containing silver, copper, and zinc nanoparticles from their salts. .... heptahydrate(Na2HPO4.7H2O), and potassium phosphate monobasic (KH2PO4. ...... micelle-in-nanofiber device for efficient and safe cancer therapy,â ACS Nano, vol. 9, no.
Effect of Electrospun Nanofibers on Growth Behavior of Fungal Cells Arifa Parveen North Carolina A&T State University
A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department: Joint School of Nanoscience & Nanoengineering Major: Nanoengineering Major Professor: Dr. Lifeng Zhang Greensboro, North Carolina 2018
ii The Graduate College North Carolina Agricultural and Technical State University
This is to certify that the Doctoral Dissertation of
Arifa Parveen
has met the dissertation requirements of North Carolina Agricultural and Technical State University
Greensboro, North Carolina 2018
Approved by:
Dr. Lifeng Zhang Committee Member
Dr. Dennis R. LaJeunesse Committee Member
Dr. Ajit Kelkar Committee Member
Dr. Shyam Aravamudhan Committee Member
Dr. Ajit Kelkar Department Chair
Dr. James G. Ryan Committee Member
Dr. Sanjiv Sarin Dean, The Graduate College
iii
© Copyright by Arifa Parveen 2018
iv Biographical Sketch Arifa Parveen was born and raised up in Dhaka, Bangladesh. She received her B.S in Materials Science and Metallurgical Engineering in 2004 from Bangladesh University of Engineering and Technology in Bangladesh, and completed her M.S. in Mechanical Engineering, May, 2012 from Tuskegee University, Alabama, AL.
v Dedication This work is dedicated to my parents Mohammad Azizur Rahman and MST Jahanara Aziz, my kids (Sunehra & Savaira), my husband (Dr. Kazi Al Imran) and my best friend Late Umme Salma (Died in Nepal Plane Crash on March 12, 2018) for their support, care, and endless joy. I would to thank Dr. Lifeng Zhang and Dr. Dennis LaJeunesse in grateful recognition of their boundless faith, encouragement and untiring support.
vi Acknowledgments It is my privilege to acknowledge my supervisor Dr. Lifeng Zhang, and co-supervisor Dr. Dennis LaJeunesse for their constant inspiration, encouragement, guidance and support during my graduate study. This research could not have been possible without their generous assistance and encouragement. I learned and benefit a lot not only from their knowledge but also their personalities. I also want to express my thanks to the Joint School of Nanoscience and Nanoengineering and Dean James Ryan for the financial support and facilities provided at JSNN. I would like to thank my committee member, Dr. Ajit Kelkar, his cooperation and help in my tough time have been of great value in my entire life. I would also like to thank my committee member Dr. Dennis LaJeunesse for his time, valuable discussion and suggestion to this research study. In addition, I would like to thank members of Nanomaterials lab and Genomics lab for helping me in different ways. Thanks a lot, to my colleagues and friends in JSNN for coming to help me whenever I face problem throughout my graduate studies. Finally, I wish to express my gratitude to my parents Mohammad Azizur Rahman and MST Jahanara Aziz, my Kids, husband and my friends for the love and encouragement in carrying out this challenge. I will always remember their support, encouragement and love without which the higher education would have remained a dream.
vii Table of Contents List of Figures ................................................................................................................................ xi List of Tables ................................................................................................................................ xv Abstract ........................................................................................................................................... 1 CHAPTER 1 Introduction & Literature Review ............................................................................ 2 1.1 Introduction to Microorganism .......................................................................................... 2 1.2 Introduction to Yeast/Fungus ............................................................................................. 2 1.3 Infectious Disease Caused by Fungal(Yeast) Cells ........................................................... 3 1.4 Structure of Yeast Cells ..................................................................................................... 4 1.5 Electrospinning Technique .............................................................................................. 10 1.6 Applications of Electrospun Nanofibers in Biomedical Field ......................................... 12 1.6.1 Tissue engineering ................................................................................................. 13 1.6.2 Wound dressing material ....................................................................................... 14 1.6.3 Drug delivery ......................................................................................................... 17 1.6.4 Nanofibers with antibacterial activity ................................................................... 19 1.6.5. Microbes interaction with nanostructured surfaces .............................................. 20 1.6.6 Other applications of electrospun nanofibers ........................................................ 22 1.7 Objectives and Scope of Research ................................................................................... 24 CHAPTER 2 Research Design and Methodology ........................................................................ 26 2.1 Research Goals and Outline............................................................................................. 26 2.2 Materials and Methods .................................................................................................... 27 2.2.1 Cellulose acetate (CA) & cellulose nanofibers and film ....................................... 27 2.2.1.1 Materials ...................................................................................................... 27 2.2.1.2 Methods ....................................................................................................... 27
viii 2.2.2 PAN nanofibers and film ....................................................................................... 28 2.2.2.1 Materials ...................................................................................................... 28 2.2.2.2 Methods ....................................................................................................... 29 2.2.3 PAN nanofibers with graphene ............................................................................. 31 2.2.3.1 Materials ...................................................................................................... 31 2.2.3.2 Methods ....................................................................................................... 31 2.2.4 Bicomponent polymers .......................................................................................... 32 2.2.4.1 PAN-PMMA nanofibers ............................................................................. 32 2.2.4.1.1 Materials ............................................................................................ 32 2.2.4.1.2 Methods ............................................................................................. 33 2.2.4.2 PAN-PEO nanofibers .................................................................................. 33 2.2.4.2.1 Materials ............................................................................................ 33 2.2.4.2.2 Methods ............................................................................................. 33 2.3 Yeast Strain ...................................................................................................................... 34 2.3.1 Yeast cell culture and growth on surface of nanofibers mat and solid film .......... 34 2.4 Characterization ............................................................................................................... 35 2.4.1 Characterization of nanofibers .............................................................................. 35 2.4.1.1 Contact angle measurements ....................................................................... 35 2.4.1.2 Fourier Transform Infrared Spectroscopy (FTIR) ...................................... 36 2.4.1.3 Scanning Electron Microscopy & Energy Dispersive Spectrometer .......... 36 2.4.2 Characterization of cells ........................................................................................ 36 2.4.2.1 Optical Density ............................................................................................ 36 2.4.2.2 Colony Forming Units(CFU) ...................................................................... 37 2.4.2.3 Fungal cell-nanofiber interaction test .......................................................... 37 2.4.2.4 Scanning electron microscopy .................................................................... 38
ix 2.4.2.5 Confocal laser scanning microscopy ........................................................... 38 CHAPTER 3 Interaction between Fungal Cells and Different Electrospun Nanofibers .............. 40 3.1 Abstract ............................................................................................................................ 40 3.2 Introduction...................................................................................................................... 40 3.3 Results and discussions.................................................................................................... 41 3.3.1 Viability of yeast cells on solid film and nanofibrous mat .................................... 46 3.4 Mechanism Discussion .................................................................................................... 50 3.5 Conclusions...................................................................................................................... 51 CHAPTER 4 Effect of Sizes of PAN Electrospun Nanofibers on Fungal Cell Growth............... 52 4.1 Abstract ............................................................................................................................ 52 4.2 Introduction...................................................................................................................... 52 4.3 Results and Discussions ................................................................................................... 53 4.4 Conclusions...................................................................................................................... 62 CHAPTER 5 Effect of Surface Topological Features of Electrospun Nanofibrous mats on Yeast Cells Growth ................................................................................................................................. 63 5.1 Abstract ............................................................................................................................ 63 5.2 Introduction...................................................................................................................... 63 5.3 Results & Discussions ..................................................................................................... 65 5.3.1 Rough Nanofiber Surface with Positive Curvature by Adding Graphene ............ 65 5.3.2 Rough Nanofiber Surface with Negative Curvature ............................................. 74 5.3.2.1 Rough PAN Nanofiber Surface from PAN/PMMA Nanofibers ................. 74 5.3.2.2 Rough PAN Nanofiber Surface from PAN/PEO nanofibers ...................... 79 5.4 Conclusions...................................................................................................................... 82
x CHAPTER 6 Effect of Hydrophobicity of Nanofibrous Material and Yeast Cells on Cells’ Viability ....................................................................................................................................... 83 6.1 Introduction...................................................................................................................... 83 6.2 Microbial adhesion to hydrocarbon MATH assay........................................................... 83 6.3 Structure and Dynamics of Cell Wall in Yeast Cells ...................................................... 86 6.4 Conclusions...................................................................................................................... 87 CHAPTER 7 Overall Conclusions & Future Research ................................................................ 88 7.1 Conclusions...................................................................................................................... 88 7.2 Future Work ..................................................................................................................... 89 References ..................................................................................................................................... 91
xi List of Figures Figure 1. Structure and Composition of Fungal Cell. ..................................................................... 5 Figure 2. Life cycle of SK1 and C. albicans. .................................................................................. 6 Figure 3. Lifestyles of Yeast cells (a) Scheme showing the development of colonies, biofilms, filaments, flocs and flor by cell-cell (self) and cell-substrate (foreign) adhesion from sessile or planktonic cells on solid or in liquid media. ................................................................................... 8 Figure 4. Number of publications per year (2006–2016) by using electrospinning nanofibers as keyword for literature search Data source, Science Direct. .......................................................... 10 Figure 5. (a) Electrospinning setup; (b) Diagram showing onset and development of bending instabilities. Reprinted with permission from Reneker and Fong. Copyright 2005, American Chemical Society. ......................................................................................................................... 11 Figure 6. Schematic diagram of tissue engineering and several kinds of tissue engineering such as skin, bone, vascular, nerve and cartilage tissue engineering. ................................................... 14 Figure 7. Wound dressing by electrospinning, Carpinteria, C.A. (Spotlight on wound-care technologies. MPMN. 26(5), (2010)). .......................................................................................... 16 Figure 8. Composite laminate interleaved by nanofibrous mats at plies interface [93]. .............. 23 Figure 9. The schematic representation of deacetylation of CA nanofibers is given in the following figure. ........................................................................................................................... 28 Figure 10. Aligned Electrospun Nanofibers Setup ....................................................................... 30 Figure 11 The fiber distribution of (a) CA, (b) Cellulose, (c) random PAN nanofibers and (d) PAN micro fibers .......................................................................................................................... 43 Figure 12. SEM images of (a-b) Cellulose Acetate (CA) and (c-d) Cellulose nanofibers ........... 44 Figure 13. SEM images of (a-b) PAN nanofibers and (c-d) PAN microfibers............................. 45
xii Figure 14. The Fourier Transform Infrared Spectroscopy (FTIR) Spectra of (A) as-electrospun cellulose acetate nanofibers and (B) regenerated cellulose nanofibers ........................................ 46 Figure 15. Interactions between SK1 cells and different nanofibers and films. ........................... 47 Figure 16. Interactions between C. albicans cells and different nanofibers and films. ................ 47 Figure 17. Interactions of (a-b) SK1 cells and (c-d) C. albicans on CA nanofibers (left column) and films (right column). .............................................................................................................. 48 Figure 18. Interactions of (a-b) SK1cells and (c-d) C. albicans on PAN nanofibers (left column) and films (right column). .............................................................................................................. 49 Figure 19 Interactions of (a-b) SK1cells and (c-d) C. albicans on cellulose nanofibers (left column) and films (right column). ................................................................................................ 50 Figure 20. SEM images of (a-b) PAN nanofibrous mat from 10 wt.% PAN solution (top row) and (c-d) PAN nanofibrous mats from 12 wt.% PAN solution (bottom row) ..................................... 55 Figure 21. Fiber size distribution of PAN nanofibers from (a) 10 wt.% and (b) 12 wt.% of PAN solutions ........................................................................................................................................ 55 Figure 22. Aligned electrospun PAN nanofibers from different spinning time periods: (a-c) 3hrs (left column); (b-d) 4hrs (right column) ....................................................................................... 57 Figure 23. Aligned PAN nanofibers from different spinning time periods: (a-c) 5 hrs (left column); (b-d) 6hrs (right column) ............................................................................................... 58 Figure 24. Effect of different size of nanofibers on fungal cell growth: (a) SK1; (b) C. albicans 59 Figure 25 Interactions of (a-b) SK1cells and (c-d) C. albicans on 10 wt.% and 12 wt.% of PAN nanofibers. ..................................................................................................................................... 60 Figure 26 . Effect of fiber alignment on fungal cell growth ......................................................... 61
xiii Figure 27 Interactions of (a-b) SK1 and (c-d) C. albicans cells on random 10 wt.% (left column) and aligned 10 wt.% PAN nanofibers (right column)................................................................... 61 Figure 28. SEM images of graphene embedded electrospun PAN nanofibers with submicrometer sizes (a-b) GPAN-1 (top row); (c-d) GPAN-2 (middle row); and (e-f) GPAN-3 (bottom row) .. 67 Figure 29. Fungal cell growth behavior on submicrometer electrospun PAN nanofibers with smooth and rough surface (positive curvature): (a) SK1; (b) C. albicans .................................... 68 Figure 30 SEM images of SK1(a-b) and C. albicans (c-d) cultured on smooth (left column) and rough PAN (GPAN) (right column) nanofibers............................................................................ 69 Figure 31. SEM images of Graphene embedded of electrospun PAN fibers with micrometer scale diameter: (a-b) GPAN-4 (top row); (c-d) GPAN-5 (bottom row) ................................................ 71 Figure 32. Fungal cell growth behavior on over-micrometer electrospun PAN fibers with smooth and rough surface (positive curvature): (a) SK1; (b) C. albicans ................................................. 72 Figure 33 Interactions of (a-b) SK1cells and (c-d) C. albicans on smooth(PAN) (left column) and rough PAN(GPAN) (right column) nanofibers with micrometers. .............................................. 72 Figure 34. Fungal cell growth behavior on graphene embedded electrospun PAN fibers (big diameter vs small diameter): (a) SK1; (b) C. albicans .................................................................. 73 Figure 35. Comparison of fungal cell growth behavior on electrospun PAN fibers with small size and smooth surface and electrospun PAN fibers with large size and rough surface (positive curvature): (a) SK1; (b) C. albicans .............................................................................................. 74 Figure 36. The FTIR spectra of (A) electrospun PAN/PMMA nanofibers; (B) electrospun PAN/PMMA nanofibers after chloroform treatment; (C) electrospun PAN nanofibers .............. 75 Figure 37. SEM images of PAN/PMMA nanofibers: (a-b) before chloroform treatment (top row); (c-d) after chloroform treatment (bottom row). ............................................................................ 76
xiv Figure 38. Comparing the effect of smooth ESPAN nanofibers and porous ESPAN (after removing PMMA with chloroform) nanofibers on cell growth (a) SK1 and (b) C. albicans ....... 77 Figure 39. Comparing the effect of smooth ESPAN nanofibers, ESPAN-PMMA nanofibers (before removing PMMA) and ESPAN (after removing PMMA with chloroform) nanofibers on cell growth (a) SK1 and (b) C. albicans ........................................................................................ 77 Figure 40. Interactions of (a, c, e) SK1 and (b, d, f) C. albicans cells on smooth PAN (top row), PAN-PMMA(Before treatment) (middle row) and PAN-PMMA(After treatment for removing PMMA) (bottom row) nanofibers ................................................................................................. 78 Figure 41. FTIR spectra of (A) PAN/PEO electrospun nanofibers; (B) water-treated PAN/PEO electrospun nanofibers; (C) electrospun PAN nanofibers. ........................................................... 79 Figure 42. SEM images of PAN/PEO nanofibers: (a-b) before (top row); (c-d) after treatment with 70 oC water (bottom row). .................................................................................................... 80 Figure 43. Comparing the effect of smooth ESPAN nanofibers and ESPAN (after removing PEO with 70 oC water) nanofibers on cell growth (a) SK1 and (b) C. albicans ................................... 81 Figure 44. Interactions of (a-b) SK1 and (c-d) C. albicans cells on smooth ESPAN nanofibers (left column) and porous ESPAN (after removing PEO with 70 oC water) (right porous) nanofibers. ..................................................................................................................................... 82 Figure 45. Contact angle of (a) CA nanofibers, (b) Cellulose nanofibers, and (c) PAN nanofibers ....................................................................................................................................................... 85 Figure 46. Fungal cell growth behavior on PAN and cellulose nanofibrous mat ......................... 86
xv List of Tables Table 1 Parameter for Electrospinning Cellulose Acetate Nanofibers ........................................ 28 Table 2 Parameters for Electrospinning PAN Nanofibers ............................................................ 30 Table 3 Parameter for Electrospinning Graphene Embedded PAN (GPAN) Nanofibers ............ 32 Table 4 Parameter of Electrospinning PAN/PMMA and PAN/PEO Electrospun Nanofibers ..... 34 Table 5 Average Sizes of Prepared Electrospun Nanofibers ....................................................... 43 Table 6 Processing Parameters Selected for Electrospinning of PAN Solutions at Different Concentrations .............................................................................................................................. 54 Table 7 Electrospinning Parameters for Aligned PAN Nanofibers ............................................. 56 Table 8 Percentage of Fiber Alignment from Different Spinning Time ...................................... 58 Table 9 Average Diameter of Electrospun GPAN Fibers with Submicrometer Size .................. 66 Table 10 The diameter of different composition of Graphene-PAN nanofibers mat .................. 70 Table 11 The percentage of hydrophobicity of SK1 and C. albicans at exponential and stationary stage .............................................................................................................................................. 84 Table 12 The contact angle of CA nanofibers, Cellulose nanofibers, and PAN nanofibers ......... 85
1 Abstract Electrospinning is a unique and straightforward way to prepare continuous fibers with diameters ranging from tens to hundreds of nanometers. The electrospun nanofibers exhibit unique characteristics such as high specific surface area, high porosity, easy surface modifications, and stretchability in addition to simple and cost-effective preparation. Integration of electrospun nanofibers in commercial products has been growing exponentially in recent years and electrospun nanofibers have already been exposed to our environment. The impact of electrospun nanofibers on our environment, however, has not been thoroughly addressed. Little is known about biological interaction between environmental microorganism, particularly fungal cells, and electrospun nanofibers. The primary objective of this study is to explore viability and growth behavior of two yeast cells, SK1 and Candida albicans, as model fungal cells on electrospun nanofibrous mat based on chemical composition, size, alignment, surface topological features, and surface property of electrospun nanofiber through optical density measurement, cell wall morphology, and colony forming unit (CFU). This research partly addressed concerns about the impact of nanoscale fibers on microorganism in nature and the findings are also valuable for antifungal materials.
2 1
CHAPTER 1
Introduction & Literature Review 1.1 Introduction to Microorganism Microorganisms or microbes are living organism too small to be seen with naked eye but visible under microscope. Microbes exist as unicellular, multicellular, or cell clusters. They can be divided into six major types: bacteria, archaea, fungi, protozoa, algae, and virus organisms. It is generally accepted that fungi much like bacteria can form complex protective biofilm and undoubtedly a source of pathogenesis. Some Microorganisms are widespread in nature and are beneficial to life, but some can cause serious problem. 1.2 Introduction to Yeast/Fungus Single-celled microorganisms are capable of life’s processes, independent of other cells, and have existed in earth for approximately 3.2-3.8 billion years [1][2]. Right now, thousands of tiny living things are all around us. They are close enough to touch, but they are too small to be seen. They provide an abundance of nutrients and other resources on earth. Approximately 1.5 million species in nature are known as fungi, among them about 300 species are responsible to make people sick. Many of them are parasites for animals, plants and human. Fungi are eukaryotic cell microorganisms that reproduce both sexually and asexually. Yeasts are unicellular fungi although some species are multicellular. Yeast reproduces through budding depends on the condition of environment. Some yeast species are valuable, for example, Saccharomyces cerevisiae (S. cerevisiae) is used in fermentation process. It converts carbohydrates to carbon dioxide in baking and other food processing whereas it converts carbohydrates to alcohols in the process of manufacturing alcoholic. However, some other yeast species are sources of infectious disease and therefore termed pathogenic. For example, Candida
3 is pathogenic yeast; it can affect the central nervous system in human. Pathogenic Candida albicans (C. albicans) is the fourth most frequent organism found in blood of hospitalized patient [3]. Yeast cell is also employed as a model by molecular genetic researchers because its cellular mechanism of replication, recombination, metabolism and division is the same as in large eukaryotes including mammals, plants, and protozoa. A fungus is a primitive organism. Mushrooms, mold and mildew are examples. Fungi live in air, in soil, on plants and in water. Some even live in human body. Some fungi reproduce through tiny spores in the air. People can inhale the spores or they can land on them. Most fungi are not dangerous, but some types can be harmful to health. Fungal infection becomes worse in a weakened immune system. Fungal infections occur in over a billion people each year, and recent statistics suggests the rate is increasing drastically [4]. 1.3 Infectious Disease Caused by Fungal(Yeast) Cells Fungi can infect almost any part of the body including skin, nails, respiratory tract, urogenital tract, alimentary tract, or can be systemic. Systemic Candida infections are common to immunocompromised individuals, including transplant recipients, chemotherapy patients, HIVinfected patients, and low-birth weight infants [5]. The infections caused by all species of Candida are called Candidiasis. There are over 20 species of Candida yeasts that can cause infection in humans, the most common of which is C. albicans. Candida yeasts normally reside in the intestinal tract and can be found on mucous membranes and skin without causing infection. However, overgrowth of these organisms can cause symptoms to develop. Symptoms of candidiasis depends on the area of the body that is infected. Candidiasis that develops in the mouth or throat is called “thrush” or oropharyngeal candidiasis. Candidiasis in the vagina is commonly referred to as a “yeast infection”. Invasive candidiasis occurs when Candida species
4 enter the bloodstream and spread throughout the body. The annual cost of fungal infections in the United States was estimated to be $2.6 billion, approximately 0.24 percent of total U.S. healthcare expenditures [6]. A study conducted in the United States on data from 2002 concluded that there were approximately 1.7 million Healthcare Associated Infections (HAIs) resulting in nearly 100,000 deaths [7] whereas over 250,000 cases of invasive candidiasis. According to the Center for Disease Control and Prevention (CDC), examples of such HAIs include central lineassociated bloodstream infections, catheter-associated urinary tract infections, surgical site infections, and ventilator-associated pneumonia, [8] which are a new infectious disease termed chronic polymer-associated infections [9][10]. Bloodstream infections caused by Candida are responsible for as high as 50% mortality rate among the infected patients (HAIs) are also a tremendous financial burden [11][12][13]. 1.4 Structure of Yeast Cells The size of yeast cells is 3-4 µm which is smaller than animal and plant cells, but slightly larger than bacteria. Yeast cells are egg-shaped and can only be seen with a microscope. It takes 20,000,000,000 (twenty billion) yeast cells to weigh one gram, or 1/28 of an ounce, of cake yeast. C. albicans is a diploid organism which has eight sets of chromosome pairs. Macroscopically, colonies of Candida species are cream colored to yellowish. Depending on the species, their texture may be pasty, smooth, glistening or dry, wrinkled, and dull. All species produce blastoconidia, which may be round or elongated. Most produce pseudohyphae that are long, branched, or curved. C. albicans can take on either a unicellular (yeast) or multicellular (hyphae, pseudohyphae) form. When they produce hypae based on environmental condition, their size becomes 10-12 microns across. In hypae condition, the size of C. albicans cells is about 30% greater than S. cerevisiae (baker’s yeast). Log-phase cell doubling times
5 (± standard errors) for pure cultures of C. albicans averaged 2.0 ± 0.11 h (range, 1.7 to 2.6 h) [14]. The cell wall of C. albicans maintains the structural integrity of the organism and also provides a physical contact interface with the environment. Saccharomyces has organelles such as nucleus, mitochondria, endoplasmic reticulum secretory vesicles and vacuoles. The cell wall is composed of mannoprotein and glucan and chitin on the bud scar (Figure 1). The major components of the cell wall are fibrillar polysaccharides and proteins. The proteins of the cell wall may play a role in maintaining structural integrity and in mediating adherence, whether to host or microbes, or they may have enzymatic functions, e.g., proteolysis. Additional factors that may influence these proteins are the morphology of yeast cells like pseudohyphae, and hyphae and the maintenance of either a planktonic or a sessile lifestyle [15].
Figure 1. Structure and Composition of Fungal Cell. Clearly, C. albicans has emerged as a model organism for studying fungal pathogens. It is necessary to understand the complete life style of C. albicans for developing potential antifungal drugs and tackling candida infections. Most of the fungal pathogens can primarily grow either in the form of budding yeast (e.g., Cryptococcus neoformans), or as a filamentous hyphal structure (e.g., Aspergillus spp.). However, C. albicans has a unique ability to grow at least, four kinds of forms, that is, yeast-like, hyphae, pseudohyphae, and chlamydospores have been well documented [5]. C. albicans cells are diploid (2N) and can divide asexually or can
6 undergo heterothallic or homothallic mating. Mating type-like locus a (MTLa) and MTLα cells must switch from white to opaque to become mating-competent. Opaque cells secrete pheromones that result in the formation of conjugation tubes, and subsequently, cell and nuclear fusion occur to form tetraploid (4N) cells. Homothallic mating also occurs and can be driven by loss of Bar1 protease in MTLa cells. Mating products can be induced to undergo concerted chromosome loss to return to the diploid state. Death in fungi cell could occur based on the following factors such as nutrient stress, protein folding stress, redox stress, heat stress, acid (pH) stress, and chemical substance stress. Microbes such as bacteria and fungi has more rigid compare to animal cells due to having cell walls and therefore most effective method is required to death or rupture the cells membrane [16]. S. cerevisiae contains a set of cell wall associated proteins, which contributed adhesion to diverse biotic and abiotic surfaces. Adhesion of cell to cell and cell to foreign surface is key for multicellular development, colonization and pathogenesis. Adhesive properties are predominantly conferred by specific cell surface proteins, the adhesins. The molecules in cell surface proteins that are responsible for adhesion are called adhesins, which include Als1p-Als7p and Asl9p, Hwp1p, Int1p, Mnt1p, and several others [17][18].
Figure 2. Life cycle of SK1 and C. albicans.
7 In fungi, non-pathogen S. cerevisiae and pathogen C. albicans species have shown that adhesins allow agglutination of sexual partners before cell fusion, helps to form a protective and invasive multicellular growth layer. Later it enhance adhesion to foreign biotic and abiotic surfaces as well as host cells [19]. Yeast cells are able to develop diverse multicellular growth forms named as flocs [20], flors [21], biofilms [22] and pseudohyphal filaments [23]. To date, at least eight different adhesions have been identified which include FL01, FL05, FL09, FL10, FL11, FIG2 and AGA1. These cell wall associated surface proteins are to be involved in the binding of specific ligands exists on other yeast cells or on foreign surfaces [14]. A yeast flor is available in nature in airliquid interfacial layer with floating cells that are attached to each other and form biofilm named as yeast velum or flotation. For Candida, adhesion is thought to be an essential step for colonization and establishment of candida infections. C. albicans is a very versatile pathogen and has the ability to adhere to form biofilm on variety of surfaces like endothelial cells, implanted inert materials in the host body, extracellular matrix, and epithelial cells. Biofilm starts to occur when the budding yeast cells are attached to surfaces followed by grown horizontally to form the basal layer. Then biofilms will be covered by extracellular matrix with further secretion which is mainly composed of carbohydrates and proteins. One of the most important problem of biofilm is their high-level drug resistance to different antifungal drugs [24]. Adhesion to foreign surfaces is one of the key factors that enable fungal filaments to penetrate solid substrates and to grow invasively [25]. Researchers already has shown that non adhesive diploid yeast strains are able to produce filaments by unipolar cell division, but they remain at the surface of the substrate [26].
8
Figure 3. Lifestyles of Yeast cells (a) Scheme showing the development of colonies, biofilms, filaments, flocs and flor by cell-cell (self) and cell-substrate (foreign) adhesion from sessile or planktonic cells on solid or in liquid media [19]. Yeast cell adheres to solid surfaces (nonliving surfaces) and their adhesion is influenced by the chemical composition and the topography of the solid surface. In porous surfaces, the main facts for adhesion depends on surface area, porosity, and surface roughness [27]. They could survive by adhering/penetrating on and through their host [15] and causing infectious disease. From the biological point of view, cell adhesion drives are well affected by cell biological properties such as motility, growth phase, metabolism, and the shape of the cell. In fungi cells, adhesion is influenced by the cell wall components such as proteins, lipids, and carbohydrates. These components can easily form interactions by molecular recognition, protein-lipid binding, protein-protein binding, and receptor-ligand binding. In particular hydrophobic proteins, glycoproteins, and the extracellular polysaccharides mediate adhesion in
9 ascomycetes and S. cerevisiae. These proteins stabilize cells adhesion to both natural and artificial surface [28]. The fungal cell wall is mainly composed of proteins and polycarbohydrates, which protect the inner components from outside environment, maintains morphology and also determines cell viability and pathogenicity. It is a critical site for exchange and filtration of ions and proteins. External forces can create stress on cell wall protein and carbohydrate matrix which leads a defect in its structure. Fungal cell wall composition varies among species, although in general, most cells have a common structure. Yeast cell wall (Figure1) is composed of Glucan; which are polysaccharides of glucose homopolymer (C6H12O6) linked by β-glycosidic bonds. Polysaccharides gives also mechanical strength to cell wall [29]. Besides that, the structure of cell wall provides some elasticity allowing for morphological changes during fungal growth and life cycle. In S. cerevisiae and C. albicans, glycoproteins represent in about 30–50% of the dry weight of cell wall [29]. The glycoproteins present in the cell wall are highly modified with N- and O-linked oligosaccharides, predominantly formed by mannose residues known as mannanprotein. Glycoproteins take part in several functions like the maintenance of the cellular form, transmitting signals to the cytoplasm, involved in adhesion processes, and remodeling the components of the wall. S. cerevisiae exhibit negatively charged surfaces due to the phosphorylation of the mannosyl side chains exists in the outer cell wall in the range of (19.6-1207) mV at pH of 2.5-7.0 [30]. The carboxylate group is also available in cell wall become a another evidence of negative surface charge [31]. During cell growth, fungal cells synthesizes its wall components, then the components exported across the plasma membrane, and make a new cell outside the cell.
10 1.5 Electrospinning Technique Electrospinning is the unique technique to produce nanofibers with diameters in the range between few nanometers and submicrons. Since early 1990s, electrospinning has attracted attention in scientific community as well as in industry. Electrospinning offers opportunities of technology transfer [32][33], economic development, and ultimately, employment [34]. Furthermore, the number of publications about electrospinning has been increasing exponentially every year (Figure 4) [35]. 2000
No. of Publications
1750 1500 1250 1000 750 500 250 0 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017
Year
Figure 4. Number of publications per year (2006–2016) by using electrospinning nanofibers as keyword for literature search Data source, Science Direct. The importance of electrospun nanofibers in the area of biomedical engineering is the ability to fabricate nanostructure of any kind of raw materials ranging from biocompatible and biodegradable or natural to synthetic polymers. Nanofiber scaffolds enhance better attachment, proliferation, and differentiation between cells and scaffolds [36][37]. Similarly electrospun nanofibers are also used as a drug delivery carrier to targeted sites [38]. Although there has been
11 a lot of research on the interactions between nanofibers and eukaryotic cells [31] while there has been very little research focused on the interaction of micro-organisms with nanofibers. a
b
Figure 5. (a) Electrospinning setup[39]; (b) Diagram showing onset and development of bending instabilities. Reprinted with permission from Reneker and Fong[40].Copyright 2005, American Chemical Society. The principle of electrospinning is simple; however, it is necessary to control the process as several variables have an influence on the properties of the end product. As shown in Figure. 5, a typical electrospinning setup has four essential parts: a high voltage supply, a dope driven system (a syringe pump), a spinneret and a grounded metal collector [41]. At first, in the electrospinning process, an electrical potential difference is applied between a polymer solution droplet at the tip of a spinneret and a grounded collector [42]. Once a polymer solution droplet is formed at the tip of the spinneret (which is essentially a hollow or gauge-type needle), voltage applies and causes surface charge buildup on surface of the polymer solution droplet. Then the shape of the droplet is gradually transformed into a conical shape named Taylor Cone from which a jet emanates [43]. This Taylor cone can be maintained until all of the spinning solution flowing out of the tip. The force required to initiate electrospinning is described by the following formula (Eq.1):
12
…………………(1)
[44]
The critical voltage, required to generate perfect nanofibers from a given spinning solution, is determined by the surface tension of solution, spinneret radius, and distance between spinneret tip and a grounded collector [45]. During the process of elongation and bending, the solvent in nanofibers evaporates simultaneously, resulting in solidification of nanofibers [46]. The typical instability occurring in the electrospinning process is named bending (or whipping) instability [47]: the primary jet is divided into multiple sub jets, inducing a progressive diameter (defined according to Eq. (2)[48][49]) reduction of the jet from micrometer to nanometer, as sketched in the Fig. 5(b), taken from the literature [50].
--------------------------(2) Where ε is the permittivity of the fluid (in C/V cm), ṁ0 is the mass flow rate (g/s) when r0 (cm) is calculated, κ is a dimensionless parameter related with the electric currents, σ is electric conductivity (A/V cm), and ρ is the density (g/cm3) of the electrospun material. 1.6 Applications of Electrospun Nanofibers in Biomedical Field In biomedical field, it is now established that almost all tissues and organs such as skin, collagen, dentin, cartilage and bone have some sort of resemblance to highly organized, hierarchical, nano size fibrous structures. There are many research that highlight the importance of the biomedical applications of electrospun nanofibers [51][52]. Because of their unique properties, electrospun nanofibers are considered as promising scaffold materials. Furthermore,
13 nanofibrous scaffolds have shown enhanced cell adhesion, stimulated cell growth, protein adsorption, and assisted in cell differentiation [53][54][55]. 1.6.1 Tissue engineering One of the major developments behind tissue engineering is the scaffold formation. In past decade, electrospun nanofiber systems have been tested for preparation of scaffolds for tissue engineering [56]. For the regeneration of tissue, biocompatible and biodegradable fibrous scaffolds are generally preferred over conventional scaffolds. High surface area and short diffusion passage length of nanofibers increased their use as scaffold material by higher rate of drug release than the bulk material. The electrospun scaffolds have some unique nature and ability to provide the target cells/tissues with a native environment by mimicking the extracellular matrix. Fibrous scaffolds has impact on the cell-to-cell interaction along with the interaction between the cells and matrix [57] which promotes an excellent cell growing capability [58]. Furthermore, until recently researchers have mainly focused on bio/natural polymers (hyaluronic acid, alginate, collagen, silk protein, fibrinogen, chitosan, starch, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) for tissue engineering, because these polymers showed excellent biocompatibility and biodegradability [59][60][61]. Now more research has been focused on a wide range of natural and synthetic polymers for the regeneration of new tissues, specifically cartilage tissue [62], dermal tissue [63], and bones [64]. Among the synthetic polymers, poly(lactic acid-coglycolic acid) (PLGA) is considered to be an ideal material for tissue regeneration because of its tunable and biodegradable nature, easy spinnability, and the presence of multiple focal adhesion points. However, there are some limitations in the use of electrospun nanofiber scaffolds in tissue engineering. One such hurdle is the infiltration of the cells inside the scaffolds because of the smaller intra-fiber pore size. In
14 order to overcome this problem, 3D scaffolds with a larger intra-fiber pore size have been made to provide a 3D environment instead of a 2D environment. As compare to conventional 2D electrospun scaffold, 3D scaffolds have more exposed inner surface area and pore size, and therefore show enhanced infiltration of cell. Literature shows that cells migrated approximately up to 4 mm and exhibited a spatial cell distribution. Therefore 3D electrospun scaffolds are important in tissue engineering applications such as nerve regeneration, vascular grafts, and bone regeneration [41].
Figure 6. Schematic diagram of tissue engineering and several kinds of tissue engineering such as skin, bone, vascular, nerve and cartilage tissue engineering[65]. 1.6.2 Wound dressing material The two essential requirements of wound dressings include rapid hemostasis property and good antibacterial property to prevent infections from surrounding bacteria. Conventional wound dressing materials include hydrocolloids, hydrogels and alginate salts [66]. Recently, silver nanoparticles are reported to exhibit efficient antimicrobial potential for better curing.
15 Electrospinning has attracted much interest for its versatility to fabricate nanofibrous membranes for wound dressing which can create a moist environment around the wound area to promote healing. Wound dressing materials should have properties which provide easy gaseous exchange, absorbing exudates from the wound site and providing a sterile environment which does not support microbial growth [67]. Li et al. have reported thermosensitive nanofibers loaded with ciprofloxacin as antibacterial wound dressing materials. Ciprofloxacin (broad spectrum antibiotic) was loaded in thermo-responsive electrospun fiber mats containing poly(di(ethylene glycol) methyl ether methacrylate). By virtue of their thermal sensitivity, fibers could promote the proliferation of fibroblasts, and by varying the temperature, cells could easily be attached to and detached from the fibers. In vivo investigations on rats indicated that the forementioned nanofibers to have much more potent wound healing properties than commercial gauze. In 2014, the work published by II Keun Kwon et al. has well demonstrated that the silver nanoparticles containing chitosan (CS) nanofibers were prepared using electrospinning technique. The prepared nanofibers showed chelate ion between silver and amine group of chitosan. CS nanofibers were tested against Pseudomonas aeruginosa (P. aeruginosa) and Methicillinresistant Stapylococcus aureus (S. aureus) (MRSA) which revealed that nanofibers are more effective on P. aeruginosa than MRSA [68]. This study demonstrated that the chitosan was inactive against the tested microorganisms, wherein silver nanoparticles loaded chitosan nanofibers exhibited significant activity by restricting the respiration of microorganism due to attraction of positively charged silver nanoparticles on the negatively charged cell membrane.
16
Figure 7. Wound dressing by electrospinning, Carpinteria, C.A. (Spotlight on wound-care technologies. MPMN. 26(5), (2010) [69]). Chitosan (CS) is a natural polymer with both antimicrobial activity and nanofiberforming capability. Researchers blended CS aqueous salt with polyvinyl alcohol (PVA) nanofibre mats through electrospinning. CS was dissolved with hydroxybenzotriazole (HOBt), thiamine pyrophosphate (TPP) and ethylene diamine tetra acetic acid (EDTA) in distilled water without the use of toxic or hazardous solvents. These mats exhibited nontoxic to normal human fibroblast cells and antibacterial activity against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The wound healing activity of CS-EDTA/PVA electrospun nanofiber mats was better than gauze in reducing acute wound size during the 1st week after treatment. The neat PVA nanofiber mats did not show any effect on bacteria growth. The results showed that the different antibacterial activities of the CS/PVA nanofiber mats were dependent on the type of CS salt used [70]. This mechanism consists of the interaction between the positively charged chitosan chains (due to protonation of amino groups in acidic media) with the negatively charged components of cell membranes, causing an imbalance of membrane permeability [71]. Among
17 them, the CS-EDTA/PVA nanofiber mats showed the highest antibacterial activity because of the intrinsic antibacterial activity of EDTA. Several recent publications have indicated that EDTA (alone and in combination with antibiotics) was an effective antimicrobial agent. The combination of CS acetic acid and EDTA elicited synergistic activity against S. aureus [72]. 1.6.3 Drug delivery In the early 1970s, the concept of drug delivery system was proposed [73]. The key function of designing drug delivery system is to transport various drugs to the target sites in the body in a secure way and adjust the release mechanisms by controlling the amount of drugs and treatment time [74]. Recently electrospun nanofibers have gained huge attention as new drug delivery platforms based on the increase in dissolution rates of drug due to a tremendous increase in surface area which can act as a nano cargo carrier [65][75]. The easy modifications of electrospinning nanofibers help to control the release rate owing to stimuli-sensitive nature of some polymers. The release properties of selective materials (polymers) are based on either only diffusion controlled, or diffusion controlled along with nanofiber degradation. Drug release is not solely governed by diameter and effect of porosity is to be considered at the same time. It is often shown that thicker nanofibers with very high porosity release drug faster as compared to thinner fibers with low porosity. Nanofiber alignment is another parameter known to affect drug release and in general randomized pattern is associated with faster drug release because of increased tendency of water uptake [76]. Besides this, various incorporation techniques (direct or indirect) can be used to load drug onto nanofibers. In vitro study was done by Zong et al. that an antibiotic drug loaded into nanofibrous mats[77]. Recently, several new drugs are designed to cure cancer disease efficiently. Conventional cancer treatments have many limitations such as clinical toxicity in radiotherapy, toxicity to
18 healthy cells for overdose of drugs in chemotherapy and limited distribution of drugs in blood vessels. For this reason, nanofibers extend its role in cancer therapy as easier drug carrier and control release material. Until now, many kinds of drugs, including anticancer agents, proteins, antibiotics, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), have been loaded on electrospun nanofibers [78]. Shaobing Zhou and co-workers developed a new device with coreshell nanofibers as implant material for safe and effective cancer treatment. They describe that the folate-conjugated PEG/PCL copolymer layered micelle with drug (doxorubicin) loaded nanofibers prepared by co-axial electrospinning technique. Core and shell of the nanofibers were micelle/PVA and gelatin solution respectively. These nanofibrous mats with the micelle examined for therapeutic action revealed efficient killing of tumor cells with minimal loading of drug and reduced frequency of drug administration, and thereby improved survival of affected people [79]. Wang and his coworkers also reported the encapsulation of multiple drugs within a biodegradable membrane, where two bioactive drugs were successfully incorporated into this bilayer membrane and can be independently released from nanofibrous scaffolds without losing structural integrity and functionality of the anti-adhesion membrane [80]. Though many advantages are associated with core-shell model, it suffers from limitations such as barrier to cross the blood vessels, limited solubility and nonspecific uptake of the drug. Apart from this, drug loading in polymer solution may adversely affect its viscosity and surface tension rendering it unsuitable for electrospinning [81]. Alavarse et al. have reported tetracycline hydrochlorideloaded electrospun nanofiber mats based on PVA and chitosan for wound dressing. The majority drug delivery was happened in the first 2h to allow an effective antibacterial activity on the Gram-negative E. coli as well as on the Gram-positive Staphylococci epidermidis (S. epidermidis) and S. aureus [82]. Although nanofibers have shown great potential for drug
19 delivery applications, there are still certain challenges that need to be circumvented before electrospun nanofibers can be used as drug delivery vehicle. Till date certain major factors, including drug loading, the stability of active ingredients, initial burst release, the amount of residual solvents and industrial scale-up are crucial bottlenecks, which needs to be overcome before bringing this technology into mainstream drug delivery technologies. 1.6.4 Nanofibers with antibacterial activity To date numerous types of electrospun hybrid nanofiber scaffolds with antimicrobial effect have been fabricated by various research groups. There are different methods used to incorporate active agents in the nanofibers including active agent blending with polymer solution before electrospinning, fabricating core/shell structure through coaxial electrospinning, encapsulating active agent before mixing them with electrospinning solution, post-treatment of fiber after electrospinning to convert a precursor to its active form, or attaching the fiber surface with active agent. Common antibacterial materials such as antibiotics, triclosan, chlorhexidine, quaternary ammonium compounds (QACs), biguanides, silver nanoparticles, and metal oxide nanoparticles have been reported to be used in fabricating antibacterial electrospun nanofibers. Silver is toxic to bacteria because of its affinity to proteins and nucleic acids, but non-toxic to human cells [83]. The nanoparticle form of silver is more prone than the bulk material to exhibit novel materials properties. Silver nanoparticles have been extensively studied with regard to their unique physical and chemical properties. Nanoparticles could be more efficiently formed to deliver themselves to target organisms [84]. For instance, researchers fabricated PAN/Ag composite nanofiber scaffolds for their possible antimicrobial effect. The results showed that the capability of PAN/sliver (Ag) nanofiber scaffolds to inhibit both Gram-positive (Bacillus cereus) (B. cereus) and Gram-negative (E. coli) bacterial growth. The antimicrobial effect of PAN
20 nanofiber scaffolds was further assessed by immobilizing amidoxime (having antimicrobial effect) onto PAN nanofiber scaffolds. This was evident from the fact that it completely killed the E. coli and S. aureus bacterial strains. The possible mechanism behind the killing of those bacterial strains was the binding ability of amidoxime to magnesium (Mg2+) and calcium (Ca2+) ions, which are very essential for bacterial survival. The binding of these metals to the membrane with amidoxime rather than bacterial cells disturbed the balance, which therefore rupture the cell walls resulting their ultimate death [85]. Another research group has revealed silver nanoparticle-embedded polyvinyl alcohol (PVA) nanofibers as an antimicrobial mat and surface enhanced Raman scattering (SERS) substrate [86]. The nanofibers have showed good antibacterial activities against both Grampositive S. aureus and Gram-negative E. coli microorganisms. In another work, antibacterial polyethylenimine (PEI) (10, 20 and 30% (w/w)) was blended with silk fibroin (SF) and mats were fabricated by electrospinning [87]. In addition, SF nanofibers were also functionalized with sulphate groups to evaluate the antibacterial activity. PEI/fibroin bionanotextiles showed strong antibacterial activities against Gram-positive S. aureus and Gram-negative P. aeruginosa. 1.6.5. Microbes interaction with nanostructured surfaces Recently several studies have evaluated the alternative physical methods through the contact killing mechanism is the best for microorganism over chemical killing methods. When microbe cell adheres to the biological nanostructured surfaces such as insect wings, dragon fly wing, gecko skin, lotus leaves, the nanopatterns on the wings can rupture cell membrane and wall of that cell. The bactericidal activity of a nanostructured surface depends on several parameters such as size, shape and spacing/density of the nanostructures. Nowlin et al. [88] and Kelleher et al. [89] reported the eukaryotic and prokaryotic microorganism adhesion respectively
21 on different types of cicada and dragonfly wings with different nanopillar height to width (h/w) ratios. The nanopillars began to penetrate the cells immediately upon cell attachment, killing most of them within 5 mins. It was thus suggested that allowing bacteria to adhere to the nanostructured surface and killing them physically could be a more effective strategy for antibacterial surface design than repelling the bacteria from the surface [90]. The strength of adhesion between the bacteria and the nanostructured surface is a vital element in the nanostructures induced rupturing of the microbes. Meanwhile adhesion of the bacteria with the nanostructured surface depends on the hydrophobicity/hydrophilicity of the surface and the cell membrane composition. When bacteria try to settle on the nanostructured surfaces, the multiple contact points increase. In this process of stretching, the cell wall reaches a threshold limit of strain acting on it followed by cell wall rupturing. Surface hydrophobicity or superhydrophobicity is more critical in water-immersed conditions (entailing air entrapment) than in air. To mimic dragonfly wing, Ivanova et al. [91] developed black silicon surfaces using reactive ion etching of silicon. Unlike cicada wing, the nanostructures present on the dragonfly wing are randomly distributed in terms of shape, size and distribution (Fig. 3D) and show a sigmoidal distribution below 90 nm. The nanopillar diameters on the black silicon surface showed a bimodal distribution spanning 20–80 nm. Such nanotextured black silicon could effectively kill minimum infective doses of S. aureus and P. aeruginosa in very short time. Some of research has done by making electrospun nanofibers loaded with novel antimicrobial agents. Quirós et al. made electrospun polyvinylpyrrolidone (PVP) nanofibers containing silver, copper, and zinc nanoparticles from their salts. High molecular weight PVP formed uniform fibers with a narrow distribution of diameters around 500 nm. They converted
22 the fibers into an insoluble network using ultraviolet irradiation crosslinking. The efficiency of metal-loaded mats against the bacteria E. coli and S. aureus was tested. All metal-loaded fibers displayed antimicrobial effect, among them silver loaded fibers had shown a strong inhibition ageing cell growth. Functionalized electrospun nanofibers have demonstrated very good antimicrobial property. Zhang and his group [85] used polyacrylonitrile (PAN) electrospun nanofiber membrane to grow E. coli and S. aureus. Their membrane was treated with a hydroxylamine (NH2OH) aqueous solution to form amidoxime nanofiber membrane, then the membrane immersed in AgNO3 aqueous solution to coordinate Ag+ ions on the membrane surface. The surface modified nanofibrous membrane showed good antimicrobial activity. The membranes with Ag+ and Ag nanoparticles had highly antimicrobial capability (E. coli and S. aureus) than that of amidoxime surface modified membrane. 1.6.6 Other applications of electrospun nanofibers Electrospun nanofibers also demonstrated their uses in polymer composite materials. They reinforces polymer composite materials due to their special properties such as irregular pore structure, dimensionally stability, mechanical interlocking and large specific surface area, which promotes stiffness and strength [92]. The best idea to design advanced and innovative fiber reinforced polymer (FRP) composites is incorporation of reinforcing nanofibers into traditional micro-sized fibers or replace the traditional fibers. This combination usually enhance physical and chemical properties, as well as mechanical performances, of the polymer matrix [93][94][95][96].
23
Figure 8. Composite laminate interleaved by nanofibrous mats at plies interface [93]. The aviation industry is much interested in using nanofibers-incorporated composites for light and strong materials. It also uses electrospun nanofibers to reduce noise pollutant. The aviation industry is currently using traditional materials such as perforated panel, foam, and fibers for noise reduction with limited results. In 2009, Researchers at Wichita State University have discovered that electrospun nanofibrous materials displayed significant enhanced noise absorption coefficients at the nanoscale. The sound absorption coefficient of electrospun fibers is about 100%, even at high frequencies of sound (7,000 Hz). The sound absorption coefficient can be further improved by emerging carbon nanotubes. The idea is individual nanotubes oscillate with sound waves which help to absorb more sound energy [97].
24 Molnar et al. studied thermal and electrical properties of epoxy composites reinforced with carbonized electrospun nanofiber mats. They embedder carbon nanotubes into the composite structure to reduce the mass of lightning-protective layers of composite aircraft structures. Thermal conductivity of the samples has increased by approximately three times with inclusion of MWCNTs in the carbonized nanofibers. The thermal conductivity of the carbonizednanofiber-reinforced composites is almost twice of the composites with standard carbon fibers [98]. The polymer nanocomposite made with functionalized graphene platelets also offer excellent electrical and thermal conductivity [99][100]. 1.7 Objectives and Scope of Research Microbes, both pathogenic and nonpathogenic, impact human lives everyday intimately. Our bodies are hosts to trillions of microbes. In this sense, we are metagenomic that our genome is a composite that consists of Homo sapiens genes along with our residing microbes [101]. Both pathogenic and nonpathogenic microbial exist in environment from various sources like air, insects and the hydrogel cycle. Pathogen is evolving and now provides defense against metal toxicity, dehydration, salinity, phagocytosis as well as antimicrobial agents. It is time to find out new route to treat against pathogenic microorganism without adding any kind of antimicrobial agent. Very little research has been reported regarding the interaction between fungal cells and nanostructure surfaces. It is still largely unknown area. Particularly biological interaction between fungal cells and electrospun nanofibers has never been thoroughly addressed even though the application of electrospun nanofibers has been growing exponentially in recent years. The primary objective of this study is to explore fungal cell viability and growth behavior on electrospun nanofibrous mat and understand the interaction mechanism. Model electrospun nanofibers including polyacrylonitrile (PAN), cellulose acetate, and cellulose are chosen based
25 on their chemical structure and surface property. Model fungal cells including two types of yeast, S. cerevisiae (nonpathogen) and C. albicans (pathogen), were selected. S. cerevisiae is employed as a model by molecular genetic researchers because its cellular mechanism of replication, recombination, metabolism and division is the same as in large eukaryotes including mammals [102]. Cerevisiae yeast culture is a simple, safe and rapid process. It occurs in solid or liquid media. The most common used media is yeast extract, peptone, and dextrose (YPD). YPD is the source of amino acids, nucleotide precursors, vitamins, and the metabolites needed for cell growth. C. albicans is an opportunistic human fungal pathogen that causes candidiasis. C. albicans is the most abundant and significant species in human beings. Moreover, C. albicans possesses most of the characteristics of S. cerevisiae and more than 80% genes are similar in both the organisms. It acts as a model for studying fungal pathogens. The medium used for C. albicans is SBD (Sabouraud Dextrose Agar). We can handle this cell in laboratory without any problem. The cell viability and growth behavior on electrospun nanofibers based on chemical composition, size, alignment, surface topological features, and surface property were thoroughly investigated through optical density measurement, cell wall morphology, and colony forming unit (CFU).
26 2 3
CHAPTER 2
Research Design and Methodology
2.1 Research Goals and Outline This project aims to (i) find out whether different electrospun nanofibers have same effect specifically inhibition effect on growth of different yeast species; (ii) check how nanofibers surface morphology affects yeast cell growth; (iii) explore mechanism and realistic application. To achieve the research goals, the statement of work is divided into three segments: Phase I: Nanofibers synthesis: ➢ Task 1: Make cellulose acetate, cellulose and polyacrylonitrile(PAN) nanofibers and their corresponding film and also some bicomponent nanofibers ➢ Task 2: Modify the fiber diameter in size ➢ Task 3: Change the topography of electrospun nanofibers Phase II: Characterization of nanofibers ➢ Task 1: Perform characterization tests to understand nanofibers’ physical properties and chemical composition Phase III: Study cell interaction on nanofibers ➢ Task 1: Cell culture (SK1 and C. albicans) ➢ Task 2: Interact the cells with different nanostructured surfaces ➢ Task 3: Perform tests to understand the mechanism of cell interaction on nanofibers
27 2.2 Materials and Methods 2.2.1 Cellulose acetate (CA) & cellulose nanofibers and film 2.2.1.1 Materials Cellulose acetate, CA (39.8% acetyl content, with the number average molecular weight (Mn) of ∼30,000 by GPS (catalog number: 180955), Tetrahydrofuran (THF), Dimethyl Sulfoxide (DMSO), and Sodium Citrate (SC), Sodium Hydroxide (NaOH), and distilled water (DI) were purchased from Sigma-Aldrich. THF and DMSO were used as solvent for electrospinning of CA nanofibers, while NaOH were utilized for deacetylation of CA. All the chemicals were used without further purification 2.2.1.2 Methods A spinning solution of 20 wt.% CA was prepared using THF: DMSO (1:1) solvents and stirred for overnight using magnetic stirrer in order to prepare homogenous solution. Then the solution was filled into a plastic syringe having an 18 gauge 90o blunt end stainless steel needle (inner diameter 1.02 mm). A positive electrode was clamped on the needle and connected to a power supply (PS/FX3OP1O OGE9, Glassman High Voltage Inc, High bridge, New Jersey, USA). The grounded counter electrode was connected to a laboratory produced roller with a diameter of 10 inch. The voltage applied was 22 KV and the distance between needle tip and collector was 15 cm. The flow rate of 1.2 ml/hr was maintained by a syringe pump (NE-4000, New Era Pump Systems Inc., NY, USA). Cellulose acetate nanofibers were collected as the randomly overlaid felt on the electrically grounded aluminum foil that covered the roller for 12 hrs. The rotational speed of the roller during electrospinning was set at 300 rpm. The nanofibers were then dried in air for overnight. CA film was made by casting solution onto a Teflon plate followed by being dried in a fume hood at room temperature.
28 The as-electrospun cellulose acetate nanofiber mats were first hydrolyzed/deacetylated by being immersed in a 0.05 M NaOH aqueous solution for 24 h (Figure 9). The products (regenerated cellulose nanofiber mats) were then rinsed in distilled water for three times and dried in fume at room temperature. Cellulose film was carried out by treating CA film with sodium hydroxide aqueous solution for overnight followed by being washed and dried at room temperature [103].
Figure 9. The schematic representation of deacetylation of CA nanofibers is given in the following figure [104]. Table 1 Parameter for Electrospinning Cellulose Acetate Nanofibers Nanofibers
Applied
Flow Rate
Voltage 20%
22KV
1.2 ml/hr
Tip to collector
Types of Collector
Spinning
distance
and speed
Duration
20 cm
Drum, 300 rpm
12 hrs
Cellulose Acetate
2.2.2 PAN nanofibers and film 2.2.2.1 Materials Polyacrylonitrile (PAN, Mw = 150 kDa), N, N-Dimethylformamide (DMF), poly(ethyleneoxide) (PEO, Mn = 10 kDa) graphene platelets, sodium phosphate dibasic
29 heptahydrate(Na2HPO4.7H2O), and potassium phosphate monobasic (KH2PO4.H2O) were purchased from Sigma-Aldrich. Phosphate buffered saline (PBS) is prepared by mixing 1 M Na2HPO4 and 1 M KH2PO4 aqueous solutions to make a pH of 7.4. 2.2.2.2 Methods 10 wt.% and 12 wt.% PAN DMF solutions were prepared for electrospinning. The electrospinning of was performed at a voltage of 15 KV and feeding rate of 1.0 ml/hr. The nanofibers were collected on a metal collector wrapped with aluminum foil and kept at a fixed distance of 20 cm away from the needle tip of the spinneret. The nanofibers were then dried in air for overnight. The as-spun mat was rinsed three times with DI water and dried at room temperature. PAN film was obtained by casting the spin dope onto a Teflon plate followed by being dried in a fume hood at room temperature. Each nanofiber was washed 3x with DI water prior to biological experiments. Aligned electrospun PAN Nanofibers were prepared by electrospinning 10 wt.% PAN DMF solution. Herein an in-house-build collector was used to collect align nanofibers. 100% alignment is a big challenge in electrospinning, whereas a lot of factors need to be considered. Therefore, different spinning times were applied to get maximally aligned PAN nanofibers.
30
Figure 10. Aligned Electrospun Nanofibers Setup Table 2 Parameters for Electrospinning PAN Nanofibers Nanofibers
Random
PAN
Applied
Flow
Tip to
Types of
Spinning
Concentration Voltage
Rate
Collector
Collector and
Duration
Distance
Speed
10 wt.%
15KV
1.0 ml/hr
20cm
Drum, 300rpm
12 hrs
12 wt.%
15KV
1.0 ml/hr
20 cm
Drum, 300 rpm
12 hrs
10 wt.%
20KV
1.5 ml/hr
15 cm
4 small
3 hrs
cylindrical rotor,
4 hrs
1500 rpm
5 hrs
PAN Random PAN Aligned PAN
6 hrs
31
2.2.3 PAN nanofibers with graphene 2.2.3.1 Materials Polyacrylonitrile (PAN, Mw~ 150,000) powder and N, N-dimethylformamide (DMF, 99%) were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). As-grown, highly graphitic Graphene Nanoplatelets(GnP) were supplied by XG Sciences, Inc. (Lansing, MI, USA). All the chemicals were used as received without further purification or modification. 2.2.3.2 Methods The purpose of this part is to generate rough surface on nanofibers. Graphene-beaded PAN (G/PAN) nanofibers were produced by electrospinning a solution consisting of graphene nanosheets and PAN in DMF. PAN powder was first dissolved in DMF to prepare clear PAN solution at 70 oC. Then graphene nanosheets were well dispersed in DMF by sonication for 30 seconds[105][106]. After that, both solution was mixed to achieve the concentrations of 10 wt.% PAN and 2 wt.% graphene nanosheets in DMF. The solution was kept at a temperature of 70 oC and under continuous stirring on a hotplate for 24 h. During the electrospinning process, the asprepared solution was placed into a 30-ml plastic syringe installed with a stainless spinneret, which was connected to a positive high-voltage DC power supply (PS/FX3OP1O OGE9, Glassman High Voltage Inc, High bridge, New Jersey, USA). A laboratory-made rotary aluminum disk with the diameter of 33 cm was electrically grounded and used as the nanofiber collector. A drum collector rotating at a speed of 300 rpm was placed between them. The applied voltage and flow rate were fixed to 20 kV and 1.1 or 1.5 mL h−1, respectively through a digital flow controller. The distance between the tip of the needle (18 G) and the collector (rotating
32 drum, 300 rpm) was 20 cm. After electrospinning, the gained nonwoven G/PAN nanofiber mats were peeled off the aluminum foil on the rotary disk. Table 3 Parameter for Electrospinning Graphene Embedded PAN (GPAN) Nanofibers Nanofibers
Applied
Flow Rate
Voltage
Tip to
Types of
Spinning
collector
Collector Duration
distance
and speed
Without G
15KV
1.0 ml/hr
20 cm
Smaller
Drum,
12hrs
300 rpm
Diameter
25 wt % of
(GPAN)
PAN Without G
20KV
1.5ml/hr
20cm
Drum,
12hrs
300 rpm 15KV
1.0 ml/hr
20 cm
Bigger
Drum,
12hrs
300 rpm
Diameter
20 wt % of
(GPAN)
PAN
20KV
1.5ml/hr
20cm
Drum,
12hrs
300 rpm
2.2.4 Bicomponent polymers 2.2.4.1 PAN-PMMA nanofibers 2.2.4.1.1 Materials Polyacrylonitrile (PAN, Mw = 150 kDa), and poly(methyl methacrylate) (PMMA, Mw = 120 kDa by GPS) were purchased from Aldrich Chemical Company, Inc. N,N-
33 dimethylformamide (DMF) was purchased from EMD Chemicals, Inc. All materials were used as received. 2.2.4.1.2 Methods 14 wt.% of PAN and PMMA were prepared in DMF under constant stirring. Electrospinning of these solutions was conducted at 15 kV with high voltage power supplier (PS/FX3OP1O OGE9, Glassman High Voltage Inc, High bridge, New Jersey, USA) at ambient temperature. The fibers were collected on an aluminium foil collector at 20 cm distance. The obtained fibrous mats were vacuum dried for at least 24 h and detached. PMMA in the electrospun bicomponent fibers were removed by being immersed in chloroform at ambient temperature for 1 h. The treated fibrous mats were then dried and kept under vacuum for further analysis. The mass change of dried fibrous mats before and after solvent immersions was monitored to confirm morphological change. 2.2.4.2 PAN-PEO nanofibers 2.2.4.2.1 Materials Polyacrylonitrile (PAN, Mw = 150 kDa) and Poly(ethylene glycol) (PEG, Mn=10,000 flakes) were purchased from Aldrich Chemical Company, Inc. N,N-dimethylformamide (DMF) was purchased from EMD Chemicals, Inc. All materials were used as received. 2.2.4.2.2 Methods A 14 wt.% spinning solution of PAN and PEO mixtures were prepared by dissolving both at 50/50 mass ratios in DMF at 80 oC under constant stirring. Ultrafine fibers were electrospun from these solutions at 15 kV with a power supply (PS/FX3OP1O OGE9, Glassman High Voltage Inc, High bridge, New Jersey, USA) and collected on a grounded aluminum foil covered target 8 inches from the orifice at 300 rpm. The flow rate was 1 ml/hr. The obtained fibrous mats
34 were vacuumed for at least 24 h to remove the remaining solvent, then detached from collector and immersed in 70 oC deionized water for 10 mins to remove PEO. The treated nanofibrous mats were then dried at 70oC for at least 4 h and kept under vacuum for further analysis. The weight change of nanofibrous mat before and after water treatment was monitored. Selective removal of PEO by dissolution in water (70 oC for 10 min) led to nanoporous fibers with slightly enlarged sizes, potential precursors for new carbon nanofibers. Table 4 Parameter of Electrospinning PAN/PMMA and PAN/PEO Electrospun Nanofibers Nanofibers wt %(w/w)
PAN-PEO
PAN(7%)
Applied Flow Rate Tip to
Types of
Spinning
Voltage
collector
Collector and
Duration
distance
speed
20 cm
Drum, 300
15KV
1.0 ml/hr
PEO(7%) PAN-
PAN (7%)
PMMA
PMMA
12hrs
rpm 15KV
1.0 ml/hr
20 cm
Drum, 300
12hrs
rpm
(7%)
2.3 Yeast Strain C. albicans and SK1 yeast cells were obtained from American Type Culture Collection (ATCC). 2.3.1 Yeast cell culture and growth on surface of nanofibers mat and solid film S. cerevisiae yeast strains used in this study were SK1 (ATCC stock number, 204722; genotype MATa/MATalpha HO can1(r) gal2 cup(s)), and C. albicans (ATCC stock number,
35 10231; genotype: MATa ade2-1 ura3-1 his3-11 trp1-1 leu2-3 leu2-112 can1-100). Frozen stocks were maintained at -80 oC. For experiment, 5 days old colonies from freshly streaked YPD plates were used. Viability counts of each of 5 days SK1 and C. albicans colony ~0.43x107 cells/mL and 0.125x107 cells/mL were made respectively using serial dilution plating. Liquid YPD (Yeast extract, Peptone, Dextrose, water) media was inoculated with one colony per 10 mL. Liquid SBD (Sabouroud GC) media used for C. albicans cells. Liquid cultures were performed overnight with shaking ~~ 140 rpm at 30 oC to an OD600 ~~ 1.5 and 2.7 for SK1 and C. albicans respectively at which point fresh cultures were spiked to an OD600 (0.2) and incubated with shaking for 3-5 hrs at 30 oC to an OD600 ~ (0.4-0.6) indicative of mid-log phase growth [107]. OD600 measurements were made using a Thermo Scientific NANODROP 2000C spectrophotometer. Viability counts of ~1.14x107 cells/mL and 1.74x107 cells/mL of SK1 and C. albicans were respectively made using serial dilution plating to maintain that mid-log phase cells. 2.4 Characterization 2.4.1 Characterization of nanofibers To understand the nanofibers physical property, the following instruments were used 2.4.1.1 Contact angle measurements Contact angle measurements were performed using the sessile drop technique by Ramehart Model 500 Advanced Goniometer with Drop Image Advanced Software. The system is equipped with a CCD camera connected to a computer and to an automatic liquid dispenser. The contact angle was determined by placing a 5 µL drop of water on the film or nanofiber mat surface using a syringe and images were immediately sent via the CCD camera to the computer for analysis. The results represent an average angle between the right and left angles. Ten
36 consecutive measurements were made at room temperature, using the Surface Energy software mode, which allows direct measurement of contact angle (in degrees). 2.4.1.2 Fourier Transform Infrared Spectroscopy (FTIR) Fourier transform infrared spectroscopy (FTIR) is a technique which is used to obtain an infrared spectrum of absorption or transmission of a solid, liquid or gas. a Varian 600 FTIR spectrometer (Agilent Technologies) was used to perform FTIR test and get to know about the functional groups of nanofibers and films 2.4.1.3 Scanning Electron Microscopy & Energy Dispersive Spectrometer Scanning Electron Microscopy (SEM) was used to characterize nanofiber morphology and get diameters of nanofibers. Statistical analysis was performed to get average fiber diameters by measuring at least 50 individual fibers from each sample. Before SEM imaging, all surfaces were sputter-coated with gold to avoid accumulation of charge. The elemental composition of the samples was analyzed by an energy-dispersive spectrometer EDX that is attached to SEM instrument. 2.4.2 Characterization of cells 2.4.2.1 Optical Density A spectrophotometer is used to measure the concentration of cells in a suspension at 600 nm (OD600). The higher the concentration of cells in a liquid culture, the higher the optical density of that culture when measured. In this project, the growth behavior of yeast cells on different nanofibrous materials were measured using spectrophotometer. To find out the inhibition effect of cells on different nanofibers, 5-days old single colony are incubated into 10 ml medium for 16 hours with different mass of nanofibrous material at 25 oC.
37 2.4.2.2 Colony Forming Units(CFU) CFU is used to measure the viability of microorganism cells in a sample. It counts the number of viable fungal cells in a sample per mL after certain incubation time and presented as CFU/ml. The CFU/ml can be calculated using the formula: Cfu/ml =
An advantage to this method is that different microbial species may give rise to colonies that are clearly different from each other, both microscopically and macroscopically. The colony morphology determines the presence of microorganism. To assess viability of yeast cells on different surfaces, CFU experiment was performed after certain time incubation starting with single colony with surfaces at 140 rpm. After 16 hrs incubation, a serial dilution was made in order to obtain at least one plate with a countable number of cells. 2.4.2.3 Fungal cell-nanofiber interaction test The OD test was started with different weights of various nanofibers and corresponding films like 5 g, 10 g, 15 g, 20 g, and 25 g. The certain weight of different nanofibers was introduced into culture solution, which contained 5-day old single colony cells approximately 4.3 ×106 and 3.3 × 106 colony-forming units (CFU) of SK1 and C. albicans respectively. 10 mL of the inoculated YPD was transferred to electrospun nanofiber mats with 5-day old single colony of each type of yeast cells and incubated (30 oC, 140 rpm) for up to 16 h. for the CFU experiment, 100 µL of the broth culture after 16 hrs was taken and a serial dilution was performed. One hundred microliters of each of these cell solutions was seeded onto agar plate using a surface
38 spread plate technique. The plates were incubated at 30 oC for 48 hours. The numbers of fungal colonies (CFU) were counted. Pure culture medium with cells were also tested as blank control. The counts were used to calculate the surviving number of fungal cells. 2.4.2.4 Scanning electron microscopy SEM images were also used to explore initial fungal cell adhesion, progressive spreading and colonization of SK1 and C. albicans cells on electrospun nanofibers mats after being cultures for certain incubation time. Nanofibrous mat and solid films were cut into 6 mm 4 mm pieces and glued individually to glass chips. The glass chips were further glued onto Petri dishes. Cultures of SK1 and C. albicans strains were transferred into corresponding Petri dishes for certain time of contact. SEM preparation began with removal of the respective substrates from the dish after incubation time. Immediately following removal, the substrates were gently washed 3x in PBS to remove the fungi cells which were not adhered onto the fibers and then fixed in a 2.5% gluteraldehyde/2% formaldehyde solution in 0.1M cacodylate buffer (pH 7.4) for overnight. Samples were then washed 3x in DI and immediately followed by an alcoholic dehydration series of 35%, 50%, 70%, 90%, for 10 minutes and 3x of 95% and 100% for 10 minutes at each concentration. After drying, the samples had a 5nm gold layer applied using a Leica EM ACE200 with real time thickness monitoring through a quartz crystal microbalance (QCM). Scanning electron micrographs were obtained using a Zeiss Auriga FIB/SEM. 2.4.2.5 Confocal laser scanning microscopy A Zeiss Axiom Plan spinning disc confocal scanning microscope was used to view the light emission in which red and blue spots indicates dead and live cells respectively. The study is conducted by using a mixture of two nucleic acid fluorescent stains Calcofluor White(CFW) and
39 Propidium Iodide(PI) that is a blue and red fluorescent dye respectively which stain cell wall of live or dead cells and DNA of dead cells, In this assay, 500 µl of overnight yeast cells was stained with 0.5 µl of CFW and transferred to electrospun surfaces as well as the control surface. To view the effect of surfaces on cells inhibition, SK1and C. albicans strains were cultured overnight before adding any strain. The culture was diluted to OD600 of 0.15 by using YPD media and then incubated with shaking for 3-5 hrs at 30 oC to an OD600 ~ (0.4-0.6) indicative of mid-log phase growth. The culture was then incubated with nanofibrous mats and films for 10 mins, 30mins. Images from confocal microscope were processed by Image J for cell counting.
40 4 5
CHAPTER 3
Interaction between Fungal Cells and Different Electrospun Nanofibers
3.1 Abstract Polyacrylonitrile (PAN) and cellulose acetate (CA) electrospun nanofibers were prepared via electrospinning using N, N -dimethylformamide (DMF) and Tetrahydrofuran (THF)/ Dimethyl Sulfoxide (DMSO), as a solvent respectively. Cellulose electrospun nanofibers were prepared by deacetylation of CA nanofibers. Corresponding films were made by casting solution onto Teflon plate. The interaction between the three types of nanofibers and two fungal cells, i.e. S. cerevisae (Baker Yeast) and C. albicans (Pathogenic yeast) were evaluated. 3.2 Introduction Today electrospinning technology is widely used to make high-quality and well-defined fibers with submicron or nanoscale diameters. The resultant fibers have unique properties, i.e., high surface area-to-volume ratio, small pore sizes, high porosity, and the potential for controlled release of active materials [108][109]. Cellulose is a naturally occurring polymer having particular interest due to its abundant availability, biodegradability, compatibility with biological systems, CA is one of the most commercially important cellulose derivatives. It has been used in various applications such as textile fibers, plastics, films, sheeting, membrane separation, cigarette industries, and lacquers. In biomedical applications, CA had been used in drug delivery system [110][111] [112][113][114], wound dressing [115][116][114], and tissue engineering [117][118]. Conventionally, cellulose fibers are produced via wet spinning. Unfortunately, processing of cellulose is restricted by its limited solubility in common solvents and its inability to melt. Unlike cellulose, cellulose acetate is soluble in many common solvents such as acetone. Ultrafine CA fibers has been fabricated via electrospinning [119]. CA as well as
41 polyacrylonitrile (PAN) fibrous membranes have been widely adopted in filtration due to thermal stability, high mechanical properties, and chemical resistivity [120][121]. The filters of nanofibrous membranes with antimicrobial functionality have attracted growing attentions due to the concerns about qualities of purified water and/or filtered air as well as the processing cost [122][123][124][125][126]. The Tar/PAN/Ag nanofibers showed higher antimicrobial activities (up to 39%) against Gram-positive S. aureus and Gram-negative E. coli in comparison with the neat PAN nanofibers [127]. In the research of Zhang et al. [128], amidoxime surface modified PAN nanofibrous membranes with fiber diameters of ∼450 nm were made by electrospinning followed by treatment with hydroxylamine (NH2OH) aqueous solution. Later Ag+ ions and silver nanoparticles (AgNP) with sizes being tens of nanometers were incorporated with amidoxime PAN nanofibrous mat. The combination of amidoxime groups with silver ions/nanoparticles into one system was proposed as an effective strategy to achieve highly antimicrobial properties for water filtration applications. Yeast is eukaryotic cell microorganism and recognized as unicellular fungus while some yeast species are multicellular. In this study, the interaction between fungal cells (baker and pathogenic cells) and different nanofibrous mats like CA, cellulose and PAN was investigated. The yeast cell viability on nanofibrous mats were evaluated and the results indicated that PAN nanofibrous mats exhibit the most inhibition effect on fungal cells growth. 3.3 Results and discussions Uniform PAN, CA and cellulose nanofibers were obtained from electrospinning with an average diameter of 760 nm, 1212 nm, and 960 nm respectively. Electrospun PAN nanofibrous mats were fluffy and composed of PAN nanofibers with diameters of ∼760 nm (Table 5). The PAN molecule consists of methyl (CH3) and nitrile (C≡N) groups in a linear arrangement [129].
42 The FTIR spectrum of electrospun PAN nanofibers showed the peak centered at 2240 cm-1 is due to nitrile stretching, indicating the presence of acrylonitrile [130]. As shown in Figure 12(a-b), almost no beads and/or beaded-nanofibers [131] could be microscopically identified in the aselectrospun cellulose acetate nanofibers. The nanofibers, however, were not uniform; the diameters were in the range from tens of nanometers to microns. This may be desirable since the thicker fibers could provide greater mechanical support while the thinner fibers could result in larger specific surface area. After hydrolysis/deacetylation, the electrospun cellulose acetate nanofiber mats were converted into the regenerated cellulose nanofiber mats. As shown in Figure 12(c-d), it appeared that the hydrolysis/deacetylation reaction slightly affected the morphology of the nanofiber mats, and the regenerated nanofibers were slightly curved and fused together, particularly the ones with relatively small diameters. Furthermore, the hydrolysis/deacetylation appeared to result in the shrinkage of the mats; in other words, the nanofibers in the mats after the reaction seemed to be closer together. SEM photographs were used to measure the number average fiber diameter of each of electrospun nanofibrous mats (Figure 11). The average diameters of CA, cellulose and PAN nanofibers as well as PAN commercial microfibers were shown in Table 5.
43 a
Cellulose Acetate(CA)
b
Cellulose(C) 35
20 18
30 25
14
Frequency(%)
Frequency(%)
16 12 10 8
6
20 15 10
4
5
2 0
0 400
600
800
1000
1200
1400
1600
More
400
600
800
Fiber Diameter (nm)
c
Random PAN NFs(10%)
1200
More
45
18
40
16
35 Frequency(%)
12 10
8 6
30 25 20 15
4
10
2
5
0
d
Commercial PAN μFs
20
14 Frequency(%)
1000
Fiber Diameter(nm)
0 200
400
600
800
1000
1200
More
Fiber Diameter(nm)
8
10
12
14
More
Fiber Diameter(μm)
Figure 11 The fiber distribution of (a) CA, (b) Cellulose, (c) random PAN nanofibers and (d) PAN micro fibers Table 5 Average Sizes of Prepared Electrospun Nanofibers Nano and Micro Fibers
Average Size
Cellulose acetate nanofibers
1212 nm
Cellulose nanofibers
960 nm
10% random PAN nanofibers
760 nm
PAN microfibers
13.52 μm
44
a
b
c
d
Figure 12. SEM images of (a-b) Cellulose Acetate (CA) and (c-d) Cellulose nanofibers
45
a
b
c
d
Figure 13. SEM images of (a-b) PAN nanofibers and (c-d) PAN microfibers. To better understand the chemical changes involved in the sample preparation, FT-IR was employed to study the hydrolysis/deacetylation of CA nanofibers to cellulose nanofibers. As shown in Figure 14, the characteristic ester band (C=O at ∼1700 cm-1) completely disappeared after hydrolysis/deacetylation, indicating the reaction successfully converted cellulose acetate into regenerated cellulose. Additionally, the hydroxyl band (O-H at ∼3300–3500 cm-1) was stronger after the reaction, indicating more hydroxyl groups were presented in the regenerated cellulose. The wavenumber of hydroxyl groups before the reaction (curve A) was higher than that after the reaction (curve B); presumably, this was because the hydroxyl groups in cellulose acetate were less hydrogen-bonded than those in cellulose [103].
46
1700 cm-1
3300~3500 cm-1 A O-H
C=O
B
4000
3500
3000
2500
2000
1500
1000
500
Wavelength(cm-1)
Figure 14. The Fourier Transform Infrared Spectroscopy (FTIR) Spectra of (A) as-electrospun cellulose acetate nanofibers and (B) regenerated cellulose nanofibers 3.3.1 Viability of yeast cells on solid film and nanofibrous mat Two strains of S. cerevisiae, SK1, and C. albicans, were used to examine the cell growth behavior on the different nanofibrous mat and corresponding films. Figures 15 and 16 had shown cells viability of SK1 and C. albicans on different surfaces with varied mass after 16 hours of contact. For SK1 strain, CA nanofibers showed the most inhibition effect than cellulose and PAN nanofibers. For C. albicans strain, PAN nanofibers showed the most inhibition effect among all the surfaces while CA nanofibers had the second most inhibition effect after PAN nanofibers and cellulose had the least inhibition effect on the cell growth.
47
Cells Viability(SK1) 4 3.5
Optical Density
3 2.5
25mg 20 mg
2
15 mg
1.5
10 mg 1
5 mg
0.5 0 CA NF
CA Film
Cellulose NF Cellulose Film
PAN NF
PAN Film
Blank
Surfaces
Figure 15. Interactions between SK1 cells and different nanofibers and films.
Cells Viability(C. albicans) 4 3.5
Optical Density
3 2.5
25mg
2
20mg 15mg
1.5
10mg 1
5mg
0.5 0 CA NF
CA Film
Cellulose NF
C Film
PAN NF
PAN Film
Blank
Surfaces
Figure 16. Interactions between C. albicans cells and different nanofibers and films. Both yeast cells showed similar ellipsoidal shape. These yeast cells are haploid and sometimes diploid and grow by mitosis (budding) with a doubling time of ~90 min [107]. Both strains grew healthy with contact of CA film for 1 hr. These yeast cells after mid-log phase growth showed a range of sizes from 0.5 m to 3.5 µm in width and 0.5 m to 7 m in length.
48 However, shrinkage appeared on the outer surface of both cells when they came into contact to nanofibers mats that linked to cell death (Figure 17 and 18).
a
b
c
d d
Figure 17. Interactions of (a-b) SK1 cells and (c-d) C. albicans on CA nanofibers (left column) and films (right column).
49
a
b
c
d
Figure 18. Interactions of (a-b) SK1cells and (c-d) C. albicans on PAN nanofibers (left column) and films (right column). Size variation of cultured yeast cells on PAN film surface was observed and it is typical because cells generally grow bigger as they age. Yeast flocculation was also observed in which yeast cells clump and adhere to each other through protein-protein interactions [132]. Both yeast strains grew normally with contact of PAN film for 1hr except some cells showed little elongations (Figure 18(b and d)). In other words, yeast cells were still alive and healthy after 1 h contact with PAN film. The viability of yeasts cells on electrospun nanofibrous mat was completely different from that on solid film. Both SK1 and C. albicans cells flattened and shrank, losing their vitality after 1hr contact with the surface of nanofibrous mat (Figure 18(a and
50 c)). In other words, all yeast cells died after even 1 hr contact with PAN electrospun nanofibrous(ESPAN) mats. However, both types of yeast cells were still alive and healthy after 1hr contact with Cellulose nanofibers and films which support the previous statement (Figure 19).
a
b
c
d
Figure 19 Interactions of (a-b) SK1cells and (c-d) C. albicans on cellulose nanofibers (left column) and films (right column). 3.4 Mechanism Discussion It was observed that yeast cells on nanofibrous mat had limited and asymmetrical contact with surrounding nanofibers due to micrometer scale inter-fiber porosity and random orientation of nanofibers mat. Internal tension may be induced in a yeast cell on nanofibrous mat due to
51 unbalanced contact with multiple nanofibers followed by the death of cells. However, cellulose nanofibers did not show much of this effect. There must be some other factors particularly material properties and cell growth behavior that also play a role for the inhibition effect of electrospun nanofibrous mat on fungal cells. Further investigation is under way. 3.5 Conclusions In this study, different types of nanofibrous mats were prepared by electrospinning. It has been shown that electrospun nanofibrous mats have inhibition effect on yeast cells than corresponding films. After 1 hr contact with the surface of PAN and CA nanofibrous mat, both SK1 and C. albicans cells shrunk or flattened and lost their vitality while both types of yeast cells were still alive and growing on solid films. Unbalanced cell contact with electrospun nanofibers in nanofibrous mat may be the reason for the inhibition of yeast cells. However, there must be some other factors particularly material properties that also play a role here. These findings help to discover a new antifungal material without using any antifungal agents.
52 6 7
CHAPTER 4
Effect of Sizes of PAN Electrospun Nanofibers on Fungal Cell Growth
4.1 Abstract In this chapter, effect of PAN nanofiber size and alignment on the growth behavior of two fungi stains (S. cerevisae and C. albicans) was assessed through a combination of optical density, CFU and scanning electron microscopy (SEM). The results indicated that PAN nanofiber size and alignment do affect fungal cell growth. The data suggested that simple adjustment of morphological structure of electrospun fibers may be an effective strategy to control fungi cells’ growth. 4.2 Introduction In the past decade, electrospinning technique has been recognized as the most popular technique to produce continuous ultrafine fibers with diameters ranged from microns down to nanometers [133]. The resultant fibers have unique properties, i.e., high surface area-to-volume ratio, small pore sizes, high porosity, and potential for controlled release of active compounds [108][109]. However, there is little data regarding the interaction between fungal cells and electrospun nanofibers with different morphology. There are multiple factors in electrospinning such as solution parameters, processing parameters, and ambient parameters, which can affect morphology of the obtained fibers. By properly controlling these parameters, we can fabricate electrospun fibers with certain range of diameters and desired morphologies and. Electrospun nanofibers are also carriers for many additives including vitamins, growth factors, and natural compounds [134], which have great potential for new opportunities. Fiber diameter, inter fiber porosity, and fiber alignment have all been found to significantly affect the ability of cells to adhere onto corresponding electrospun scaffolds and
53 proliferate within the fibrous network [135] [136]. Despite side effects of antifungal agents, most of the research so far focused on loading antifungal agents in electrospun nanofibers. It has been shown that cell-abiotic adhesion depends on not only cell surface characteristics but also surface morphology, surface chemistry, and roughness. Mitik-Dineva et al. has reviewed the mechanisms that bacteria use to adhere to flat surfaces with different chemistries and nanotopographies [137]. Kargar et al. has discovered that the adhesion mode of P. aeruginosa on electrospun nanofiber surface was dependent on fiber diameter and spacing, suggesting that strategically designed curvatures can reduce bacterial adhesion process [138]. A recent study has also suggested that fiber diameter have an impact on bacteria attachment, proliferation and growth [139]. In the meantime, various polymers, such as poly (vinyl alcohol) (PVA) [140], poly(ethylene oxide) PEO [141][142], and polyacrylonitrile (PAN) [143], have been examined to investigate parameters to control fiber formation. In this part, we synthesized PAN nanofibrous mats with different size and morphology followed by investigation on growth behavior of yeast cells on these PAN nanofibrous mats. 4.3 Results and Discussions Multiple PAN-DMF solutions were electrospun to fabricate nanofibrous mats with controlled morphology and fiber sizes. Polymer concentration is a significant parameter that affects fiber diameter. Electrospun PAN nanofibers with significantly different sizes were acquired by changing voltage and flow rates while others parameter kept fixed (Table 6).
54 Table 6 Processing Parameters Selected for Electrospinning of PAN Solutions at Different Concentrations Nanofibers Polymer
Applied
Flow Rate
Needle to
Types of
Spinning
(% w/w)
Voltage
(ml/hr)
Collector
Collector
Duration
Distance (cm)
and Speed
(kV) 10% PAN
10
15
1.0
20 cm
Drum,
12hrs
300 rpm 12% PAN
12
15
1.0
20 cm
Drum,
12hrs
300 rpm
Two types of PAN nanofibers were prepared by electrospinning. Figure 20 showed SEM images of electrospun PAN nanofibrous mats from solutions at different concentrations. The increase of polymer concentration from 10 wt.% to 12 wt.% resulted in increase of average fiber diameter and formation of uniform fibers with no defects. Electrospun PAN nanofibers from 12 wt.% PAN solution is much bigger in diameter (~1808 nm) than those from 10 wt.% PAN solution (~760 nm) (Figure 21).
55
a
b
c
d
Figure 20. SEM images of (a-b) PAN nanofibrous mat from 10 wt.% PAN solution (top row) and (c-d) PAN nanofibrous mats from 12 wt.% PAN solution (bottom row) a
Random PAN NFs(10%)
18
18
16
16
14
Frequency(%)
Frequency(%)
14 12 10
8 6
12 10 8 6
4
4
2
2
0
b
PAN (12%)(Bigger Diameter)
20
0 200
400
600
800 Fiber Diameter(nm)
1000
1200
More
800
1000
1200
1400
1600
1800
2000
2200
More
Fiber Diameter(nm)
Figure 21. Fiber size distribution of PAN nanofibers from (a) 10 wt.% and (b) 12 wt.% of PAN solutions
56
Aligned PAN nanofibers were prepared by electrospinning 10 wt.% PAN-DMF solution and collected on an in-house-built collector (Table 7). This special collector has four small cylindrical rods which are attached in peripheral of a circular base. Aligned nanofibers and random nanofibers were collected between the rods and on the rod, respectively. It is really difficult to get 100% aligned nanofibrous mat. In this study, spinning times were varied from 3 hrs to 6 hrs (Figure 22 and Figure 23). Finally, the most aligned nanofibrous mat was obtained with 4 hrs spinning time (Figure 22(b and d), Table 8). Table 7 Electrospinning Parameters for Aligned PAN Nanofibers Nanofibers Polymer
Applied
Flow Rate
Needle to
Types of
Spinning
conc.
Voltage
(mL/hr)
Collector
Collector and Duration
(% w/w)
(KV)
Distance,
Speed
(cm) Aligned
10
20
1.5
15
4 small
3hrs
PAN
cylindrical
4hrs
nanofibers
rod, 1500
5hrs
rpm
6hrs
57
a
b
9
9
c
d
8 7
7
Frequency of Fibers(%)
Frequency of Fibers(%)
8
6 5 4 3
6 5 4 3 2
2 1
1 -90
-45
0
45
Direction (°)
90
-90
-45
0
45
90
Direction (°)
Figure 22. Aligned electrospun PAN nanofibers from different spinning time periods: (a-c) 3hrs (left column); (b-d) 4hrs (right column)
58
a
b
4.5
6
c
4.0
d 5
Frequency of Fibers(%)
Frequency of Fibers(%)
3.5 3.0 2.5 2.0 1.5
4
3
2
1.0 1 0.5 -90
-45
0
45
90
-90
-45
Direction (°)
0
45
90
Direction (°)
Figure 23. Aligned PAN nanofibers from different spinning time periods: (a-c) 5 hrs (left column); (b-d) 6hrs (right column) Table 8 Percentage of Fiber Alignment from Different Spinning Time Spinning
Goodness (%)
Time 3hrs
83
4hrs
95
5hrs
83
6hrs
88
59 To investigate the effect of fiber diameter on fungal cell’s growth behavior, electrospun PAN nanofibers from 10 wt.% PAN solution and 12 wt.% PAN solution were respectively exposed to a solution culture of SK1 and C. albicans. The OD result showed that PAN nanofibers with smaller diameter had more inhibition effect on fungi cells growth compare to PAN nanofibers with larger diameter (Figure 24). a
SK1: Comparing Different sizes of PAN NFs (10% PAN vs 12% PAN NFs)
Candida Albicans: Comparing Different sizes of PAN NFs (10% PAN vs 12% PAN NFs) 3.5
3
3
2.5
2.5
Optical Density
Optical Density
3.5
b
2 1.5 1 0.5
2 1.5 1 0.5
0
0
760nm PAN(10%)
1808 nm PAN(12%)
PAN film
ESPAN NFs & Film
Control
760 nm PAN(10%)
1808 nm PAN(12%)
PAN film
Control
ESPAN NFs & Film
Figure 24. Effect of different size of nanofibers on fungal cell growth: (a) SK1; (b) C. albicans These results revealed that fiber size does influence the capacity of cells to adhere, proliferate, and grow. Both yeast strains like SK1 and C. albicans are oval shaped and have almost similar size about 3~5µm. It was observed that yeast cells on nanofibrous mat had asymmetrical points of contact from all directions with surrounding nanofibers due to smaller size and random orientation of nanofibers. Cells had less points of contact from all directions with bigger fiber size and concurrent bigger inter-fiber pores and could reduce reduce internal stress of cells. The following SEM images supported the optical density results (Figure 25).
60
a
b
c
d
Figure 25 Interactions of (a-b) SK1cells and (c-d) C. albicans on 10 wt.% and 12 wt.% of PAN nanofibers. Random PAN and aligned PAN nanofibers in this research have similar size. However, random nanofibrous mats have more inhibition effect on cell growth than aligned nanofibers. It may be caused by the fact that cells feel like on flat solid surfaces [139] when they come into contact with bundles of aligned nanofibers. Cells have less points of contact from all directions on flat surface which helps cell to proliferate and is supported by SEM images (Figure 27).
61 SK1: Comparing Different Arrangement of 10% PAN NFs (Random PAN NFs mat vs Aligned PAN NFs mat)
a
Candida albicans: Comparing Different Arrangement of 10% PAN NFs (Random PAN NFs mat vs Aligned PAN NFs mat)
0.6 1.4
0.5
1.2 0.4
Optical Density
Optical Density
b
0.3 0.2 0.1
1 0.8 0.6 0.4 0.2
0
0
Random PAN 760 nm
Aligned PAN 760 nm
Random PAN 760 nm
Electrospun PAN NFs
Aligned PAN 760 nm Electropspun PAN NFs
Figure 26 . Effect of fiber alignment on fungal cell growth
a
b
c
d
Figure 27 Interactions of (a-b) SK1 and (c-d) C. albicans cells on random 10 wt.% (left column) and aligned 10 wt.% PAN nanofibers (right column).
62 4.4 Conclusions The diameter of electrospun PAN nanofibers showed influence on how fungal species proliferate and colonize on nanofibrous substrate. Fungal cells spread throughout the nanofibrous mats depending on fiber diameter and alignment. Cells had less points of contact from all directions on the nanofibrous mat with bigger fibers or aligned fibers, which helps the cells to survive, spread and proliferate. The presented results showed the possibility of using fiber size and alignment as a tool to control fungal cell growth on electrospun nanofibrous mats.
63 8 9
CHAPTER 5
Effect of Surface Topological Features of Electrospun Nanofibrous mats on Yeast Cells Growth
5.1 Abstract In this part of research, polyacrylonitrile (PAN) nanofibers with different topological features were prepared using electrospinning technique followed by post treatment. Graphene embedded PAN (GPAN) nanofibers were obtained by dispersing Graphene platelets into PANDMF solution followed by electrospinning. These nanofibers demonstrated surface roughness with a positive curvature. Furthermore bicomponent nanofibers were prepared by electrospinning PAN-PMMA and PAN-PEO DMF solutions. PMMA and PEO were then selectively removed from the bicomponent nanofibers by chloroform and DI water treatment, respectively. These PAN nanofibers possessed surface roughness with a negative curvature (nanoporous structure). The cell growth behavior on these electrospun nanofibrous mats with different topological features indicated that roughness of electrospun nanofiber surface can change their inhibition effect on fungal cells. PAN nanofibers with negative curvature demonstrated more inhibition effect than those with surface positive curvature. 5.2 Introduction It is known that attachment and biofilm formation by pathogenic cells on abiotic surfaces in nature, industrial and hospital settings lead to infections, illness and even death. Cell attachment to surface could be minimized by controlling surface topography and thus reduce the incidence of illness and subsequent human and financial losses [144] [145]. There are many cellular and environmental factors affecting cell’s attachment and biofilm formation, among which surface roughness plays a very important role. Cells may attach to rough surfaces in
64 greater quantities due to their higher surface area for attachment while protecting the cells from shear forces. The role of surface roughness (i.e. the presence of surface irregularities) at micrometric scale on bacterial attachment has been investigated by several groups. Boyd et al. [146] found that enhanced adhesion of S. aureus occurred on rougher surface as compared to smooth surfaces. Surfaces with features on the same scale as the S. aureus cells (1 µm) appeared to promote the strongest attachment due to maximal cell-substrate contact area. Electrospinning is a useful one-step and straightforward process for fabrication of nanofibers. Surface morphology of electrospun nanofibers can be tuned for complicated structures such as core-shell or multiple layers [140][147], surface-attached nanoparticles [148] [99], porous structure by selectively removing bicomponent polymers [149][150]. Electrospun nanofibers have been widely investigated in the past few decades as candidates in biomedical fields such as drug delivery, tissue engineering, and wound dressing applications. However, there are still very limited data available for interaction between electrospun nanofibers and fungal cells. Particularly the effect of fiber surface topography (i.e. the specific arrangement of the physical features on a surface) at the nanoscale on fungal cell growth has been far less investigated. Polyacrylonitrile (PAN) is a kind of polymer material with functional cyano-groups (– CN) on its macromolecular chain. The preparation of PAN nanofibers by electrospinning has attracted wide attention, including preparation conditions of PAN nanofibers [151], PAN nanocomposite, and so forth [152][153][154]. In this research, the interactions between two types of yeast cells (non-pathogen SK1 and pathogen C. albicans) and PAN electrospun nanofibrous mats with different fiber surface morphological features were investigated. To get PAN nanofibers with positive surface curvature, graphene platelets were included into PAN
65 nanofibers where graphene was covered with PAN. To obtain PAN nanofibers with negative curvature, PAN-PMMA and PAN-PEO bicomponent solution were electrospun followed by selectively removal of PMMA and PEO, respectively. The knowledge obtained herein allows for a better understanding on the interaction between electrospun PAN nanofibers and fungal cells. 5.3 Results & Discussions 5.3.1 Rough Nanofiber Surface with Positive Curvature by Adding Graphene The study by Seyam et al showed that beaded cyanoethyl chitosan nanofibers is less effective than smooth fibers in the inhibition of the bacteria [155]. The inhibition ability of chitosan derivatives is dependent on the amount of contact between the bacteria and the material [155][156]. Based on this finding, roughness of PAN nanofiber surface should have effect on growth of fungal cells. Herein PAN nanofibers with rough surface were obtained from including different percentage of graphene platelets in PAN-DMF solution followed by electrospinning. Comparison of fungal cell growth between rough surface and smooth surface with similar diameter was conducted. Table 9 and Figure 28 showed the size and morphology of PAN nanofibers with graphene integrated.
66 Table 9 Average Diameter of Electrospun GPAN Fibers with Submicrometer Size Nanofibers
Average Diameter of Single Nanofiber
GPAN-1
476 nm
From spinning solution containing 6.7% PAN with graphene at 20 wt% of PAN GPAN-2
668 nm
From spinning solution containing 7.3% PAN with graphene at 20 wt.% of PAN GPAN-3
891 nm
From spinning solution containing 8 wt.% PAN with graphene at 25 wt.% of PAN PAN From spinning solution containing 10 wt.% PAN
760 nm
67
a
b
c
d
e
f
Figure 28. SEM images of graphene embedded electrospun PAN nanofibers with submicrometer sizes (a-b) GPAN-1 (top row); (c-d) GPAN-2 (middle row); and (e-f) GPAN-3 (bottom row)
68 The growth behavior of fungal cells was characterized on these electrospun PAN nanofibers with rough surface caused by graphene (positive curvature on surface). In the case of sub-micrometer diameter, PAN nanofibers with smooth surface showed more inhibition effect on fungal cell growth than those with rough surface from graphene inclusion (Figure 29). The inhibition ability of nanofibers is dependent on the amount of contact between the cells and the PAN nanofibers. In the nanofibrous mat, graphene platelets appeared on nanofiber surface. The relatively large graphene platelets (based on submicrometer size of nanofibers) on surface increased average fiber diameter and also increased inter fiber pores, which generated relatively less points of contact from all directions between the cells and the nanofibers when compared to the smooth nanofibers. This may partially relieve asymmetric tension from the contact and allow cells grow. A consistent result was also directly obtained from corresponding SEM images (Figure 30). SK1: Comparing Smooth Surface vs Rough Surface of PAN NFs (10% PAN vs 8% PAN+G NFs)
a
Candida albicans: Comparing Smooth Surface vs Rough Surface of PAN NFs (10% PAN vs 8% PAN+G NFs)
3.5
3.5 3
Optical Density
Optical Density
3 2.5 2 1.5 1 0.5
2.5 2 1.5 1 0.5
0
0
760 nm PAN(10%)
891nm PAN+G
Control
760 nm PAN(10%)
Electrospun NFs
891nm PAN+G
Control
Electrospun NFs
Figure 29. Fungal cell growth behavior on submicrometer electrospun PAN nanofibers with smooth and rough surface (positive curvature): (a) SK1; (b) C. albicans
b
69
a
b
c
d
Figure 30 SEM images of SK1(a-b) and C. albicans (c-d) cultured on smooth (left column) and rough PAN (GPAN) (right column) nanofibers. Further investigation was performed to check fungal cell growth behavior on micrometer scale electrospun PAN nanofibers with positive surface curvature from graphene. To get effective rough surface in micrometer size, the amount of graphene was increased. The diameter of PAN nanofibers with graphene was compared with bigger size but smooth ESPAN nanofibers (Table 10, Figure 31).
70 Table 10 The diameter of different composition of Graphene-PAN nanofibers mat Nanofibers(Bigger Size)
Diameter
GPAN-4
1580 nm
From spinning solution containing 10 wt.% PAN with graphene at 10 wt.% of PAN GPAN-5
1572 nm
From spinning solution containing 10 wt.% PAN with graphene at 20 wt.% of PAN PAN From spinning solution containing 12 wt.% PAN
1808 nm
71
a
b
c
d
Figure 31. SEM images of Graphene embedded of electrospun PAN fibers with micrometer scale diameter: (a-b) GPAN-4 (top row); (c-d) GPAN-5 (bottom row) Evaluation of yeast cell growth on the electrospun PAN and GPAN fibers with diameter over one micrometer (Figure 32) indicated that smooth surface has less inhibition effect on cell growth than rough surface which is opposite to those results from electrospun PAN and GPAN fibers with submicrometer diameters. In the case of over-micrometer fiber diameter, graphene platelets are relatively small. Surface roughness from graphene on these big diameter fibers just increased points of contact from all directions between fungal cells and electrospun nanofibers when compared to the smooth nanofibers, which disturbed the growth of cells.
72 SK1: Comparing Smooth surface vs Roughen Surface of PAN NFs (12% PAN vs 10% PAN+G NFs)
a
Candida albicans: Comparing Smooth surface vs Roughen Surface of PAN NFs (12% PAN vs 10% PAN+G NFs) 3.5
3
3
2.5
2.5
Optical Density
Optical Density
3.5
b
2 1.5 1 0.5
2 1.5 1 0.5
0
0
1808 nm PAN(12%)
1572 nm PAN(10%)+G
Control
1808 nm PAN(12%)
Electrospun PAN Fibers
1572 nm PAN(10%)+G
Control
Electrospun PAN Fibers
Figure 32. Fungal cell growth behavior on over-micrometer electrospun PAN fibers with smooth and rough surface (positive curvature): (a) SK1; (b) C. albicans
a
b
c
d
Figure 33 Interactions of (a-b) SK1cells and (c-d) C. albicans on smooth(PAN) (left column) and rough PAN(GPAN) (right column) nanofibers with micrometers.
73 Graphene embedded PAN nanofibers with smaller diameter and bigger diameter were further compared from the point of view of fungal cell growth. The result showed that electrospun GPAN fibers with bigger diameter (over-micrometer) had more inhibition effect on fungal cells’ growth than those with smaller diameter (sub-micrometer) (Figure 34). The size of bigger diameter is almost double of that of smaller nanofibers. The graphene basically has the same size in both cases. The relatively small graphene sheet on bigger fibers seemed to generate more disturbance to the fungal cells than those on smaller fibers. SK1:Comparing two types of PAN+G(smaller diameter and big diameter) (Both are Rough Surface)
a
Candida albicans:Comparing two types of PAN+G(smaller diameter and big diameter) (Both are Rough Surface) 3.5
3.5 3
3
2.5
2.5
Optical Density
Optical Density
b
2 1.5 1
2 1.5 1 0.5
0.5
0
0 891 nm PAN(8%)+G
1572 nm PAN(10%)+G
Control
Electrospun PAN Fibers
891 nm PAN(8%)+G
1572 nm PAN(10%)+G
Control
Electrospun PAN Fibers
Figure 34. Fungal cell growth behavior on graphene embedded electrospun PAN fibers (big diameter vs small diameter): (a) SK1; (b) C. albicans Furthermore, cell growth data showed that electrospun PAN nanofibers with smooth and small diameter had more significant inhibition effect on cell growth than those with rough and big diameter (Figure 35). Inter-fiber porosity is smaller in small diameter electrospun PAN nanofiber mat. When cells enter into inter-fiber space, they would feel more points of contact from all direction and thus asymmetric tension, which prohibited their growth.
74 a
SK1:Smaller and Smooth PAN vs PAN (Bigger Diameter)+G
Candida albicans:Smaller and Smooth PAN vs PAN (Bigger Diameter)+G 3.5
3
3
2.5
2.5
Optical Density
Optical Density
3.5
b
2 1.5 1 0.5
2 1.5 1 0.5
0
0
760 nm PAN(10%)
1572 nm PAN(10%)+G
Control
760 nm PAN(10%)
Electrospun PAN Fibers
1572 nm PAN(10%)+G
Control
Electrospun PAN Fibers
Figure 35. Comparison of fungal cell growth behavior on electrospun PAN fibers with small size and smooth surface and electrospun PAN fibers with large size and rough surface (positive curvature): (a) SK1; (b) C. albicans 5.3.2 Rough Nanofiber Surface with Negative Curvature 5.3.2.1 Rough PAN Nanofiber Surface from PAN/PMMA Nanofibers Figure 36 showed the FTIR absorption spectra of PAN/PMMA nanofibers, chloroform treated PAN/PMMA and pure PAN nanofibers. The characteristic peak of PMMA at 1732 cm-1 indicates presence of the acrylate carboxyl group. After being immersed in chloroform, the characteristic peak of PMMA was significantly reduced, indicating PMMA removal that expected to generate porous structure on PAN nanofiber surface. The diameter of PAN/PMMA nanofibers before chloroform treatment and porous PAN nanofibers after chloroform treatment are 828 nm and 708 nm, respectively.
75 A-PAN-PMMA B-PAN(Treated) C-PAN A
1732 cm 2244 cm
-1
-1
1452 cm
-1
B
C
4000
3500
3000
2500
2000
1500
1000
500
Wavelength(cm-1)
Figure 36. The FTIR spectra of (A) electrospun PAN/PMMA nanofibers; (B) electrospun PAN/PMMA nanofibers after chloroform treatment; (C) electrospun PAN nanofibers
76
a
b
c
d
Figure 37. SEM images of PAN/PMMA nanofibers: (a-b) before chloroform treatment (top row); (c-d) after chloroform treatment (bottom row). Based on cell growth data (Figure 38 and Figure 39), chloroform treated PAN nanofibers (with negative surface curvature) showed more inhibition effect on fungal cell growth than those pure PAN electrospun nanofibers. The negative curvature on nanofiber surface could create obstacles for cells’ growth due to creation of more points of contact. SEM images in Figure 40 showed the comparison of cell growth behavior on smooth PAN, PAN-PMMA and porous PAN nanofibers and the results supported the previous statement.
77 a
SK1: Comparing Smooth PAN vs Treated PAN-PMMA(Porous) NFs
Candida albicans: Comparing Smooth PAN vs Treated PAN-PMMA(Porous) NFs 3.5
3
3
2.5
2.5
Optical Density
Optical Density
3.5
b
2
1.5 1 0.5
2
1.5 1 0.5
0
0
760 nm PAN(10%)
708 nm PAN-PMMA (After Treated)
Control
760 nm PAN(10%)
Electrospun NFs
708 nm PAN-PMMA (After Treated)
Control
Electrospun NFs
Figure 38. Comparing the effect of smooth ESPAN nanofibers and porous ESPAN (after removing PMMA with chloroform) nanofibers on cell growth (a) SK1 and (b) C. albicans a
SK1:Comparing Smooth PAN, PAN-PMMA, PAN-PMMA(Treated)
3.5
3.5 3
3
2.5
2.5
Optical Density
Optical Density
b
Candida albicans :Comparing Smooth PAN, PAN-PMMA, PAN-PMMA(Treated)
2
1.5 1
2 1.5 1 0.5
0.5
0
0
760 nm PAN(10%)
828 nm PAN-PMMA (Before Treated)
708 nm PAN-PMMA (After Treated)
Electrospun NFs
Control
760 nm PAN(10%)
828 nm PAN-PMMA (Before Treated)
708 nm PAN-PMMA (After Treated)
Control
Electrospun NFs
Figure 39. Comparing the effect of smooth ESPAN nanofibers, ESPAN-PMMA nanofibers (before removing PMMA) and ESPAN (after removing PMMA with chloroform) nanofibers on cell growth (a) SK1 and (b) C. albicans
78
a
b
c
d
e
f
Figure 40. Interactions of (a, c, e) SK1 and (b, d, f) C. albicans cells on smooth PAN (top row), PAN-PMMA(Before treatment) (middle row) and PAN-PMMA(After treatment for removing PMMA) (bottom row) nanofibers
79
5.3.2.2 Rough PAN Nanofiber Surface from PAN/PEO nanofibers Figure 41 showed FTIR absorption spectra of PAN/PEO nanofibers, water treated PAN/PEO nanofibers and pure PAN nanofibers. The characteristic peak of electrospun PAN fibers showed stretching –C≡N bond vibration at 2244 cm-1 and strong C–H in-plane deformation vibration of –CH2– group at 1452 cm-1. PEO exhibited strong peaks at around 950 and 842 cm-1, both attributed to the C–H out-of-plane deformation vibration in the –CH2–CH2– O– unit. Strong peak at 1109 cm-1 with two shoulder peaks at 1147 and 1061 cm-1 were attributed to C–C/C–O stretching vibration. The diameters of PAN/PEO nanofibers before water treatment and PAN/PEO nanofibers after water treatment are 1044 nm and 1012 nm, respectively. A:PAN-PEO B:PAN(Treated) C:PAN
1109 cm-1
2244 cm-1
A
1452 cm
-1
950 cm-1 -1 842 cm
B
C
4000
3500
3000
2500
1500
2000
1000
500
-1
Wavelength(cm )
Figure 41. FTIR spectra of (A) PAN/PEO electrospun nanofibers; (B) water-treated PAN/PEO electrospun nanofibers; (C) electrospun PAN nanofibers.
80
a
b
c
d
Figure 42. SEM images of PAN/PEO nanofibers: (a-b) before (top row); (c-d) after treatment with 70 oC water (bottom row). Fungal cell growth data showed that porous PAN nanofibers from water-treated PAN/PEO nanofibers had more inhibition effect than pure PAN nanofibers on the cell growth (Figure 43). SEM images showed consistent results with the OD test (Figure 44). Cells felt more points of contact on nanofiber surface with porous surface feature and these points of contact became obstacles to cells’ proliferation.
81 a
SK1:Comparing Smooth PAN vs PAN-PEO(Treated & Porous) NFs 3.5
3.5
3
3
Optical Density
Optical Density
b
Candida albicans:Comparing Smooth PAN vs PAN-PEO(Treated & Porous) NFs
2.5 2 1.5 1 0.5
2.5 2 1.5 1 0.5
0
0
760 nm PAN(10%)
1012 nm PAN-PEO (After Treated)
Control
760 nm PAN(10%
Electrospun NFs
1012 nm PAN-PEO (After Treated)
Control
Electrospun NFs
Figure 43. Comparing the effect of smooth ESPAN nanofibers and ESPAN (after removing PEO with 70 oC water) nanofibers on cell growth (a) SK1 and (b) C. albicans
a
b
c
d
82 Figure 44. Interactions of (a-b) SK1 and (c-d) C. albicans cells on smooth ESPAN nanofibers (left column) and porous ESPAN (after removing PEO with 70 oC water) (right porous) nanofibers. 5.4 Conclusions Rough surface of electrospun PAN nanofibers demonstrated inhibition effect on fungal cell growth. The negative surface curvature on electrospun PAN nanofibers particularly facilitated the inhibition effect on fungal cell growth. However, fiber size (micrometer vs submicrometer) played a role here. Further investigation is needed.
83 10 CHAPTER 6 11 Effect of Hydrophobicity of Nanofibrous Material and Yeast Cells on Cells’ Viability 6.1 Introduction In this part, mechanism of the interaction between yeast cells and electrospun nanofibers were briefly explored. Viability of a series of yeast cells on electrospun nanofibrous mat surface including polyacrylonitrile (PAN), cellulose acetate and cellulose was investigated. The surface properties of electrospun nanofibrous mats were evaluated by contact angle measurements. The growth behavior of yeast cells was evaluated by electron microscopes (cell morphology) and biological assays including colony forming unit and MATH assay. 6.2 Microbial adhesion to hydrocarbon MATH assay Microbial adhesion to hydrocarbons (MATH) is a very common method to measure the microbial cell surface hydrophobicity [157]. The MATH test normally is done by suspending microbial cell in a hydrocarbon solution and optical density of the microbial cell decreases due to adhesion of some cells to the hydrocarbon. Hydrophobic strains adhere well to hydrocarbon and hydrophilic strain has less adherence to hydrocarbon [158]. Microbial cell hydrophobicity is a good determination of the ability of cells to adhere, invade, degrade and cause diseases [159]. In this part of research, MATH tests were performed on log-phase and stationary phase cells of the two strains (SK1 and C. albicans). Cells were first grown to the desired phase: OD ~ 0.4 and OD ~ 2 were taken for log phase and stationary phase, respectively. For log phase cells, 50 ml cell suspension were taken into 50 ml plastic bottle and was centrifuged at 3000 rcf for 5 minutes. Then the solution was decanted and washed in 50 mM EDTA (PH 8.0) to inhibit flocculation, and next suspended in 25 ml 0.9% NaCl (pH~6) to an OD ~ (0.8-1.0). In a glass tube, 2 mL of cell suspension was carefully overlaid with 400 μL Octane. 1 mL of aqueous phase
84 was carefully removed and the remainder was vortexed for 60 seconds, followed by 10 minutes of rest for phase separation. After separation, the other 1 mL aqueous phase was carefully removed and OD measurements were made on both samples. Relative cell hydrophobicity measurements was calculated by using the following equation. At least 10 samples were measured and averaged.
Hydrophobicity of yeast cells increased with the change of cells growth stage. MATH assay result showed that the percentage of hydrophobicity of SK1 cells is higher than that of C. albicans at stationary stage as well as exponential stage (Table 11). Table 11 The percentage of hydrophobicity of SK1 and C. albicans at exponential and stationary stage Strain SK1
C. albicans
Surface
n
% hydrophobicity
Exponential
10
36.2
Stationary
10
45.94
Exponential
10
9.7
Stationary
10
13.82
Contact angles of CA nanofibrous mat, cellulose nanofibrous mat and PAN nanofibrous mat were measured (Figure 45 and Table 12). Growth behavior of SK1 and C. albicans on PAN and cellulose nanofibrous mats were evaluated (Figure 46). It seems that more hydrophobic PAN nanofibrous mat showed more inhibition effect on more hydrophobic SK1 cells. However, more hydrophilic cellulose nanofibrous mat also showed more inhibition effect on more hydrophobic SK1 cells. The results indicated that this is a complicated case. There must be other factors that affect the inhibition effect. The potential reason may be related to cell wall structure.
85 a
b
c
Figure 45. Contact angle of (a) CA nanofibers, (b) Cellulose nanofibers, and (c) PAN nanofibers Table 12 The contact angle of CA nanofibers, Cellulose nanofibers, and PAN nanofibers Nanofibers
Contact Angle (o)
Cellulose Acetate
128
Cellulose
