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 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.

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.

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.

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.

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.