Colloids and Surfaces A: Physicochemical and Engineering Aspects
Microgravity biosynthesized penicillin loaded electrospun polyurethane–dextran nanofibrous mats for biomedical applications
Graphical abstract
Introduction
A report shows that it costs about 800 million USD and nearly a decade to bring a new 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, 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 walls [3]. Since Fleming's discovery of penicillin, research has been carried out on their 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. 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 improvement of P. chrysogenum has never been stopped. The productivity of the industrial strain is far better than their ancestor, and the development was achieved by classical 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].
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) designed by Synthecon, Inc., USA. The important components of HARV are the oxygenator membrane, disk shaped culture chamber and the rotator base. The culture chamber can hold 50 ml of liquid medium. Medium is filled using fill port while sample collection is done through the sample ports with the help of three way stop cock. The oxygenator membrane is a thin layer consisting of silicone rubber, covering a polyester cloth backing. This membrane forms one side 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 user. The shear stress on any particle in the fluid is the product of the fluid velocity gradient and the viscosity of the medium [9]. The whole setup is shown in Fig. 2. The spores when 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 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].
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 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 production by fungi under microgravity, for the sake of health of space crew as most of the 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 modeled microgravity condition. Even though, penicillin production is observed under microgravity, penicillin concentration is not increased when compared to the normal gravity conditions (data not shown). The obtained result is in accordance with our previously published work stating microgravity does not pose stress on P. chrysogenum [14].
Electrospinning is a simple, versatile, cost-effective, and scalable system, which uses high voltage electrical field to generate aligned or random nanofibers from several synthetic and natural polymers [15], [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 unique electrospinning process allows as impregnating the nanofiber membranes with antibacterial and therapeutic agents [17], [18], [19].
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 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 and tissue culture [21] and molecular arms [22] etc. Dextran-based hydrogels can serve as instructive scaffolds to promote neovascularization and skin regeneration in chronic wounds. Dextran based scaffolds are soft 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 blended easily with hydrophobic polymers by making use of the unique solubility characteristic. Dextran can be directly blended with biodegradable hydrophobic polymers such as polyurethane (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].
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]. Staphylococcus aureus and Enterococcus faecalis are known to cause skin and wound infection, abscess, bacteremia, endocarditis, urinary tract infection, peritonitis and nosocomial infections [2]. The nanofiber currently made was found to be effective on both S. aureus and E. faecalis which could be used as a wound dressing material.
Section snippets
Fungal strain and medium
P. chrysogenum (KACC 425892) was purchased from Korean Agricultural Culture Collection (Suwon, South Korea). Fungal cultures were maintained on potato dextrose agar (BD Difco, Sparks, MD) supplemented with streptomycin (100 mg/L) (Sigma–Aldrich, St. Louis, MO). Spores from 7 days old culture were used in this experiment. Culture medium contains yeast extract 3 gm/L (BD Difco, MN, USA), Lactose monohydrate 3 gm/L (Samchun chemical, South Korea) and 1,3 diamino propane 5 mM/L (Sigma–Aldrich, St.
Results and discussion
The simulated microgravity condition was provided to the P. chrysogenum by adjusting the speed of the vessel as the fungal mass grows bigger to maintain the free flow. HARV was rotated on an 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 shown in Fig. 2. Care was taken so that fungal mass stays in suspension as illustrated in Fig. 3. The rotation was
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 like a topical
Acknowledgements
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 Chonbuk National University.
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These two authors equally contributed to the work.