Microfluidic Synthesis of Ultrasmall Chitosan/Graphene Quantum Dots Particles for Intranasal Delivery in Alzheimer's Disease Treatment.

Nanoparticles (NPs) based therapies for Alzheimer's disease (AD) attract interest due to their ability to pass across or bypass the blood-brain barrier. Chitosan (CS) NPs or graphene quantum dots (GQDs) are promising drug carriers with excellent physicochemical and electrical properties. The current study proposes the combination of CS and GQDs in ultrasmall NP form not as drug carriers but as theranostic agents for AD. The microfluidic-based synthesis of the CS/GQD NPs with optimized characteristics makes them ideal for transcellular transfer and brain targeting after intranasal (IN) delivery. The NPs have the ability to enter the cytoplasm of C6 glioma cells in vitro and show dose and time-dependent effects on the viability of the cells. IN administration of the NPs to streptozotocin (STZ) induced AD-like models lead to a significant number of entrances of the treated rats to the target arm in the radial arm water maze (RAWM) test. It shows the positive effect of the NPs on the memory recovery of the treated rats. The NPs are detectable in the brain via in vivo bioimaging due to GQDs as diagnostic markers. The noncytotoxic NPs localize in the myelinated axons of hippocampal neurons. They do not affect the clearance of amyloid ß (Aß) plaques at intercellular space. Moreover, they showed no positive impact on the enhancement of MAP2 and NeuN expression as markers of neural regeneration. The memory improvement in treated AD rats may be due to neuroprotection via the anti-inflammation effect and regulation of the brain tissue microenvironment that needs to be studied.

Study on the antibacterial effect of CuO nanoparticles on Klebsiella pneumonia bacteria: Efficient treatment for colorectal cancer.

Colorectal cancer (CRC) is a widespread type of cancer across the world. One efficient therapy approach is the use of antibiotic agents, but one of the main issues related to treating CRC is microbial resistance to antibiotics. As microbes are becoming more resistant to antibiotics and other traditional antimicrobial agents, nanobiotechnology has made it possible to employ nanomaterials with the aim of creating a new generation of antimicrobial agents. In the present study, we have assessed the antimicrobial potential of CuO nanoparticles (NPs) against gram-negative bacteria like Klebsiella pneumoniae carrying PKS genes responsible for encoding colibactin as the key factor for CRC development. For this purpose, the antibacterial effects of conventional antibacterial agents, including erythromycin, piperacillin, and ampicillin, as well as CuONPs, were compared on isolated strains from cancerous candidates. The obtained results revealed that isolates (K. pneumoniae) showed resistance toward the mentioned conventional antibiotics, but CuONPs showed efficient antibacterial properties against K. pneumonia with a MIC = 62 µg/mL. On the other hand, a synergistic antibacterial effect was obtained when CuONPs were used in combination with conventional antibiotics, which are ineffective when used alone. Therefore, CuONPs can be introduced as an excellent antimicrobial agent against K. pneumoniae bacteria in CRC, especially when they are combined with other antibiotics since they can activate the antimicrobial activity of the conventional antibiotics.

Graphene family in cancer therapy: recent progress in cancer gene/drug delivery applications.

In the past few years, the development in the construction and architecture of graphene based nanocomplexes has dramatically accelerated the use of nano-graphene for therapeutic and diagnostic purposes, fostering a new area of nano-cancer therapy. To be specific, nano-graphene is increasingly used in cancer therapy, where diagnosis and treatment are coupled to deal with the clinical difficulties and challenges of this lethal disease. As a distinct family of nanomaterials, graphene derivatives exhibit outstanding structural, mechanical, electrical, optical, and thermal capabilities. Concurrently, they can transport a wide variety of synthetic agents, including medicines and biomolecules, such as nucleic acid sequences (DNA and RNA). Herewith, we first provide an overview of the most effective functionalizing agents for graphene derivatives and afterward discuss the significant improvements in the gene and drug delivery composites based on graphene.

The detection of the captured circulating tumor cells on the core-shell nanofibrous membrane using hyaluronic acid-functionalized graphene quantum dots.

In recent years, cancerous cases have increased remarkably worldwide, and metastasis is the leading cause of death. Therefore, research on the early detection of cancer and metastasis has expanded to aid successful cancer treatment. Here in this paper, at the first step, an electrospun nanofibrous membrane (NFM) with a core-shell structure was fabricated from PCL and HA to achieve cancer cell capturing (about 75% of cells). On the other hand, hyaluronic acid (HA)-functionalized graphene quantum dots (GQDs) were used to detect captured cancer cells on NFM through the changes in photoluminescence intensity. Therefore, CD44 receptor-HA interaction is the main principle used for both entrapment and detection of cancer cells. Results demonstrated the GQD-HA fluorescent intensity of solution decreased through the increase of the captured cancer cell numbers on NFM, which is related to the more adsorption of GQD nanocomposites to the CD44 receptors. In contrast, this intensity for noncancerous cells was steady with any cell concentrations. This difference shows the system's remarkable selectivity and specificity, which can be crucial in fluorescent imaging for accurate cancer diagnosis.

A review of graphene quantum dots and their potential biomedical applications.

Today, nanobiotechnology is a pioneering technology in biomedicine. Every day, new nanomaterials are synthesized with elevated physiochemical properties for better diagnosis and treatment of diseases. One advancing class of materials is the Graphene family. Among different kinds of graphene derivatives, graphene quantum dots (GQDs) show fantastic optical, electrical, and electrochemical features originating from their unique quantum confinement effect. Due to the distinct properties of GQD, including large surface-to-volume ratio, low cytotoxicity, and easy functionalization, this nanomaterial has gone popular in biomedical field. Herein, a short overview of different strategies developed for GQD synthesis and functionalization is discussed. In the following, the most recent progress of GQD based nanomaterials in different biomedical fields, including bio-imaging, drug/gene delivery, antimicrobial, tissue engineering, and biosensors, are reviewed.

Hyaluronic acid-graphene quantum dot nanocomposite: Potential target drug delivery and cancer cell imaging.

Nowadays, the use of nanoparticle-based drug delivery systems has received much more attention. In this regard, here, graphene quantum dots (GQD) were used as drug carriers as well as imaging agents for cancer cells. In order to optimize the dose of the drug and reduce its side effects for healthy cells, hyaluronic acid was decorated on the surface of GQD to target cancer cells. The morphology and size of the synthesized nanoparticles alone and conjugated with hyaluronic acid were investigated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM); TEM images revealed a particles size of ∼5.67 and ∼8.69 nm, respectively. In the presence of 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS), hyaluronic acid was bounded to dopamine hydrochloride and was prepared to react with GQD. After synthesis of graphene quantum dot-hyaluronic acid nanocomposite, curcumin (CUR) as a drug model was loaded on the synthesized nanocarriers, and its loading percentage was measured. The results showed that 98.02% of the drug was loaded on the nanocarriers. Also, the conjugation of each agent on the nanocarrier was approved by photoluminescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), and UV-visible absorption techniques, and the results showed that the reactions were performed correctly. The effect of GQD, graphene quantum dot-hyaluronic acid, CUR, graphene quantum dot-hyaluronic acid-CUR on the viability of HeLa and L929 cells was evaluated by the MTT test. The results showed that the synthesized nanocarrier is completely biocompatible, and the drug nanocarriers reduce HeLa cell viability significantly due to the mediation of hyaluronic acid-CD44 for drug cell uptake. Simultaneously with drug delivery, the other goal of these nanocarriers is to image cancer cells by emitting fluorescent light. Fluorescent microscopy showed that these nanocarriers were adsorbed on HeLa cells, unlike L929 cells.

Brain Homogenate of a Rat Model of Alzheimer's Disease Modifies the Secretome of 3D Cultured Periodontal Ligament Stem Cells: A Potential Neuroregenerative Therapy.

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disease leading to neuronal cell death and manifested by cognitive disorders and behavioral impairment. Mesenchymal stem cells (MSCs) are one of the most promising candidates to stimulate neuroregeneration and prevent disease progression. Optimization of MSC culturing protocols is a key strategy to increase the therapeutic potential of the secretome. Objectives: Here, we investigated the effect of brain homogenate of a rat model of AD (BH-AD) on the enhancement of protein secretion in the secretome of periodontal ligament stem cells (PDLSCs) when cultured in a 3D environment. Moreover, the effect of this modified secretome was examined on neural cells to study the impact of the conditioned medium (CM) on stimulation of regeneration or immunomodulation in AD. Methods: PDLSCs were isolated and characterized. Then, the spheroids of PDLSCs were generated in a modified 3D culture plate. PDLSCs-derived CM was prepared in the presence of BH-AD (PDLSCs-HCM) and the absence of it (PDLSCs-CM). The viability of C6 glioma cells was assessed after exposure to different concentrations of both CMs. Then, a proteomic analysis was performed on the CMs. Results: Differentiation into adipocytes and high expression of MSCs markers verified the precise isolation of PDLSCs. The PDLSC spheroids were formed after 7 days of 3D culturing, and their viability was confirmed. The effect of CMs on C6 glioma cell viability showed that both CMs at low concentrations (> 20 mg/mL) had no cytotoxic effect on C6 neural cells. The results showed that PDLSCs-HCM contains higher concentrations of proteins compared to PDLSCs-CM, including Src-homology 2 domain (SH2)-containing PTPs (SHP-1) and muscle glycogen phosphorylase (PYGM) proteins. SHP-1 has a role in nerve regeneration, and PYGM is involved in glycogen metabolism. Conclusions: The modified secretome derived from 3D cultured spheroids of PDLSCs treated by BH-AD as a reservoir of regenerating neural factors can serve as a potential source for AD treatment.

Highly efficient detection of cancer-derived exosomes using modified core-shell electrospun nanofibers as a capture substrate and antibody immobilized-graphene quantum dots as a signaling agent.

In the past few years graphene quantum dots (GQDs) have been used as a signaling agent for medical diagnosis. They can be modified and labeled with different macromolecules to give them potential to be attached to a specific target. Herein GQDs were labeled with an antibody which is specific for cancer-derived exosomes, isolated from blood serum by using a specialized PCL-gelatin core-shell NFM. This membrane showed excellent sensitivity for isolating exosomes from a complex mixture such as serum, and the GQD-antibody complex detected the isolated exosomes with great sensitivity. The final results allow this method to be considered as one that can be used to quantify the concentration of a desired analyte in a mixture.

A promising approach toward efficient isolation of the exosomes by core-shell PCL-gelatin electrospun nanofibers.

Exosomes as cell-derived vesicles are promising biomarkers for noninvasive and early detection of different types of cancer. However, a straightforward and cost-effective technique for isolation of exosomes in routine clinical settings is still challenging. Herein, we present for the first time, a novel coaxial nanofiber structure for the exosome isolation from body fluids with high efficiency. Coaxial nanofiber structure is composed of polycaprolactone polymer as core and a thin layer of gelatin (below 10 nm) as the shell. The thermo-sensitive thin layer of gelatin can efficiently release the captured exosome by specific antibody namely, CD63, whenever its temperature raised to the physiological temperature of 37 °C. Moreover, the thin layer of gelatin induces less contamination to separated exosomes. The interconnected micro-pores of electrospun nanofibrous membrane insurances large surface area for immobilization of specific antibody for efficient exosome capturing. The efficacy of exosome isolation is determined by direct ELISA and compared with ultracentrifugation technique. For the exosome isolation, it was observed that over 87% of exosomes existed in the culture medium can be effectively isolated by coaxial electrospun nanofibers with the average thickness of 50 µm. Therefore, this promising technique can be substituted for the traditional techniques for exosome isolation which are mostly suffering from low efficacy, high cost, and troublesome process.

Electrospun nanofibers: a promising horizon toward the detection and treatment of cancer.

Due to the increase in the number of cancer patients, because of environmental parameters, high stress, low immunity, etc., there is an urgent need to develop cost-effective sensors for early targeted detection of cancerous cells with adequate selectivity and efficiency. Early disease diagnosis is important, as it is necessary to start treatments before disease progression. On the other hand, we need new, more efficient cancer treatment approaches with minimized side effects, more biocompatibility, and easy disposal. Nanobiotechnology is a field that can assist in developing new diagnostic and treatment approaches, specifically in fatal cancers. Herein, a study on the different applications of nanofibers in cancer detection as well as its treatment has been done. Here, a very brief survey on the main structure of biosensors and their different categories has been conducted and will precede the discussion of the study to serve as a reference and guide the reader's understanding.

Pivotal role of electrospun nanofibers in microfluidic diagnostic systems - a review.

Recently, the usage of electrospinning technology for the fabrication of fine fibers with a good deal of variation in morphology and structure has drawn the attention of many researchers around the world. These fibers have found their way in the many fields of science including medical diagnosis, tissue engineering, drug delivery, replica molding, solar cells, catalysts, energy conversion and storage, physical and chemical sensors and other applications. Among all applications, biosensing with the aim of rapid and sensitive biomarker detection is an area that warrants attention. Electrospun nanofibrous membranes enjoy numerous factors which benefit them to be used as potential candidates in biosensing platforms. Some of these factors include a high surface to volume ratio, analogous scale compared to bioactive molecules and relatively defect-free properties of nanofibers (NFs). In this review, we focused on the recent advances in electrospun nanofibrous membrane-based micro-analytical devices with an application as diagnostic systems. Hence, a study on the electrospun nanofiber usage in lab-on-a-chip and paper-based point-of-care devices, with an opening introduction to biosensors, nanofibers, the electrospinning method, and microfluidics as the principles of the intended subject, is provided. It is anticipated that the given examples in this paper will provide sufficient evidence for the potential of electrospun NFs for being used as a substrate in the commercial fabrication of highly sensitive and selective biosensors.

Natural Compounds for Skin Tissue Engineering by Electrospinning of Nylon-Beta Vulgaris.

Natural compounds containing polysaccharide ingredients have been employed as candidates for treatment of skin tissue. Herein, for the first time, electrospinning setup was proposed to fabricate an efficient composite nanofibrous structure of Beta vulgaris (obtained from Beet [Chenopodiaceae or Amaranthaceae]) belonged to polysaccharides and an elastic polymer named nylon 66 for skin tissue engineering. Both prepared scaffolds including noncomposite and composite types were studied by Scanning electron microscope (SEM), Fourier transform infrared (FTIR) spectroscopy, mechanical assay, and contact angle. Scanning electron microscope examinations have approved the uniform and homogeneous structure of composite nanofibers containing nylon polymer and B. vulgaris extract. FTIR spectroscopy was endorsed the presence of B. vulgaris extract within the interwoven mat of nanofibers. Also, measurement of mechanical property with cell-laden composite scaffolds approved the desirable similarity between corresponding scaffold and native skin tissue. To our surprise, it was found that compared with nylon nanofibrous scaffold, composite sample containing B. vulgaris extract has lower contact angle indicating a higher hydrophilic surface. After cell seeding process of keratinocyte cells on composite and noncomposite scaffolds, SEM and 3[4,5-dimethylthiazoyl-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assays approved higher number of attached cells onto the corresponding composite electrospun membrane. Epidermal gene expression such as involucrin, cytokeratin 10, and cytokeratin 14 was observed through real-time polymerase chain reaction (PCR) technique. Furthermore, immunocytochemistry results (cytokeratin 10 and loricrin) approved that the original property of keratinocytes was strongly preserved using composite scaffold. The corresponding study tries to introduce a new type of natural-based scaffolds for dermal tissue engineering that exhibits an elastic behavior similar to native skin tissue.

Electrospun polyethersolfone nanofibrous membrane as novel platform for protein immobilization in microfluidic systems.

In the present study, the feasibility of electrospun polyethersolfone (PES) nanofibrous membrane as the solid substrate for microfluidic based immunoassays to enhance the density of immobilized antibody on the surface of membrane was assessed. Conversely, the efficacy of antibody immobilization was compared by two different strategies as 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) coupling chemistry and hydrophobic interaction. Compared to conventional immunoassays carried out in plates or gels, microfluidic based immunoassays grant a lot of advantages such as a consumption of little samples and reagents, shorter analysis time, and higher efficiency. Therefore, microfluidic immunoassays can be efficiently used as a point-of-care device in medical diagnosis. Surprisingly, we found the increase of specific surface areas of the microfluidic channels improve density of immobilized proteins and leads to higher signal strength. Anti-staphylococcus enterotoxin B (anti-SEB) was used as an analyte model to demonstrate the utility of our proposed platform. Fluorescent microscopy, Fourier transform infrared spectroscopic (FTIR), gas adsorption, contact angle, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), Uv-Vis spectrophotometer and atomic force microscopy (AFM) techniques were used to assess the efficacy of antibody immobilization on the surface. To understand dominant mechanism of protein immobilization, zeta potential measurement was also carried out and it was found electrostatic attraction play significant role in antibody immobilization running into micro- channels containing through EDC/NHS. Moreover, incorporation of nanofibrous membrane causes significant improvement in the signal detection of microfluidic based immunoassay. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1108-1120, 2018.

Ammonia plasma-treated electrospun polyacrylonitryle nanofibrous membrane: the robust substrate for protein immobilization through glutaraldhyde coupling chemistry for biosensor application.

The surface of polyacrylonitrile electrospun nanofibrous membrane (PAN NFM) was aminated by the ammonia plasma treatment. The content of amine groups has been estimated for different time of plasma treatment. The newly generated amine groups were successfully activated by glutaraldehyde (Ga) for the covalent attachment of the protein molecules on the NFM surface. Bio-functionalization of ammonia plasma treated PAN NFM was carried out by the primary antibodies (Ab) immobilization as a protein model through Ga coupling chemistry. For comparison, the immobilization of Ab was also performed through physical interactions. Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) was used for the characterization of surface functional groups of PAN NFM after different modifications. The surface morphology of the NFM after immobilization was characterized using scanning electron microscope (SEM). The efficacy of Ab immobilization was estimated by enzyme-linked immuno sorbent assay (ELISA) method. X- Ray photoelectron spectroscopy (XPS) was performed to confirm the covalent immobilization of Ab on the modified PAN NFM. Results show that ammonia plasma treatment effectively increased the amount of Ab immobilization through Ga coupling chemistry. Our findings suggest that this is a versatile model for the preparation of stable bio-functionalized NFM which is applicable in different field of biomedical science.

Study of epithelial differentiation and protein expression of keratinocyte-mesenchyme stem cell co-cultivation on electrospun nylon/B. vulgaris extract composite scaffold.

Employing of the composite electrospun scaffold containing herbal extract in conjugation with co-culturing of cells can open up new window to the design of efficient biomaterials for skin tissue regeneration. Here, we introduce the synergistic effect of composite electrospun nanofibrous scaffold of nylon66 loaded with Beta vulgaris (B. vulgaris) (extract of beet roots, a plants whose widely used in Iranian folk medicine as wound healing medicine) and co-culture of mesenchymal stem-cells (MSCs)-human keratinocyte (H-keratino) differentiation towards epithelial lineage. In vitro biocompatibility was examined through MTT assay and epithelial differentiation checked by real-time PCR and immunocytochemistry (ICC) assay after co-culturing of MSCs and H-keratino on proposed scaffold. Significant enhancement in cell proliferation was detected after cell culturing on the composite type of electrospun scaffold containing B. vulgaris. Moreover, after 14days of co-culturing process, gene expression results revealed that both composite and non-composite nylon66 electrospun scaffold promote epithelial differentiation compared to mono-cell culturing of H-keratino in terms of several markers as Cytokeratin 10, Cytokeratin 14 and Involucrin and ICC of some dermal proteins like Cytokeratin 14 and Loricrin. To the best of our knowledge, findings of this study will introduce new way for the generation of novel biomaterials for the development of current skin tissue engineering.

The nanofibrous PAN-PANi scaffold as an efficient substrate for skeletal muscle differentiation using satellite cells.

Among polymers, polyaniline (PANi) has been introduced as a good candidate for muscle regeneration due to high conductivity and also biocompatibility. Herein, for the first time, we report the use of electrospun nanofibrous membrane of PAN-PANi as efficient scaffold for muscle regeneration. The prepared PAN-PANi electrospun nanofibrous membrane was characterized by scanning electron microscopy (SEM), Attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) and tensile examination. The softer scaffolds of non-composite electrospun nanofibrous PAN govern a higher rate of cell growth in spite of lower differentiation value. On the other hand, PAN-PANi electrospun nanofibrous membrane exposed high cell proliferation and also differentiation value. Thank to the conductive property and higher Young's modulus of composite type due to the employment of PANi, satellite cells were induced into more matured form as analyzed by Real-Time PCR. On the other hand, grafting of composite nanofibrous electrospun scaffold with gelatin increased the surface stiffness directing satellite cells into lower cell proliferation and highest value of differentiation. Our results for first time showed the significant role of combination between conductivity, mechanical property and surface modification of PAN-PANi electrospun nanofibers and provid new insights into most biocompatible scaffolds for muscle tissue engineering. The schematic figure conveys the effective combination of conductive and surface stiffness on muscle tissue engineering.

The different fate of satellite cells on conductive composite electrospun nanofibers with graphene and graphene oxide nanosheets.

Electrospinning of composite polymer solutions provides fantastic potential to prepare novel nanofibers for use in a variety of applications. The addition of graphene (G) and graphene oxide (GO) nanosheets to bioactive polymers was found to enhance their conductivity and biocompatibility. Composite conductive nanofibers of polyaniline (PANI) and polyacrylonitrile (PAN) with G and GO nanosheets were prepared by an electrospinning process. The fabricated membranes were investigated by physical and chemical examinations including scanning electron microscopy (SEM), Raman spectroscopy, x-ray diffraction (XRD) and tensile assay. The muscle satellite cells enriched by a pre-plating technique were cultured in the following and their proliferation and differentiation behavior studied by MTT, Real-Time PCR assays and 4', 6-diamidino-2-phenylindole (DAPI) staining. The cultured cells on composite nanofibrous PAN/PANI-CSA/G confirmed a higher proliferation and differentiation value compared to other groups including PAN/PANI-CSA/GO and PAN/PANI-CSA scaffolds. Furthermore, the higher stiffness of the former scaffold showed a lower cell spreading as a function of stem cell activation into more proliferative cells. It is supposed that the enhanced conductivity value in addition to relative higher stiffness of the PAN/PANI-CSA/G composite nanofibers plays a favorable role for proliferation and differentiation of satellite cells.

Efficient protein immobilization on polyethersolfone electrospun nanofibrous membrane via covalent binding for biosensing applications.

In this paper we introduce novel strategy for antibody immobilization using high surface area electrospun nanofibrous membrane based on ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling chemistry. To present the high performance of proposed biosensors, anti-staphylococcus enterotoxin B (anti-SEB) was used as a model to demonstrate the utility of our proposed system. Polymer solution of polyethersolfone was used to fabricate fine nanofibrous membrane. Moreover, industrial polyvinylidene fluoride membrane and conventional microtiter plate were also used to compare the efficiency of antibody immobilization. Scanning electron microscopy images were taken to study the morphology of the membranes. The surface activation of nanofibrous membrane was done with the help of O2 plasma. PES nanofibrous membrane with carboxyl functional groups for covalent attachment of antibodies were treated by EDC/NHS coupling agent. The quantity of antibody immobilization was measured by enzyme-linked immuno sorbent assay (ELISA) method. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy was performed to confirm the covalent immobilization of antibody on membrane. Atomic force microscopy, scanning electron microscopy and invert fluorescence microscopy were used to analyze the antibody distribution pattern on solid surfaces. Results show that oxygen plasma treatment effectively increased the amount of antibody immobilization through EDC/NHS coupling chemistry. It was found that the use of nanofibrous membrane causes the improved detection signal of ELISA based biosensors in comparison to the standard assay carried out in the 96-well microtiter plate. This method has the potential to improve the ELISA-based biosensor and we believe that this technique can be used in various biosensing methods.