carboxyl groups (CQD-COOH) with Photinus pyralis firefly luciferase enzyme were investigated by experimental and computational approaches. The structural changes and reduction in activity of the luciferase upon treatment with CQDs were experimentally proved. CQD-NH2 induced more reduction in enzyme activity (15 %) compared to CQD-COOH (7.4 %). The interactions CQD-NH2 with luciferase led to higher affinity of the enzyme for its substrate. It was found by molecular dynamic simulations that CQD-NH2 binds to multiple regions on the surface of luciferase. Secondary structure analysis showed that CQD-NH2 had more profound effects on the active site amino acids, the adjacent amino acids to the active site and the residues involved in ATP binding site. In addition, CQD-NH2 interactions with luciferase were suggested to be stronger than CQD-COOH based on the number of hydrogen bonds and the binding energies.

Aptasensor for ovarian cancer biomarker detection using nanostructured gold electrodes.

The development of an electrochemical aptasensor for the detection of CA125 as an ovarian cancer biomarker using gold nanostructures (GNs) modified electrodes is reported. The GNs were deposited on the surface of fluorine-doped tin oxide electrodes using a simple electrochemical method and the effects of pH and surfactant concentration on the topography and electrochemical properties of the resulting GNs modified electrodes were investigated. The electrodes were characterized using field-emission scanning electron microscopy and X-ray diffraction, cyclic voltammetry, and electrochemical impedance spectroscopy. The best electrode, in terms of the uniformity of the deposited GNs and the increase in electroactive surface area, was used for development of an aptasensor for CA125 tumor marker detection in human serum. Signal amplification was done by using aptamer-conjugated gold nanorods resulting in the detection limit of 2.6 U/ml and a linear range of 10 to 800 U/ml. The results showed that without the need for expensive antibodies, the developed aptasensor could specifically measure the clinically relevant concentrations of the tumor marker in human serum.

Evaluation of combined carbon dots and ciprofloxacin on the expression level of pslA, pelA, and ppyR genes and biofilm production in ciprofloxacin-resistant Pseudomonas aeruginosa isolates from burn wound infection in Iran.

OBJECTIVES: Antimicrobial resistance and biofilm formation are increasingly significant public health concerns. This study aimed to examine the antibacterial and antibiofilm properties of carbon dots (C-dots) alone and in combination with antibiotics against biofilm-forming isolates of Pseudomonas aeruginosa. METHODS: The antibacterial property of C-dots was investigated by broth microdilution method against ATCC PAO1 and P. aeruginosa clinical isolates. The antibacterial effect of the C-dots and ciprofloxacin combination was investigated using the checkerboard method. The antibiofilm effect of the C-dots alone and its combination with ciprofloxacin was evaluated using the microtiter plate method. Subsequently, the toxicity of each agent was tested on L929 fibroblast cells. In the end, the effects of C-dots on the expression levels of pslA, pelA, and ppyR genes were determined using real-time quantitative PCR. RESULTS: The combination of C-dots and ciprofloxacin exhibited a synergistic effect. Additionally, this compound substantially decreased bacterial growth (P < 0.0001) and inhibited biofilm formation at MIC (96 µg/mL) and sub-MIC (48 µg/mL) concentrations (P < 0.0053, P < 0.01). After being exposed to C-dots at a concentration of 1mg/mL for 24 hours, the survival rate of L929 cells was 87.3%. The expression of genes pslA, pelA, and ppyR, associated with biofilm formation in P. aeruginosa, was significantly reduced upon exposure to C-dots (P < 0.0023). CONCLUSIONS: The findings demonstrate a promising new treatment method for infections. Furthermore, reducing the dosage of antibiotics can lead to an improvement in the toxic effects caused by dose-dependent antibiotics and antimicrobial activity.

A turn-off fluorimetric -aptasensor for early detection of apoptosis inside the cells.

To detect cytochrome c (Cyt c) as an important biomarker of apoptosis inside the cells, a simple, label-free, fluorometric detection method has been presented. For this purpose, an aptamer/gold nanocluster probe (Aptamer@AuNCs) was produced which could specifically bind to Cyt c leading to fluorescence quenching of AuNCs. The developed aptasensor showed two linear ranges of 1-80 µM and 100-1000 µM and a detection limit of 0.77 µM and 297.5 µM, respectively. This platform was successfully used to assay Cyt c release inside the apoptotic cells and their cell lysate. Aptamer@AuNC due to its enzyme-like properties could replace antibodies in Cyt c detection by conventional blotting techniques.

DNA binding properties and cytotoxic effects of two double rollover cycloplatinated (II) complexes on cancer cell lines.

In this study, the DNA binding capacity and cytotoxic effects of two double rollovers cycloplatinated complexes, [Pt2(µ-bpy-2H)(CF3COO)2(PPh3)2] and [Pt2(µ-bpy-2H)(I)2(PPh3)2] denoted as C1 and C2, respectively, were evaluated. By using UV-Visible spectroscopy the intrinsic binding constant (Kb) of C1 and C2 to DNA were determined as 2.9 × 105 M-1, and 5.4 × 105 M-1, respectively. Both the compounds were able to quench the fluorescence of ethidium bromide as a well known DNA intercalator. The calculated Stern-Volmer quenching constants (Ksv) for C1 and C2 were 3.5 × 103 M-1, and 1.2 × 104 M-1, respectively. Upon interaction of both the compounds with DNA, increase in viscosity of DNA solution were observed, further confiming the involvement of intercalative interactions between the complexes and DNA. The cytotoxic effects of complexes in compare to cisplatin were evaluated on different cancer cell lines by MTT assay. Interestingly, C2 showed the highest cytotoxicity on A2780R, a cisplatin resistant-cell line. Induction of apoptosis by the complexes was proved by flowcytometry. In all the studied cell lines, the extent of apoptosis induced by C2 was comparable or higher than cisplatin. Cisplatin induced more necrosis in all the cancer cell lines in the tested concentration.

Spectroscopic analysis of recombinant human growth hormone in the presence of sucrose and trehalose.

Recombinant human growth hormone (rhGH) is a therapeutic protein, associated with various human diseases, such as growth hormone deficiency. One of the interesting issues in the formulation of therapeutic proteins is excipients like disaccharides. In the current study, we try to compare the effect of sucrose and trehalose on the structure of rhGH in the liquid state at 25°C and 55°C. We use spectroscopic techniques including intrinsic and extrinsic fluorescence, Fourier-transform infrared (FTIR), circular dichroism (CD), dynamic light scattering (DLS), and time-resolved fluorescence. FTIR shows a slight change in the secondary structure of rhGH in presence of the sugars as sucrose is more effective than trehalose. Fluorescence investigations also confirm the enhancements of folding of rhGH and fluorescein isothiocyanate (FITC)-rhGH in presence of sucrose (1.5-fold more than trehalose). Also, we studied sucrose's effect on the rete of aggregation of rhGH using spectroscopy of Congo red, and fluorescence imaging of thioflavin T (ThT)-treated samples. It can be suggested that sucrose facilitates the amyloid formation of rhGH during 20 days of incubation at 37°C. This study will help to understand the growth hormone structural behavior in the liquid state in the presence of sucrose and trehalose in vitro.

Review of electrochemical and optical biosensors for testosterone measurement.

Testosterone is an anabolic steroid and a major sex hormone in males. It plays vital roles, including developing the testis, penis, and prostate, increasing muscle and bone, and sperm production. In both men and women, testosterone levels should be in normal ranges. Besides, testosterone and its analogs are major global contributors to doping in sport. Due to the importance of testosterone testing, novel, accurate biosensors have been developed. This review summarizes the various methods for testosterone measurement. Also, recent optical and electrochemical approaches for the detection of testosterone and its analogs have been discussed.

Label-Free and Bioluminescence-Based Nano-Biosensor for ATP Detection.

A bioluminescence-based assay for ATP can measure cell viability. Higher ATP concentration indicates a higher number of living cells. Thus, it is necessary to design an ATP sensor that is low-cost and easy to use. Gold nanoparticles provide excellent biocompatibility for enzyme immobilization. We investigated the effect of luciferase proximity with citrate-coated gold, silver, and gold-silver core-shell nanoparticles, gold nanorods, and BSA-Au nanoclusters. The effect of metal nanoparticles on the activity of luciferases was recorded by the luminescence assay, which was 3-5 times higher than free enzyme. The results showed that the signal stability in presence of nanoparticles improved and was reliable up to 6 h for analytes measurements. It has been suggested that energy is mutually transferred from luciferase bioluminescence spectra to metal nanoparticle surface plasmons. In addition, we herein report the 27-base DNA aptamer for adenosine-5'-triphosphate (ATP) as a suitable probe for the ATP biosensor based on firefly luciferase activity and AuNPs. Due to ATP application in the firefly luciferase reaction, the increase in luciferase activity and improved detection limits may indicate more stability or accessibility of ATP in the presence of nanoparticles. The bioluminescence intensity increased with the ATP concentration up to 600 µM with a detection limit of 5 µM for ATP.

Curcumin carbon dots inhibit biofilm formation and expression of esp and gelE genes of Enterococcusfaecium.

The increasing prevalence of vancomycin-resistant Enterococcus faecium, along with the ability of this bacterium to form biofilm on biotic surfaces and medical devices, has created a serious challenge. Therefore, the development of new antibacterial agents is an urgent need. In this study, curcumin carbon dots (Cur-CDs) were synthesized by a one-step hydrothermal method, and its antibacterial and antibiofilm effects were investigated. By broth microdilution method, the minimal inhibitory concentration (MIC) against vancomycin-resistant and sensitive clinical isolates of Enterococcus faecium (two clinical isolates in total) and standard strain of Enterococcus faecalis ATCC 29212 was determined, which were 1000, 1000, and 125 µg/ml, respectively. The inhibitory effect of Cur-CDs on biofilm formation of vancomycin-resistant E. faecium clinical isolates were evaluated by microtiter plate assay. Cur-CDs (1000 µg/ml) significantly prevented (p = 0.009) the biofilm formation of E. faecium isolates. Real-time PCR results showed that Cur-CDs (1000 µg/ml) significantly downregulated the expression of esp and gelE genes (p = 0.001 and p = 000000002, respectively) in clinical isolates of E. faecium, while Cur-CDs did not affect acm gene expression (p = 0.086). This study revealed that Cur-CDs can be effective antibacterial and antibiofilm agents against vancomycin-resistant and biofilm producer E. faecium, which makes them interesting candidates for treating or preventing bacterial infections.

Assessment of Helicobacter pylori positive infected patients according to Clarithromycin resistant 23S rRNA, rpl22 associated mutations and cyp2c19*1, *2, *3 genes pattern in the Early stage of Gastritis.

OBJECTIVE: Clarithromycin resistant Helicobacter pylori (CAM-R) is the main cause of standard triple therapy eradicating failure. Proton pump inhibitors (PPIs) directly pose bacteriocidic activity and prepare the optimum condition for Clarithromycin's best function. In counter with Poor metabolizer subjects, Homozygote Extensive Metabolizers have well characterized by treatment failure. Eventually, determination of CAM-R profile and estimation of PPIs metabolization rate support clinicians in better prescription. So, we explored Helicobacter pylori'mutations in 23S rRNA and rpl22 resistant genes, and cyp2c19 *1, *2, *3 allele variations, and PPIs metabolization patterns in patients, consequently the results reported to the physician. RESULTS: Sixteen out of 96 patients considered to be CAM-R Helicobacter pylori. A2143C (1/16), rpl22 insertion (16/16), and GTG deletion (2/16) recorded in CAM-R strains. P450 2C19 human genotyping demonstrated that the highest proportion of the H. pylori- positive strains infected patients 43/61(70.49%) categorized in Homozygote extensive metabolizer class. The rest (12/61)19.67% classified as Poor metabolizers, and 6/61(9.83%) distinct from Heterozygote extensive metabolizer group. Proportion of poor metabolizers and Heterozygote extensive metabolizer phenotypes between CAM-R strains mentioned to be 10/16(62.5%), and 6/16(37.5%). Cross points between the most frequently distributed allele in CAM-R strains indicated 81.25% for *2, and w2 for 18.75%.

Betanin alleviates oxidative stress through the Nrf2 signaling pathway in the liver of STZ-induced diabetic rats.

BACKGROUND: Continuing hyperglycemia causes and exacerbate oxidative stress. Betanin as the principal pigment of red beet root has antioxidant, anti-inflammatory, and anti-diabetic properties. The purpose of this study was to investigate the potency of betanin on antioxidant defense in STZ-induced diabetic rats' livers. METHODS: STZ at a single dose of 60 mg/kg body weight was intraperitoneally injected and betanin (10, 20, and 40 mg/kg/day) was administered orally for 28 days. Malondialdehyde (MDA), total antioxidant capacity (TAC), protein carbonyl (PC) levels, and the enzyme activity of superoxide dismutase (SOD), catalases and glutathione peroxidases (GPx) were evaluated in the liver. Furthermore, gene expression of Nrf2 and mentioned antioxidant enzymes were measured by Real-time PCR. RESULTS: Betanin (10 and 20 mg/kg) significantly reduced PC levels and increased antioxidant enzyme activity in diabetic rats compared to the control diabetic group (P < 0.01). In comparison to the diabetic control group, all studied genes expression in diabetic rats were increased significantly with betanin at doses of 10 and 20 mg/kg (P < 0.02). The increase in gene expression at 20 mg/kg of betanin was significantly stronger than others (P < 0.015) except for the catalase (P = 0.201), that was almost the same. Moreover, treatment of diabetic rats with 20 mg/kg of betanin could significantly increase TAC levels (P < 0.05) and decrease MDA levels (P < 0.001) compared to diabetic control group. CONCLUSIONS: Betanin could increase the antioxidant capacity of liver tissue associated with the Nrf2-mediated pathway in a dose-dependent manner.

A FRET-based aptasensor for the detection of α-synuclein oligomers as biomarkers of Parkinson's disease.

Soluble oligomers of α-synuclein (α-syn) are known to be responsible for neuronal death in the early stages of Parkinson's disease (PD). Thus, the development of a simple, rapid, and inexpensive method for the detection of α-syn oligomers can help PD diagnosis before irreversible damage to the brain tissue occurs. The present study is aimed at developing a FRET-based aptasensor for the selective and sensitive detection of α-syn oligomers. Herein, FAM-labeled aptamer adsorption on graphene oxide (GO) resulted in fluorescence quenching of FAM. The binding of α-syn oligomers to the aptamer led to the recovery of the fluorescence intensity. Under optimized conditions, the developed biosensor showed two linear response ranges from 10-100 nM and 250 nM to 2 µM in α-syn oligomer detection. The limit of detection (LOD) and limit of quantification (LOQ) were calculated to be 6.3 nM and 19.3 nM, respectively. Furthermore, the performance of the sensing platform in the detection of the target analyte in biological matrices was demonstrated by the assay of α-syn oligomers in spiked human saliva samples. According to the obtained results, this sensing platform has a good performance for α-syn oligomer detection and it can be considered as a promising candidate for the early diagnosis of PD.

TRAIL/S-layer/graphene quantum dot nanohybrid enhanced stability and anticancer activity of TRAIL on colon cancer cells.

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), known as a cytokine of the TNF superfamily, is considered a promising antitumor agent due to its ability to selectively induce apoptosis in a wide variety of cancer cells. However, failure of its successful translation into clinic has led to development of nano-based platforms aiming to improve TRAIL therapeutic efficacy. In this regard, we fabricated a novel TRAIL-S-layer fusion protein (S-TRAIL) conjugated with graphene quantum dots (GQDs) to benefit both the self-assembly of S-layer proteins, which leads to elevated TRAIL functional stability, and unique optical properties of GQDs. Noncovalent conjugation of biocompatible GQDs and soluble fusion protein was verified via UV-visible and fluorescence spectroscopy, size and ζ-potential measurements and transmission electron microscopy. The potential anticancer efficacy of the nanohybrid system on intrinsically resistant cells to TRAIL (HT-29 human colon carcinoma cells) was investigated by MTT assay and flow cytometry, which indicated about 80% apoptosis in cancer cells. These results highlight the potential of TRAIL as a therapeutic protein that can be extensively improved by taking advantage of nanotechnology and introduce S-TRAIL/GQD complex as a promising nanohybrid system in cancer treatment.

Design and preparation of a theranostic peptideticle for targeted cancer therapy: Peptide-based codelivery of doxorubicin/curcumin and graphene quantum dots.

Although chemotherapy has been known as a powerful medication for cancer treatment over the years, there is an important necessity for designing a novel targeted drug delivery system to overcome the drawbacks of this conventional method including undesired side effects on normal cells and drug resistance. The structural differences between the surface of cancerous and normal cells allow to design and engineer targeted drug delivery systems for cancer treatment. Integrins as one of the cell surface receptors over-expressed in cancer cells could potentially be suitable candidates for targeting cancer cells. In the present study, the novel nano-carriers based on designed MiRGD peptides and graphene quantum dots (GQDs) have been used for targeted delivery of doxorubicin (Dox) and curcumin (Cur) as hydrophilic and hydrophobic drug models, respectively. The prepared nano-composites were characterized by UV-vis and photoluminescence (PL) spectroscopies, Zeta-Sizer and transmission electron microscopy (TEM). Altogether, the results of cellular uptake and fluorimetric assays performed in HUVEC and HFF cells as models of αv integrin-over-expressed cancer and normal cells, respectively, besides in-vivo study on breast cancer bearing BALB/c mice, demonstrated that the prepared nano-composites can be considered as suitable multifunctional theranostic peptideticles for targeted drug delivery and tracking.

Identification of NLRP3PYD Homo-Oligomerization Inhibitors with Anti-Inflammatory Activity.

Inflammasomes are multiprotein complexes that represent critical elements of the inflammatory response. The dysregulation of the best-characterized complex, the NLRP3 inflammasome, has been linked to the pathogenesis of diseases such as multiple sclerosis, type 2 diabetes mellitus, Alzheimer's disease, and cancer. While there exist molecular inhibitors specific for the various components of inflammasome complexes, no currently reported inhibitors specifically target NLRP3PYD homo-oligomerization. In the present study, we describe the identification of QM380 and QM381 as NLRP3PYD homo-oligomerization inhibitors after screening small molecules from the MyriaScreen library using a split-luciferase complementation assay. Our results demonstrate that these NLRP3PYD inhibitors interfere with ASC speck formation, inhibit pro-inflammatory cytokine IL1-ß release, and decrease pyroptotic cell death. We employed spectroscopic techniques and computational docking analyses with QM380 and QM381 and the PYD domain to confirm the experimental results and predict possible mechanisms underlying the inhibition of NLRP3PYD homo-interactions.

A dual responsive robust human serum albumin-based nanocarrier for doxorubicin.

Targeted drug therapy against cancer has been introduced as a smart strategy to combat the unwanted side effects due to systemic administration of chemotherapeutics. A human serum albumin (HSA)-based nanocarrier was fabricated with the aim to target reductive media and acidic pH of the tumor tissues. α-Lipoic acid (LA) was applied to increase the number of disulfide bonds in the nanocarrier to target higher glutathione concentrations present in tumor tissues and polyethylene glycol was used to target the acidic pH of tumors. UV illumination, ethanol desolvation, oxygen bubbling, and a mixture of redox buffers were employed to prepare doxorubicin-loaded HSA-LA nanoparticles. The nanocarrier was supposed to release the loaded doxorubicin in reductive and acidic pH media. Fourier-transform infrared spectroscopy and energy dispersive X-ray analysis indicated successful attachment of LA to HSA. The prepared nanoplatform presented improved doxorubicin loading efficiency and content and successfully released the loaded doxorubicin in the expected conditions. Protein corona study indicated that positively charged plasma proteins with molecular weights of nearly 80 kDa are absorbed to the surface of the nanoparticles. Furthermore, it showed desirable UV and storage stability, which implied its robustness and improved shelf life if applied in nanomedicine.

Design of an optical nanobiosensor for detection of Legionella pneumophila in water samples.

Background and Objectives: Legionella spp. is a causative agent of Legionnaires' disease that creates public health problems. Isolation of these bacteria from water sources is essential to identify outbreak origins and prevent disease. Diagnostic biosensors for water quality control to protect consumers from water-borne infections can predict many outbreaks. Gold nanoparticles conjugated probes are a new generation of diagnostic tools. In this study, an optical nano biosensor was designed and characterized to detect Legionella pneumophila in water samples rapidly. Materials and Methods: Thiolated probes designed for the mip gene were attached to gold nanoparticles and then water samples containing Legionella pneumophila were examined. Results: The limit of detection for PCR and biosensor was 104 and 103 copy numbers/µl, respectively. Biosensor sensitivity and PCR were reported to be 90% (18 out of 20) and 85% (17 out of 20), respectively. Specificity 100% has been reported for both methods. Conclusion: According to the obtained results, this method has the potential to diagnose L. pneumophila with high sensitivity and specificity. This system can be employed as a practical tool for rapid, accurate, high-sensitivity, and acceptable detection of Legionella pneumophila in contaminated water, which is cost-effective in terms of cost and time.