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Microgravity biosynthesized penicillin loaded electrospun polyurethane–dextran nanofibrous mats for biomedical applications
Accepted Manuscript Title: Microgravity Biosynthesized Penicillin Loaded Electrospun Polyurethane-Dextran Nanofibrous Mats for Biomedical Applications Author: Yesupatham Sathish Kumar Afeesh Rajan Unnithan Dwaipayan Sen Cheol Sang Kim Yang Soo LeeThese two authors equally contributed to the work.
S0927-7757(15)00093-X http://dx.doi.org/doi:10.1016/j.colsurfa.2015.01.065 COLSUA 19712
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
13-10-2014 22-1-2015 26-1-2015
Please cite this article as: Y.S. Kumar, A.R. Unnithan, D. Sen, C.S. Kim, Y.S. Lee, Microgravity Biosynthesized Penicillin Loaded Electrospun Polyurethane-Dextran Nanofibrous Mats for Biomedical Applications, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2015), http://dx.doi.org/10.1016/j.colsurfa.2015.01.065 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical abstract
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Highlights Extended application of Bioreactors for penicillin production Enhanced bactericidal nanofiber
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Therapeutic and cosmetic applications
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Microgravity Biosynthesized Penicillin Loaded Electrospun Polyurethane-Dextran Nanofibrous Mats for Biomedical
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Applications
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Cheol Sang Kim2, Yang Soo Lee1**
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Yesupatham Sathish Kumar1#, Afeesh Rajan Unnithan2#, Dwaipayan Sen3,
1 Department of Forest Science and Technology, College of Agriculture and Life Sciences,
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Chonbuk National University, Jeonju 561-756, Republic of Korea
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2 Bionano Systems Engineering Department, Chonbuk National University, Jeonju 561-756, Republic of Korea
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3 School of Biosciences and Technology, VIT University, Vellore 632014, India
#These two authors equally contributed to the work.
Corresponding Author:
Prof. Yang Soo Lee ([email protected])
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Abstract: Electrospinning is a fabrication process that uses an electric field to control the deposition of
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polymer fibers onto a target substrate. Nanofiber possess the characteristic features of high length-to-diameter ratio and specific surface areas, enabling it to be used for protective clothing,
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filter, antibacterial membrane, reinforced composite, and tissue engineering. We have adopted a method of direct in situ electrospinning to produce mats composed of the culture filtrate from
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Low Shear Modeled Microgravity (LSMMG) grown Penicillium chrysogenum with a suitable
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carrier polymer to aid in its electrospinning. The antibacterial activity of the mat is attributed to the presence of penicillin in the culture filtrate. The presence of penicillin was confirmed by
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using Liquid chromatography–mass spectrometry (LC-MS/MS). The mat was found to be effective against gram positive bacteria like Staphylococcus aureus and Enterococcus faecalis.
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The process, stability and characterization of the biological properties of such nanofibrous
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scaffolds are demonstrated.
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Keywords: Electrospinning; Low shear modeled microgravity; Penicillium chrysogenum; Liquid chromatography–mass spectrometry.
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1. Introduction: A report shows that it costs about 800 million USD and nearly a decade to bring a new
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drug to the market [1]. Despite advancement in the medicinal chemistry, most of the drugs that are currently in use are semisynthetic modifications of natural compounds like penicillin,
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cephalosporins and the carbapenems [2] . Penicillin plays an imperative role in the human medical history. Penicillin cause lysis of bacterial cells by inhibiting the biosynthesis of their cell
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walls [3] . Since Fleming’s discovery of penicillin, research has been carried out on their
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biosynthesis and regulation. An interesting aspect of the metabolism of Penicillium chrysogenum is that it will express metabolic genes differentially when grown in a different medium.
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Metabolic engineering of P chrysogenum to obtain higher penicillin yields has become the primary objective of the industrial research on antibiotics [4] . For commercial reasons, the P chrysogenum has never been stopped. The productivity of the industrial
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improvement of
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strain is far better than their ancestor, and the development was achieved by classical
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mutagenesis and screening methods [5]. Apart from penicillin, P. chrysogenum can produce industrially important enzymes like alpha-amylase, glucose oxidase. Interestingly, fungi are reported to be sensitive to the gravity vector [6]. While numerous environmental stimuli have been examined for their effect on microorganisms, effects due to changes in the gravitational force are also becoming increasingly important [7]. The changes in the physical forces of hydrostatic pressure, gravity, and fluid shear plays an important role in the evolution and microbial physiology. Very little is known about how fungal cells convert these mechanical signals into molecular and biochemical responses [8].
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Space Microbiology focuses on how microbial consortia carried as contaminants to the space ships like International Space Stations behave in that particular environment. Microgravity conditions can be achieved at the laboratory scale using High Aspect Ratio Vessel (HARV)
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designed by Synthecon, Inc, USA.. The important components of HARV are the oxygenator membrane, disc shaped culture chamber and the rotator base. The culture chamber can hold 50
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ml of liquid medium. Medium is filled using fill port while sample collection is done through the
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sample ports with the help of 3 way stop cock. The oxygenator membrane is a thin layer consisting of silicone rubber, covering a polyester cloth backing. This membrane forms one side
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of the chamber enabling gas transfer to the cells cultured in the HARV. The HARV is then attached to the Rotator base which can rotate the vessel at an appropriate speed selected by the
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user. The shear stress on any particle in the fluid is the product of the fluid velocity gradient and
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the viscosity of the medium [9]. The whole setup is shown in the figure.2. The spores when
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suspended in the culture medium experience shear force due to fluid velocity gradient and viscosity of the medium.The constant reorientation due to the rotation effectively nullifies the
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cumulative sedimentation thus providing the low shear environment. HARV system does not remove the gravitational force, but creates a state of “functional weightlessness” by randomizing the gravitational vector and minimizing turbulence (shear) over the surface of the cell [10]. HARV is essentially an optimized form of suspension culture and consists of a hollow disk or cylinder that is completely filled with medium without any visible bubbles, i.e., “zero headspace” and rotates on an axis parallel to the ground to provide low shear microgravity condition. The same vessel if rotated on an axis perpendicular to ground it confers normal gravity [11] .
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The HARV bioreactor system enables sufficient movement of the cells to allow continuous exchange of dissolved gases through a permeable membrane [8]. There is no sedimentation of organism inside the vessel and turbulent motion of the organism is greatly
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minimized. Hence, it is believed that the organism is placed in the environment close to the space condition [12]. It is of prime importance to evaluate the ability of secondary metabolite
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production by fungi under microgravity, for the sake of health of space crew as most of the
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fungal metabolites are toxic and allergic to humans.[13] . To the best of our knowledge, this is the first report on Eukaryotic fungal secondary metabolites under low-shear modelled
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microgravity condition. Even though, penicillin production is observed under microgravity, penicillin concentration is not increased when compared to the normal gravity conditions (Data
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not shown). The obtained result is in accordance with our previously published work stating
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microgravity does not pose stress on P chrysogenum [14].
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Electrospinning is a simple, versatile, cost-effective, and scalable system, which uses
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high voltage electrical field to generate aligned or random nanofibers from several synthetic and natural polymers [15] and [16]. Antibacterial dressings from electrospun nanofibers potentially offer many advantages over conventional processes. Generally, the ultimate goal of the nanofiber design is to provide an ideal structure that can replace the natural extracellular matrix until the host cells can grow and synthesize a new natural cellular matrix since the environment changes dynamically over time as the polymer nanofibers grade, allowing the seeded cells to proliferate and produce their own ECM. Due to this huge surface area and microporous structure, the nanofibers could quickly start signaling pathway and attract fibroblasts to the dermal layer, which can secrete essential extracellular matrix components, such as collagen and several cytokines e.g. Growth factors and angiogenic factors to repair damaged tissue. In addition, the
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unique electrospinning process allows as impregnating the nanofiber membranes with antibacterial and therapeutic agents [17] [17] [18] and [19].
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Dextran is a biopolymer produced by a variety of lactic acid bacteria with numerous known applications. Dextran is highly biocompatible and biodegradable and is also
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suitable polymer to be developed as hydrogels. The uses of dextran are becoming increasingly important in biomedical applications, such as carriers for drug delivery [20], scaffolds for cell
etc. Dextran-based hydrogels can serve as instructive scaffolds to promote
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[22]
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and tissue culture [21] et al., 2008) and molecular arms [21] et al., 2008) and molecular arms
neovascularization and skin regeneration in chronic wounds. Dextran based scaffolds are soft
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and pliable, offering opportunities to improve the management of burn wound treatment [23]. Most importantly, dextran is soluble in both water and organic solvents. Dextran could be
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blended easily with hydrophobic polymers by making use of the unique solubility characteristic.
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Dextran can be directly blended with biodegradable hydrophobic polymers such as polyurethane
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(PU) to prepare composite nanofibrous membranes by electrospinning the mixed solution in organic solvents. The mechanical strength of dextran, its swelling properties in water, and the biological activity could thereby be modulated [24]. Previous studies on few bacteria revealed that secondary metabolite production is inhibited under microgravity like decreased production of peptide antibiotic cephalosporin and microcin B17 by Streptomyces clavuligerus and
Escherichia coli respectively.[25] .
Interestingly, the production of gramicidin S by Bacillus Brevis was unaffected by Low shear modeled microgravity (LSMMG). These findings suggest that the LSMMG does not have the same effects on all microorganisms[26].
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Natural penicillins have few drawbacks like limited solubility and instability in acidic and basic environments in stomach and intestine respectively[27]. Previous studies have used antibacterial electrospun nanofibrous scaffolds as
a wound dressing material [28] .
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Staphylococcus aureus and Enterococcus faecalis are known to cause skin and wound infection, abscess, bacteremia, endocarditis, urinary tract infection, peritonitis and nosocomial infections
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[2]. The nanofiber currently made was found to be effective on both Staphylococcus aureus and
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Enterococcus faecalis which could be used as a wound dressing material.
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2. Materials and methods 2.1 Fungal strain and medium
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P chrysogenum (KACC 425892) was purchased from Korean Agricultural Culture Collection
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(Suwon, South Korea). Fungal cultures were maintained on potato dextrose agar (BD Difco,
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Sparks, MD) supplemented with streptomycin (100 mg l-1) (Sigma-Aldrich, St. Louis, MO). Spores from 7 days old culture were used in this experiment. Culture medium contains yeast
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extract 3gm/L (BD Difco, MN, USA), Lactose monohydrate 3gm/L (Samchun chemical, South Korea) and 1,3 diamino propane 5mM/L (Sigma-Aldrich, St. Louis, USA). 2.2 High aspect ratio vessel
Rotatory cell culture system (RCCS-1) was purchased from Synthecon, Incorporated (Houston, Texas 77054 USA) with Autoclavable High Aspect Ratio Vessel (HARV) of 50 ml capacity. Spores from 7 days old culture were used. Approximately 1012 spores were added to the HARV and rotated at 25 rpm in the horizontal axis, inside the chamber with 90 % humidity and maintained at 25˚C..
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2.3. Electrospinning Polyurethane, 10wt% (PU, Estane® Skythane® X595A-11, Lubrizol) was prepared by
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dissolving overnight in a solvent mixture of N, N dimethylformamide (DMF, Samchun, Korea) and methyl ethyl ketone/2-butanone (MEK, extra pure, Samchun, Korea) (50/50, wt:wt%).
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2wt% The Dextran (from Leconostoc mesenteroides, average MW = 8500~11500, Sigma Aldrich) has been added to the solution along with culture filtrate. The obtained solutions were
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placed in a plastic syringe tube and fed through a metal capillary (nozzle) with a diameter di =
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0.21 mm (21 G) attached to a 1-D robot-system that moves laterally and is controlled by the LabVIEW 9.0 software program (National Instrument). The feeding rate was maintained at 0.5
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ml/h via a controllable syringe pump. Electrospinning was carried out at a voltage of 18 KV and
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2.4. Characterizations
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working distance of 15 cm at room temperature.
The morphology of the electrospun composite mats was observed by using field-emission
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scanning electron microscopy (FE-SEM, Hitachi S-7400, Hitachi, Japan). The bonding configurations of the samples were characterized by means of Fourier-transform infrared (FT-IR) spectroscopy.
2.5. Liquid chromatography/ Electrospray Ionization- Mass spectrometry. Agilent 6410B Triple Quadrupole LC/MS (Agilent Technologies, Wilmington, USA) equipped with an ESI source was employed for the analysis. Penicillin G (Lot#SLBG4604V) was purchased from Sigma-Aldrich (St. Louis, USA) and used as a reference standard. 100 µl of culture media was mixed with 900 µl of methanol and centrifuged. Aliquots of 3 µl of the processed samples were injected into the HPLC system (1200 Series LC, Agilent Technologies,
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Wilmington, USA) fitted with Phenomenex Kinetex™ 2.6 µm C8 100 Å 50 x 2.1 mm column, maintained
at 30˚C. ESI was operating at +3000V and the source temperature of
380˚C.Capillary voltage, cone voltage and source offset were set at 3kV, 30kV and 30V
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respectively. The gas flow of desolvation and the cone were set at 650L/Hr,150 L/Hr with a nebulizer pressure of 15 bar. A mobile phase composed of 0.1% formic acid in distilled water
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(Buffer A) and 0.1% formic acid in acetonitrile (Buffer B) was used to separate the analyte and
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pumped into the ESI chamber at a flow rate of 0.5ml/min for 20 min. Fragmentor voltage and collision voltage were set at 70V and 10V respectively. Detection of the ion’s was carried out in
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the multiple-reaction monitoring mode (MRM), by observing the transition pairs of m/z 335.1
MassHunter Software (Version B.04.00).
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2.6 Bacterial growth curve test:
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precursor ion to the m/z 176 ion for penicillin G. Data acquisition was performed with the
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Staphylococcus aureus (KACC 10768), and Enterococcus faecalis (KACC 11304) used for this
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study were purchased from Korean type culture collection. All cultures were maintained on nutrient agar plates and stored at 4˚C.1ml of nutrient broth, and 10µl of the overnight broth was added to Eppendorf tubes. Sterile nutrient broth (BD Difco, MD, USA) was used for blanking. Optical density was measured at 4-hour interval for 12 hours
using a spectrophotometer
(Shimadzu UV-1800, Tokyo, Japan) wavelength set at 600nm. Nanofiber discs of 6mm width were made using punching machine. Discs were placed into the tubes and kept in a shaker incubator set at 200 RPM. Dextran mat without culture filtrate acts as a negative control. Experiments were performed in triplicate.
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2.7 Antimicrobial activity by disc diffusion method. Overnight culture of Staphylococcus aureus (KACC 10768) and Enterococcus faecalis (KACC
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11304) was used to perform the experiment. Lawn culture was made by streaking the bacterial culture on to Muller Hinton Agar (Oxoid, Hampshire, England). Discs of 6mm width were made
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from nanofiber sheet and immediately placed on the agar plate and kept for incubation at 37˚C for 18 hours. The antibacterial activity of the nanofiber was evaluated based on the size of Zone
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of Inhibition. Experiments were performed in duplicates. Nanofiber made without culture filtrate
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is kept as negative control. 3. Results and discussion:
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The simulated microgravity condition was provided to the P chrysogenum by adjusting the speed
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of the vessel as the fungal mass grows bigger to maintain the free flow. HARV was rotated on an
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axis perpendicular to the gravity vector as illustrated in Fig.1. Air bubble formation due to the fungal metabolism was avoided by placing the HARV inside a humidity chamber set at 90% as
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shown in the Fig. 2. Care was taken so that fungal mass stays in suspension as illustrated in Fig.3. The rotation was adjusted to 25 rpm as the spore aggregates formed a fluid orbit within the vessel. Continual fall without contacting the vessel wall was attained according to the manufacturer's instruction (Operation Manual, The Rotary Cell Culture System, Synthecon, Inc, USA). Lactose was used in this study as early reports suggest that if glucose is replaced with lactose as a carbon source in the medium, penicillin production is increased drastically[29]. Prolonged incubation may lead to attachment of fungal mass onto the wall of HARV, which will dampen the effect of fluid dynamics resulting in decreased low shear microgravity condition. In order to prevent this 5mM 1,3 diamino propane is added to the media, which is shown to induce
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the penicillin production in a short period [30]. P chrysogenum has the potential of producing at least 20 secondary metabolites including polyketides, non-ribosomal peptides, isoprenoid compounds and hybrid polyketide-non-ribosomal peptides [31]. After 72 hours of incubation,
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medium turned yellowish indicating the diffusion of secondary metabolites into the medium.
and vacuum dried to 5 ml before proceeding with electrospinning.
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Since the total volume of the bioreactor is only 50ml, culture filtrate from 5 batches was pooled,
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LC-MS/MS is considered to be more sensitive and accurate method for the potential
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identification of chemical of a particular mass in a complex mixture of culture filtrate. Analytical grade Penicillin was used as a standard reference. Fig.4A represents the chromatogram of
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standard penicillin showing retention time at 5.587. Culture filtrate was analyzed in the Selected Reaction Monitoring (SRM), Parent mass → Fragment mass (335.1→176). Very clean
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chromatogram with negligible noise background was obtained. The peak was observed at 5.503
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as shown in Fig.4B confirming the presence of penicillin in the culture filtrate.
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Fig. 5(A)–(C) shows the FESEM images of electrospun PU, PU-dextran and PU-dextran-Extract loaded composite nanofibers respectively. It can be observed that these randomly oriented asspun nanofibers exhibited bead-free, smooth surface with almost uniform diameters along their lengths. The diameters of these composite nanofibers were determined to be in the range of 600– 700 nm. For PU electrospun mat (Fig. 5A), the fibers appear well-defined without any interconnection among the fibers. However, the composite mats containing extracts and dextran showed minor variations in fiber morphology (Fig. 5 B&C). As seen in Figure 5C, no beaded nanofibers and no extra structures were detected by FESEM on the surface of the fibers loaded with the dextran and extract, demonstrating an excellent compatibility of drug–polymer–solvent. The resulting nanofibers also showed that, the incorporation of dextran and extract into the
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nanofibers not only decreased their average diameter, but also reduced the diameter distribution of electrospun nanofibers (Fig. 5C). The addition of dextran and extract can significantly change solution viscosity; however, it did not affect solution conductivity. Solution viscosity influences
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the morphological structure and average size of the resulting fibers [32]. A possible explanation for the decreasing viscosity of the polymeric solution by the addition of dextran and the extract is
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may be due to the trapping of dextran between polymeric chains, and thus acts as a plasticizer
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[33]. The composite electrospun nanofiber mat produced in this study was desirably smooth and
implantation and a comfortable texture for use.
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flexible. This flexibility, in addition to hydrophobicity, provides easy handling during
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FT-IR spectroscopy was used to investigate the changes of the functional groups that occur during the blending of drug and dextran with PU nanofibers. Fig.6 illustrates the FTIR spectra of
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PU, PU-dextran-extract loaded nanofibers. The spectroscopy of electrospun polyurethane has a
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characteristic absorption band at 3320, 2960, 1710, 1530, 1220, 1110 and 777 cm−1, which
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represents υ(N–H), υ(C–H), υ(C-O), υ(C-C),υ(C–C), υ(C–O), υ(C–H) on substituted benzene, respectively (Jiang, Yuan, Li &Chow, 2006). In addition to that, several characteristic bands of dextran are located at 759 and 849 cm–1 (CH bend); 1272cm–1 (C–O stretch); 1435 cm–1 (CH3 bend); 2922 cm–1 (CH stretch); 3117 cm–1 (CH3 stretch); 3335cm–1 (OH stretch, end group), 1140, 1122 and 1033 cm–1 (saccharide structure) [34].[34]. It can be seen that all the characteristic peaks of PU and dextran were visible in PU-dextran nanofibers, and some peaks were being overlapped. The spectra of Penicillin were shown in the range of 1500–1800 cm−1 as the most relevant spectral range to the concentration of penicillin. The region 1710–1800 cm−1due to the contribution of carbonyl stretching of the carboxylate group and 1580–1614 cm−1 corresponding to the carbonyl stretching of amide III group [35]. Most of the peaks of PU,
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dextran and penicillin have been overlapped in the PU-dextran-extract loaded nanofibers due to the relative similarity and shifting.
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The bacterial growth curve test was performed to show the bacteriostatic activity of the nanofiber synthesized. The optical density of a medium gradually increases along with an increase in the
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bacterial cells. However, S aureus and E faecalis could not multiply properly in the presence nanofiber containing culture filtrate. As shown in the Graph 1 & 2, the optical density is less
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compared to the negative control. This result shows that the nanofiber is active against
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Staphylococcus aureus and Enterococcus faecalis.
Antimicrobial susceptibility testing was performed by the Kirby-Bauer method. Overnight broth
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of Staphylococcus aureus and Enterococcus faecalis was adjusted to the opacity of 1 McFarland unit. Approximately 108 bacteria were plated onto the agar, and a lawn culture was made by
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streaking with a sterile swab. Discs made from the nanofiber were placed immediately and kept
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for overnight incubation. The nanofiber containing only the dextran did not show any activity
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while the nanofiber containing the culture filtrate showed zone of inhibition as shown in Fig.7, indicating the antimicrobial activity of the nanofiber. Hence, the mat is shown to be effective against both S aureus and E faecalis. Conclusion:
In this study, we looked for the ability of P. chrysogenum to produce penicillin under LSMMG conditions and its further incorporation into nanofibers and showed biological and antimicrobial properties. Hereby, we report that production of penicillin by P. chrysogenum was not hampered by microgravity condition. Further, we exploited the antibacterial activity of penicillin by incorporating it into nanofibers with the scope of increasing the bioavailability of the drug more
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like a topical administration of the antibiotic near the wound site. The nanofiber currently made was found to be effective on both Staphylococcus aureus and Enterococcus faecalis. This study represents useful information to improve the production of nanofibers with many other bioactive
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secondary metabolites not only in P. chrysogenum, but in other filamentous fungi as well, which would be very preferable for tissue engineering and other cosmetic and therapeutic application
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such as wound dressing.
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Acknowledgement:
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This research was supported by the National Research Foundation of Korea (NRF) Grant No.1201002578 funded by the Korean Government and university research grants from the
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Chonbuk National University.
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Natl Acad Sci USA, 108 (2011) 20976-20981. [24] H.L. Jiang, D.F. Fang, B.S. Hsiao, B. Chu, W.L. Chen, Optimization and characterization of dextran membranes prepared by electrospinning, Biomacromolecules, 5 (2004) 326-333. [25] Q. Gao, A. Fang, D.L. Pierson, S.K. Mishra, A.L. Demain, Shear stress enhances microcin B17 production in a rotating wall bioreactor, but ethanol stress does not, Appl Microbiol Biot, 56 (2001) 384-387. [26] A. Fang, D.L. Pierson, S.K. Mishra, D.W. Koenig, A.L. Demain, Gramicidin S production by Bacillus brevis in simulated microgravity, Curr Microbiol, 34 (1997) 199-204.
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[27] K. Bauer, W. Kaufmann, H. Offe, Enzymatische Synthese von α-Phenoxypropionyl-6aminopenicillansäure, Naturwissenschaften, 47 (1960) 469-469. [28] A.R. Unnithan, G. Gnanasekaran, Y. Sathishkumar, Y.S. Lee, C.S. Kim, Electrospun
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antibacterial polyurethane-cellulose acetate-zein composite mats for wound dressing, Carbohydr Polym, 102 (2014) 884-892.
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[29] N. Castillo, F. Fierro, S. Gutiérrez, J. Martín, Genome-wide analysis of differentially
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expressed genes from Penicillium chrysogenum grown with a repressing or a non-repressing carbon source, Curr Genet, 49 (2006) 85-96.
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[30] J. Martin, C. Garcia-Estrada, K. Kosalkova, R.V. Ullan, S.M. Albillos, J.F. Martin, The inducers 1,3-diaminopropane and spermidine produce a drastic increase in the expression of the
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penicillin biosynthetic genes for prolonged time, mediated by the LaeA regulator, Fungal Genet
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Biol, 49 (2012) 1004-1013.
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[31] M.A. van den Berg, R. Albang, K. Albermann, J.H. Badger, J.M. Daran, A.J.M. Driessen, C. Garcia-Estrada, N.D. Fedorova, D.M. Harris, W.H.M. Heijne, V. Joardar, J.A.K.W. Kiel, A.
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Kovalchuk, J.F. Martin, W.C. Nierman, J.G. Nijland, J.T. Pronk, J.A. Roubos, I.J. van der Klei, N.N.M.E. van Peij, M. Veenhuis, H. von Dohren, C. Wagner, J. Wortman, R.A.L. Bovenberg, Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum, Nat Biotechnol, 26 (2008) 1161-1168.
[32] C.J. Thompson, G.G. Chase, A.L. Yarin, D.H. Reneker, Effects of parameters on nanofiber diameter determined from electrospinning model, Polymer, 48 (2007) 6913-6922. [33] M. Zamani, M. Morshed, J. Varshosaz, M. Jannesari, Controlled release of metronidazole benzoate from poly epsilon-caprolactone electrospun nanofibers for periodontal diseases, Eur J Pharm Biopharm, 75 (2010) 179-185.
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[34] J. Zhi, K.J. Yuan, S.F. Li, W.K. Chow, Study of FTIR spectra and thermal analysis of polyurethane, Spectrosc Spect Anal, 26 (2006) 624-628. [35] Z. Talebpour, R. Tavallaie, S.H. Ahmadi, A. Abdollahpour, Simultaneous determination of
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penicillin G salts by infrared spectroscopy: Evaluation of combining orthogonal signal correction with radial basis function-partial least squares regression, Spectrochim Acta A, 76 (2010) 452-
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Figure captions
Graphical representation of
HARV rotated about the axis
perpendicular to the gravity vector.
High aspect ratio vessel placed inside humidity and temperature
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Figure. 2.
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Figure. 1.
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controlled chamber. The right panel shows a closer view of the HARV rotated on a perpendicular axis to the gravity vector with
The simulated microgravity condition is applied to the fungal
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Figure. 3.
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3-way stopcock attached to the sample ports.
spore by the fluid dynamics created by the HARV, nullifying the
(A) Multiple reaction mode Chromatogram of penicillin G
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Figure. 4.
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gravitational force.
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standard. (B) Multiple reaction mode Chromatogram of culture filtrate.
Figure. 5.
SEM images of electrospun (A) PU, (B) PU-Dextran and (C) PUdextran-extract nanofibrous mat
Figure. 6.
FTIR spectra of PU and ) PU- dextran-extract nanofibrous mat
Figure. 7.
(A) Antibacterial activity of nanofiber containing penicillin on MHA plate. A zone of inhibition is seen near nanofibers made from Polyurethane -dextran -culture filtrate (NF+CF). No zone of
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inhibition was observed near the disc made only with Polyurethane –dextran which acts as a negative control.
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(B) Staphylococcus aureus broth O.D reading at 600nm is decreased in the presence of Nanofiber with culture filtrate
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(NF+CF) compared to Blank (BLK) and NF (Nanofiber without
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Culture filtrate).
(C) Enterococcus faecalis broth O.D reading at 600nm is decreased
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in the presence of Nanofiber with culture filtrate (NF+CF)
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filtrate)
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compared to Blank (BLK) and NF (Nanofiber without Culture
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S0927-7757(15)00093-X http://dx.doi.org/doi:10.1016/j.colsurfa.2015.01.065 COLSUA 19712
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
13-10-2014 22-1-2015 26-1-2015
Please cite this article as: Y.S. Kumar, A.R. Unnithan, D. Sen, C.S. Kim, Y.S. Lee, Microgravity Biosynthesized Penicillin Loaded Electrospun Polyurethane-Dextran Nanofibrous Mats for Biomedical Applications, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2015), http://dx.doi.org/10.1016/j.colsurfa.2015.01.065 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical abstract
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Highlights Extended application of Bioreactors for penicillin production Enhanced bactericidal nanofiber
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Therapeutic and cosmetic applications
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Microgravity Biosynthesized Penicillin Loaded Electrospun Polyurethane-Dextran Nanofibrous Mats for Biomedical
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Applications
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Cheol Sang Kim2, Yang Soo Lee1**
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Yesupatham Sathish Kumar1#, Afeesh Rajan Unnithan2#, Dwaipayan Sen3,
1 Department of Forest Science and Technology, College of Agriculture and Life Sciences,
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Chonbuk National University, Jeonju 561-756, Republic of Korea
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2 Bionano Systems Engineering Department, Chonbuk National University, Jeonju 561-756, Republic of Korea
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3 School of Biosciences and Technology, VIT University, Vellore 632014, India
#These two authors equally contributed to the work.
Corresponding Author:
Prof. Yang Soo Lee ([email protected])
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Abstract: Electrospinning is a fabrication process that uses an electric field to control the deposition of
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polymer fibers onto a target substrate. Nanofiber possess the characteristic features of high length-to-diameter ratio and specific surface areas, enabling it to be used for protective clothing,
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filter, antibacterial membrane, reinforced composite, and tissue engineering. We have adopted a method of direct in situ electrospinning to produce mats composed of the culture filtrate from
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Low Shear Modeled Microgravity (LSMMG) grown Penicillium chrysogenum with a suitable
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carrier polymer to aid in its electrospinning. The antibacterial activity of the mat is attributed to the presence of penicillin in the culture filtrate. The presence of penicillin was confirmed by
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using Liquid chromatography–mass spectrometry (LC-MS/MS). The mat was found to be effective against gram positive bacteria like Staphylococcus aureus and Enterococcus faecalis.
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The process, stability and characterization of the biological properties of such nanofibrous
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scaffolds are demonstrated.
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Keywords: Electrospinning; Low shear modeled microgravity; Penicillium chrysogenum; Liquid chromatography–mass spectrometry.
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1. Introduction: A report shows that it costs about 800 million USD and nearly a decade to bring a new
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drug to the market [1]. Despite advancement in the medicinal chemistry, most of the drugs that are currently in use are semisynthetic modifications of natural compounds like penicillin,
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cephalosporins and the carbapenems [2] . Penicillin plays an imperative role in the human medical history. Penicillin cause lysis of bacterial cells by inhibiting the biosynthesis of their cell
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walls [3] . Since Fleming’s discovery of penicillin, research has been carried out on their
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biosynthesis and regulation. An interesting aspect of the metabolism of Penicillium chrysogenum is that it will express metabolic genes differentially when grown in a different medium.
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Metabolic engineering of P chrysogenum to obtain higher penicillin yields has become the primary objective of the industrial research on antibiotics [4] . For commercial reasons, the P chrysogenum has never been stopped. The productivity of the industrial
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improvement of
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strain is far better than their ancestor, and the development was achieved by classical
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mutagenesis and screening methods [5]. Apart from penicillin, P. chrysogenum can produce industrially important enzymes like alpha-amylase, glucose oxidase. Interestingly, fungi are reported to be sensitive to the gravity vector [6]. While numerous environmental stimuli have been examined for their effect on microorganisms, effects due to changes in the gravitational force are also becoming increasingly important [7]. The changes in the physical forces of hydrostatic pressure, gravity, and fluid shear plays an important role in the evolution and microbial physiology. Very little is known about how fungal cells convert these mechanical signals into molecular and biochemical responses [8].
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Space Microbiology focuses on how microbial consortia carried as contaminants to the space ships like International Space Stations behave in that particular environment. Microgravity conditions can be achieved at the laboratory scale using High Aspect Ratio Vessel (HARV)
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designed by Synthecon, Inc, USA.. The important components of HARV are the oxygenator membrane, disc shaped culture chamber and the rotator base. The culture chamber can hold 50
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ml of liquid medium. Medium is filled using fill port while sample collection is done through the
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sample ports with the help of 3 way stop cock. The oxygenator membrane is a thin layer consisting of silicone rubber, covering a polyester cloth backing. This membrane forms one side
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of the chamber enabling gas transfer to the cells cultured in the HARV. The HARV is then attached to the Rotator base which can rotate the vessel at an appropriate speed selected by the
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user. The shear stress on any particle in the fluid is the product of the fluid velocity gradient and
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the viscosity of the medium [9]. The whole setup is shown in the figure.2. The spores when
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suspended in the culture medium experience shear force due to fluid velocity gradient and viscosity of the medium.The constant reorientation due to the rotation effectively nullifies the
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cumulative sedimentation thus providing the low shear environment. HARV system does not remove the gravitational force, but creates a state of “functional weightlessness” by randomizing the gravitational vector and minimizing turbulence (shear) over the surface of the cell [10]. HARV is essentially an optimized form of suspension culture and consists of a hollow disk or cylinder that is completely filled with medium without any visible bubbles, i.e., “zero headspace” and rotates on an axis parallel to the ground to provide low shear microgravity condition. The same vessel if rotated on an axis perpendicular to ground it confers normal gravity [11] .
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The HARV bioreactor system enables sufficient movement of the cells to allow continuous exchange of dissolved gases through a permeable membrane [8]. There is no sedimentation of organism inside the vessel and turbulent motion of the organism is greatly
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minimized. Hence, it is believed that the organism is placed in the environment close to the space condition [12]. It is of prime importance to evaluate the ability of secondary metabolite
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production by fungi under microgravity, for the sake of health of space crew as most of the
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fungal metabolites are toxic and allergic to humans.[13] . To the best of our knowledge, this is the first report on Eukaryotic fungal secondary metabolites under low-shear modelled
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microgravity condition. Even though, penicillin production is observed under microgravity, penicillin concentration is not increased when compared to the normal gravity conditions (Data
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not shown). The obtained result is in accordance with our previously published work stating
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microgravity does not pose stress on P chrysogenum [14].
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Electrospinning is a simple, versatile, cost-effective, and scalable system, which uses
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high voltage electrical field to generate aligned or random nanofibers from several synthetic and natural polymers [15] and [16]. Antibacterial dressings from electrospun nanofibers potentially offer many advantages over conventional processes. Generally, the ultimate goal of the nanofiber design is to provide an ideal structure that can replace the natural extracellular matrix until the host cells can grow and synthesize a new natural cellular matrix since the environment changes dynamically over time as the polymer nanofibers grade, allowing the seeded cells to proliferate and produce their own ECM. Due to this huge surface area and microporous structure, the nanofibers could quickly start signaling pathway and attract fibroblasts to the dermal layer, which can secrete essential extracellular matrix components, such as collagen and several cytokines e.g. Growth factors and angiogenic factors to repair damaged tissue. In addition, the
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unique electrospinning process allows as impregnating the nanofiber membranes with antibacterial and therapeutic agents [17] [17] [18] and [19].
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Dextran is a biopolymer produced by a variety of lactic acid bacteria with numerous known applications. Dextran is highly biocompatible and biodegradable and is also
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suitable polymer to be developed as hydrogels. The uses of dextran are becoming increasingly important in biomedical applications, such as carriers for drug delivery [20], scaffolds for cell
etc. Dextran-based hydrogels can serve as instructive scaffolds to promote
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[22]
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and tissue culture [21] et al., 2008) and molecular arms [21] et al., 2008) and molecular arms
neovascularization and skin regeneration in chronic wounds. Dextran based scaffolds are soft
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and pliable, offering opportunities to improve the management of burn wound treatment [23]. Most importantly, dextran is soluble in both water and organic solvents. Dextran could be
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blended easily with hydrophobic polymers by making use of the unique solubility characteristic.
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Dextran can be directly blended with biodegradable hydrophobic polymers such as polyurethane
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(PU) to prepare composite nanofibrous membranes by electrospinning the mixed solution in organic solvents. The mechanical strength of dextran, its swelling properties in water, and the biological activity could thereby be modulated [24]. Previous studies on few bacteria revealed that secondary metabolite production is inhibited under microgravity like decreased production of peptide antibiotic cephalosporin and microcin B17 by Streptomyces clavuligerus and
Escherichia coli respectively.[25] .
Interestingly, the production of gramicidin S by Bacillus Brevis was unaffected by Low shear modeled microgravity (LSMMG). These findings suggest that the LSMMG does not have the same effects on all microorganisms[26].
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Natural penicillins have few drawbacks like limited solubility and instability in acidic and basic environments in stomach and intestine respectively[27]. Previous studies have used antibacterial electrospun nanofibrous scaffolds as
a wound dressing material [28] .
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Staphylococcus aureus and Enterococcus faecalis are known to cause skin and wound infection, abscess, bacteremia, endocarditis, urinary tract infection, peritonitis and nosocomial infections
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[2]. The nanofiber currently made was found to be effective on both Staphylococcus aureus and
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Enterococcus faecalis which could be used as a wound dressing material.
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2. Materials and methods 2.1 Fungal strain and medium
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P chrysogenum (KACC 425892) was purchased from Korean Agricultural Culture Collection
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(Suwon, South Korea). Fungal cultures were maintained on potato dextrose agar (BD Difco,
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Sparks, MD) supplemented with streptomycin (100 mg l-1) (Sigma-Aldrich, St. Louis, MO). Spores from 7 days old culture were used in this experiment. Culture medium contains yeast
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extract 3gm/L (BD Difco, MN, USA), Lactose monohydrate 3gm/L (Samchun chemical, South Korea) and 1,3 diamino propane 5mM/L (Sigma-Aldrich, St. Louis, USA). 2.2 High aspect ratio vessel
Rotatory cell culture system (RCCS-1) was purchased from Synthecon, Incorporated (Houston, Texas 77054 USA) with Autoclavable High Aspect Ratio Vessel (HARV) of 50 ml capacity. Spores from 7 days old culture were used. Approximately 1012 spores were added to the HARV and rotated at 25 rpm in the horizontal axis, inside the chamber with 90 % humidity and maintained at 25˚C..
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2.3. Electrospinning Polyurethane, 10wt% (PU, Estane® Skythane® X595A-11, Lubrizol) was prepared by
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dissolving overnight in a solvent mixture of N, N dimethylformamide (DMF, Samchun, Korea) and methyl ethyl ketone/2-butanone (MEK, extra pure, Samchun, Korea) (50/50, wt:wt%).
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2wt% The Dextran (from Leconostoc mesenteroides, average MW = 8500~11500, Sigma Aldrich) has been added to the solution along with culture filtrate. The obtained solutions were
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placed in a plastic syringe tube and fed through a metal capillary (nozzle) with a diameter di =
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0.21 mm (21 G) attached to a 1-D robot-system that moves laterally and is controlled by the LabVIEW 9.0 software program (National Instrument). The feeding rate was maintained at 0.5
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ml/h via a controllable syringe pump. Electrospinning was carried out at a voltage of 18 KV and
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2.4. Characterizations
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working distance of 15 cm at room temperature.
The morphology of the electrospun composite mats was observed by using field-emission
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scanning electron microscopy (FE-SEM, Hitachi S-7400, Hitachi, Japan). The bonding configurations of the samples were characterized by means of Fourier-transform infrared (FT-IR) spectroscopy.
2.5. Liquid chromatography/ Electrospray Ionization- Mass spectrometry. Agilent 6410B Triple Quadrupole LC/MS (Agilent Technologies, Wilmington, USA) equipped with an ESI source was employed for the analysis. Penicillin G (Lot#SLBG4604V) was purchased from Sigma-Aldrich (St. Louis, USA) and used as a reference standard. 100 µl of culture media was mixed with 900 µl of methanol and centrifuged. Aliquots of 3 µl of the processed samples were injected into the HPLC system (1200 Series LC, Agilent Technologies,
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Wilmington, USA) fitted with Phenomenex Kinetex™ 2.6 µm C8 100 Å 50 x 2.1 mm column, maintained
at 30˚C. ESI was operating at +3000V and the source temperature of
380˚C.Capillary voltage, cone voltage and source offset were set at 3kV, 30kV and 30V
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respectively. The gas flow of desolvation and the cone were set at 650L/Hr,150 L/Hr with a nebulizer pressure of 15 bar. A mobile phase composed of 0.1% formic acid in distilled water
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(Buffer A) and 0.1% formic acid in acetonitrile (Buffer B) was used to separate the analyte and
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pumped into the ESI chamber at a flow rate of 0.5ml/min for 20 min. Fragmentor voltage and collision voltage were set at 70V and 10V respectively. Detection of the ion’s was carried out in
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the multiple-reaction monitoring mode (MRM), by observing the transition pairs of m/z 335.1
MassHunter Software (Version B.04.00).
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2.6 Bacterial growth curve test:
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precursor ion to the m/z 176 ion for penicillin G. Data acquisition was performed with the
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Staphylococcus aureus (KACC 10768), and Enterococcus faecalis (KACC 11304) used for this
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study were purchased from Korean type culture collection. All cultures were maintained on nutrient agar plates and stored at 4˚C.1ml of nutrient broth, and 10µl of the overnight broth was added to Eppendorf tubes. Sterile nutrient broth (BD Difco, MD, USA) was used for blanking. Optical density was measured at 4-hour interval for 12 hours
using a spectrophotometer
(Shimadzu UV-1800, Tokyo, Japan) wavelength set at 600nm. Nanofiber discs of 6mm width were made using punching machine. Discs were placed into the tubes and kept in a shaker incubator set at 200 RPM. Dextran mat without culture filtrate acts as a negative control. Experiments were performed in triplicate.
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2.7 Antimicrobial activity by disc diffusion method. Overnight culture of Staphylococcus aureus (KACC 10768) and Enterococcus faecalis (KACC
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11304) was used to perform the experiment. Lawn culture was made by streaking the bacterial culture on to Muller Hinton Agar (Oxoid, Hampshire, England). Discs of 6mm width were made
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from nanofiber sheet and immediately placed on the agar plate and kept for incubation at 37˚C for 18 hours. The antibacterial activity of the nanofiber was evaluated based on the size of Zone
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of Inhibition. Experiments were performed in duplicates. Nanofiber made without culture filtrate
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is kept as negative control. 3. Results and discussion:
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The simulated microgravity condition was provided to the P chrysogenum by adjusting the speed
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of the vessel as the fungal mass grows bigger to maintain the free flow. HARV was rotated on an
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axis perpendicular to the gravity vector as illustrated in Fig.1. Air bubble formation due to the fungal metabolism was avoided by placing the HARV inside a humidity chamber set at 90% as
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shown in the Fig. 2. Care was taken so that fungal mass stays in suspension as illustrated in Fig.3. The rotation was adjusted to 25 rpm as the spore aggregates formed a fluid orbit within the vessel. Continual fall without contacting the vessel wall was attained according to the manufacturer's instruction (Operation Manual, The Rotary Cell Culture System, Synthecon, Inc, USA). Lactose was used in this study as early reports suggest that if glucose is replaced with lactose as a carbon source in the medium, penicillin production is increased drastically[29]. Prolonged incubation may lead to attachment of fungal mass onto the wall of HARV, which will dampen the effect of fluid dynamics resulting in decreased low shear microgravity condition. In order to prevent this 5mM 1,3 diamino propane is added to the media, which is shown to induce
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the penicillin production in a short period [30]. P chrysogenum has the potential of producing at least 20 secondary metabolites including polyketides, non-ribosomal peptides, isoprenoid compounds and hybrid polyketide-non-ribosomal peptides [31]. After 72 hours of incubation,
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medium turned yellowish indicating the diffusion of secondary metabolites into the medium.
and vacuum dried to 5 ml before proceeding with electrospinning.
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Since the total volume of the bioreactor is only 50ml, culture filtrate from 5 batches was pooled,
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LC-MS/MS is considered to be more sensitive and accurate method for the potential
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identification of chemical of a particular mass in a complex mixture of culture filtrate. Analytical grade Penicillin was used as a standard reference. Fig.4A represents the chromatogram of
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standard penicillin showing retention time at 5.587. Culture filtrate was analyzed in the Selected Reaction Monitoring (SRM), Parent mass → Fragment mass (335.1→176). Very clean
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chromatogram with negligible noise background was obtained. The peak was observed at 5.503
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as shown in Fig.4B confirming the presence of penicillin in the culture filtrate.
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Fig. 5(A)–(C) shows the FESEM images of electrospun PU, PU-dextran and PU-dextran-Extract loaded composite nanofibers respectively. It can be observed that these randomly oriented asspun nanofibers exhibited bead-free, smooth surface with almost uniform diameters along their lengths. The diameters of these composite nanofibers were determined to be in the range of 600– 700 nm. For PU electrospun mat (Fig. 5A), the fibers appear well-defined without any interconnection among the fibers. However, the composite mats containing extracts and dextran showed minor variations in fiber morphology (Fig. 5 B&C). As seen in Figure 5C, no beaded nanofibers and no extra structures were detected by FESEM on the surface of the fibers loaded with the dextran and extract, demonstrating an excellent compatibility of drug–polymer–solvent. The resulting nanofibers also showed that, the incorporation of dextran and extract into the
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nanofibers not only decreased their average diameter, but also reduced the diameter distribution of electrospun nanofibers (Fig. 5C). The addition of dextran and extract can significantly change solution viscosity; however, it did not affect solution conductivity. Solution viscosity influences
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the morphological structure and average size of the resulting fibers [32]. A possible explanation for the decreasing viscosity of the polymeric solution by the addition of dextran and the extract is
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may be due to the trapping of dextran between polymeric chains, and thus acts as a plasticizer
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[33]. The composite electrospun nanofiber mat produced in this study was desirably smooth and
implantation and a comfortable texture for use.
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flexible. This flexibility, in addition to hydrophobicity, provides easy handling during
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FT-IR spectroscopy was used to investigate the changes of the functional groups that occur during the blending of drug and dextran with PU nanofibers. Fig.6 illustrates the FTIR spectra of
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PU, PU-dextran-extract loaded nanofibers. The spectroscopy of electrospun polyurethane has a
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characteristic absorption band at 3320, 2960, 1710, 1530, 1220, 1110 and 777 cm−1, which
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represents υ(N–H), υ(C–H), υ(C-O), υ(C-C),υ(C–C), υ(C–O), υ(C–H) on substituted benzene, respectively (Jiang, Yuan, Li &Chow, 2006). In addition to that, several characteristic bands of dextran are located at 759 and 849 cm–1 (CH bend); 1272cm–1 (C–O stretch); 1435 cm–1 (CH3 bend); 2922 cm–1 (CH stretch); 3117 cm–1 (CH3 stretch); 3335cm–1 (OH stretch, end group), 1140, 1122 and 1033 cm–1 (saccharide structure) [34].[34]. It can be seen that all the characteristic peaks of PU and dextran were visible in PU-dextran nanofibers, and some peaks were being overlapped. The spectra of Penicillin were shown in the range of 1500–1800 cm−1 as the most relevant spectral range to the concentration of penicillin. The region 1710–1800 cm−1due to the contribution of carbonyl stretching of the carboxylate group and 1580–1614 cm−1 corresponding to the carbonyl stretching of amide III group [35]. Most of the peaks of PU,
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dextran and penicillin have been overlapped in the PU-dextran-extract loaded nanofibers due to the relative similarity and shifting.
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The bacterial growth curve test was performed to show the bacteriostatic activity of the nanofiber synthesized. The optical density of a medium gradually increases along with an increase in the
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bacterial cells. However, S aureus and E faecalis could not multiply properly in the presence nanofiber containing culture filtrate. As shown in the Graph 1 & 2, the optical density is less
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compared to the negative control. This result shows that the nanofiber is active against
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Staphylococcus aureus and Enterococcus faecalis.
Antimicrobial susceptibility testing was performed by the Kirby-Bauer method. Overnight broth
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of Staphylococcus aureus and Enterococcus faecalis was adjusted to the opacity of 1 McFarland unit. Approximately 108 bacteria were plated onto the agar, and a lawn culture was made by
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streaking with a sterile swab. Discs made from the nanofiber were placed immediately and kept
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for overnight incubation. The nanofiber containing only the dextran did not show any activity
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while the nanofiber containing the culture filtrate showed zone of inhibition as shown in Fig.7, indicating the antimicrobial activity of the nanofiber. Hence, the mat is shown to be effective against both S aureus and E faecalis. Conclusion:
In this study, we looked for the ability of P. chrysogenum to produce penicillin under LSMMG conditions and its further incorporation into nanofibers and showed biological and antimicrobial properties. Hereby, we report that production of penicillin by P. chrysogenum was not hampered by microgravity condition. Further, we exploited the antibacterial activity of penicillin by incorporating it into nanofibers with the scope of increasing the bioavailability of the drug more
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like a topical administration of the antibiotic near the wound site. The nanofiber currently made was found to be effective on both Staphylococcus aureus and Enterococcus faecalis. This study represents useful information to improve the production of nanofibers with many other bioactive
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secondary metabolites not only in P. chrysogenum, but in other filamentous fungi as well, which would be very preferable for tissue engineering and other cosmetic and therapeutic application
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such as wound dressing.
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Acknowledgement:
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This research was supported by the National Research Foundation of Korea (NRF) Grant No.1201002578 funded by the Korean Government and university research grants from the
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Chonbuk National University.
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References: [1] J.M. Reichert, Trends in development and approval times for new therapeutics in the United States, Nat Rev Drug Discov, 2 (2003) 695-702.
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[2] F. von Nussbaum, M. Brands, B. Hinzen, S. Weigand, D. Häbich, Antibacterial Natural Products in Medicinal Chemistry—Exodus or Revival?, Angewandte Chemie International
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Edition, 45 (2006) 5072-5129.
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[3] J. Drews, Drug discovery: A historical perspective, Science, 287 (2000) 1960-1964. [4] D. Cullen, The genome of an industrial workhorse, Nat Biotechnol, 25 (2007) 189-190.
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[5] F.Q. Wang, J. Zhong, Y. Zhao, J.F. Xiao, J. Liu, M. Dai, G.Z. Zheng, L. Zhang, J. Yu, J.Y.
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Figure captions
Graphical representation of
HARV rotated about the axis
perpendicular to the gravity vector.
High aspect ratio vessel placed inside humidity and temperature
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Figure. 2.
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Figure. 1.
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controlled chamber. The right panel shows a closer view of the HARV rotated on a perpendicular axis to the gravity vector with
The simulated microgravity condition is applied to the fungal
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Figure. 3.
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3-way stopcock attached to the sample ports.
spore by the fluid dynamics created by the HARV, nullifying the
(A) Multiple reaction mode Chromatogram of penicillin G
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Figure. 4.
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gravitational force.
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standard. (B) Multiple reaction mode Chromatogram of culture filtrate.
Figure. 5.
SEM images of electrospun (A) PU, (B) PU-Dextran and (C) PUdextran-extract nanofibrous mat
Figure. 6.
FTIR spectra of PU and ) PU- dextran-extract nanofibrous mat
Figure. 7.
(A) Antibacterial activity of nanofiber containing penicillin on MHA plate. A zone of inhibition is seen near nanofibers made from Polyurethane -dextran -culture filtrate (NF+CF). No zone of
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inhibition was observed near the disc made only with Polyurethane –dextran which acts as a negative control.
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(B) Staphylococcus aureus broth O.D reading at 600nm is decreased in the presence of Nanofiber with culture filtrate
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(NF+CF) compared to Blank (BLK) and NF (Nanofiber without
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Culture filtrate).
(C) Enterococcus faecalis broth O.D reading at 600nm is decreased
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in the presence of Nanofiber with culture filtrate (NF+CF)
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filtrate)
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compared to Blank (BLK) and NF (Nanofiber without Culture
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Fig.1
Fig.2
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Fig.3
Fig.4
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Fig.6
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Fig.7
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