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GRAPHENE OXIDE AND ITS BIOMEDICAL APPLICATIONS

SITANSU SEKHAR NANDADEPARTMENT OF BIONANOTECHNOLOGY

GRADUATE SCHOOL, GACHON UNIVERSITY

UNDER SUPERVISION OF

PROF. DONG KEE YI

Presentation for Ph.D. Degree

TABLE OF CONTENTS

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Chapter.1. Introduction Chapter.2. Graphene Oxide Based Fluorometric Detection of Hydrogen Peroxide in Milk Chapter.3. Study of antibacterial mechanism of Graphene Oxide using Raman Spectroscopy Chapter.4. Oxidative stress and antibacterial properties of a graphene oxide-cystamine nanohybrid Chapter.5. Future work & perspective

Chapter.1. Introduction

Since the Nobel prize for Physics was awarded to Andre Geim and Konstantin Novoselov “for groundbreaking experiments regarding the two-dimensional material graphene”, the eyes of the scientific world have been focused on this so-called miracle material. GO is a two-dimensional material of exceptional strength, unique optical, physical, mechanical, and electronic properties. Ease of functionalization and high antibacterial activity are two major properties identified with GO. Due to its excellent aqueous process ability, surface functionalization capability, surface enhanced Raman scattering (SERS), and fluorescence quenching ability, GO chemically exfoliated from oxidized graphite is considered a promising material for biological applications.

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CHAPTER-1Graphene as exfoliated from graphite, is hydrophobic, i.e., not dispersible in water, highly reactive and non-biocompatible. However, upon oxidation to form Graphene Oxide (GO), it becomes hydrophilic and therefore water soluble and is amenable to a host of biomedical applications.  Hybridization of GO with polymers, gold and magnetic nanoparticles results in carbon-related Nano composites used in a variety of biomedical and biotechnological applications such as for phototherapy, bio-imaging, drug and gene delivery, bio sensing, and antibacterial action.

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This data depicts the relative research activity on different graphene-family of materials for biological applications indicating that more than 50%of them (62.6%) are biomedical in nature.

Due to the small size, large surface area, and useful non covalent interactions with aromatic ring molecules, GO is a promising material for drug delivery.

For this reason the subject of GO toxicity has attracted special attention in order to increase the biosafety of GO.  

The valid evaluation and actual confirmation of the biocompatibility of GO is central to many scientific investigations.

CHAPTER-1

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Marcano et al.  prepared an improved method for the synthesis of GO.

They found that excluding NaNO3, increasing KMnO4

and a mixture of H2SO4/H3PO4 can improve the oxidation process.

A schematic presentation of their results is presented here.

According to their reaction protocol, the reaction is not exothermic and produces no toxic gas. This makes it useful for biomedical applications.

CHAPTER-1

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TABLE

Some of the major research on GO related biomedical application are presented here in the table.

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Chapter.2. Graphene Oxide Based Fluorometric Detection of Hydrogen

Peroxide in Milk

The analytical feature of our proposed method includes low detection limit (10 mmol mL−1) and satisfactory recovery values for samples.

The presence of H2O2 in milk is a major concern because it constitutes a public health hazard. Many milk industries are using H2O2 as a preservative, but if the concentration increases then it causes so many health problems such as neurodegenerative disorders, cancer and diabetes.

Present methods show an easy way for detecting H2O2 generally require considerable time and laboratory facilities. The chemical tests have sufficient sensitivity to detect wide linear range of H2O2 concentration.

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CHAPTER-2 In Fig. (a) typical field emission scanning electron

microscopy (FE-SEM) images of GO are shown.

In Fig. (b) the main absorbance peak attributable to π-π* transitions of C=C in synthesized GO occurs at around 232 nm.

In Fig. (c) the XRD spectra of GO show a distinct peak at 15.10° corresponding to a d-spacing (in this case, the interlayer distance between sheets) of approximately 7.15 Å that is due to interlamellar water trapped between hydrophilic GO sheets.

In Fig. (d) the Fourier transform infrared (FTIR) spectra of the GO clearly show the presence of carboxyl (O-H deformation 1730-1700 cm-1), hydroxyl (O-H stretching vibration 3450 cm-1), epoxy (750 cm-1), and carbonyl (C=O stretching 1050 cm-1) groups.

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CHAPTER-2 In Figure (a) the XPS spectra of GO are shown.

In Figure (b) the deconvolution of the C1s peak in the XPS spectrum shows the presence of four types of carbon bonds: C–C (284.8 eV), C–O (hydroxyl and epoxy, 288.2 eV), C=O (carbonyl, 292.7 eV), and O C–O (carboxyl, 294.8 eV).

By integrating the area of the deconvolution peaks, the approximate percentage obtained for C–C is 45.36%. Similarly, In Figure 2(c) the deconvolution of the O1s peak in the XPS spectrum shows the presence of one type of oxygen bond: O–H (530 eV).

By integrating the areas of the deconvolution peaks, the approximate percentage for O–H was obtained to be 38.72%.

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CHAPTER-2 In This Figure the photoluminescence spectra (PL)

of milk containing GO and DCFH-DA are shown.

The solution was excited at 350 nm and the emission spectra were obtained at 420 nm and 480 nm, respectively. Due to the absence of H2O2, the PL is mostly similar to the spectra of GO.

Initially, they show the distinctive band-edge absorption peak at 420 nm. As the stacking deposition proceeds, the absorption reaches to the shorter wavelengths down to 480 nm and becomes more featureless.

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CHAPTER-2 In this Figure the PL spectra of milk with GO,

DCFH-DA and H2O2 are shown. The solution was excited at 350 nm and the emission spectra were obtained at 530 nm.

This newly proposed method has been used to determine H2O2 in nine milk samples; A low limit of H2O2 (10 mmol mL−1) has been detected. When H2O2 was added into the milk at nine different concentrations (10, 15, 20, 25, 30, 35, 40, 45 and 50 mmol mL−1 of H2O2 are observed for all samples.

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Chapter.3. Study of antibacterial mechanism of Graphene Oxide using Raman Spectroscopy

Here we developed a new and sensitive fingerprint approach to study the antibacterial activity of GO and underlying mechanism, using Raman spectroscopy.

Spectroscopic signatures obtained from biomolecules such as Adenine and proteins from bacterial cultures with different concentrations of GO, allowed us to probe the antibacterial activity of GO with its mechanism at the molecular level.

Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) were used as model micro-organisms for all the experiments performed.

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CHAPTER-3 In general, the Raman spectrum of graphite exhibits a ‘G

band’ at 1580 cm-1 and a ‘D band’ at 1350 cm-1. The G

band is due to the first order scattering of the E2g mode

whereas the D band is related to the defect in the graphite lattice.

The Raman spectra of GO are shown in Figure, which show the presence of a G band at 1660 cm-1 and a D band at 1380 cm-1. The G band of GO is shifted towards a higher wave number, an observation that co-relates with the oxidation of graphite which results in the formation of sp3 carbon atoms.

Furthermore, the D band in the GO is broadened due to the size reduction of the in-plane sp2 domains during oxidation.

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CHAPTER-3

(a) SEM images of E. coli (control) (b) SEM images of E. coli when treated with 50µg/mL of GO (c) SEM images of E. coli when treated with 100µg/mL of GO (d) SEM images of E. coli when treated with 150µg/mL of GO.

As the concentration of GO increases the shape of E.coli changes.

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CHAPTER-3 Among the bands displaying obvious changes,

the 729 cm−1 band was assigned to Adenine. As the concentration of GO increased, the concentration of the Adenine ring mode increased, as shown in Figure.

The conformation about these bonds is related to the structure of the protein. The S-S stretching vibration occurred at 490 cm-1 as shown in Figure. The intensity of the S-S stretching vibrations increased as the concentration of GO increased.

Here, the Amide VI band (CO-NH bending vibration) occurred at 610 cm-1 which is shown in Figure. The intensity of Amide VI band (CO-NH bending vibration) increased as the concentration of GO increased.

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CHAPTER-3 Bio- AFM images of E.coli cells, before and after

treatment with various concentrations of GO, immobilized on a glass slide.

(a) Images of untreated E.coli, showing a height profile of 150.9 nm. (b) Images of E.coli after GO of 50 µg/mL concentration was added showing an increased height profile up to 207.8 nm. (c) Images of E.coli after GO of 100µg/mL concentration was added showing an increased height profile up to 329.1 nm. (d) Images of E.coli after GO of 150 µg/mL concentration was added showing an increased height profile up to 551.9 nm. (e) Plot of bacterial height profile (y-axis) vs. GO concentration (x-axis); as the concentration of GO increased the E.coli height profile increased.

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CHAPTER-3 Bio- AFM images of E. faecalis cells, before and after

treatment with various concentrations of GO, immobilized on a glass slide.

(a) Images of untreated E. faecalis, showing a height profile of 376.2 nm. (b) Images of E. faecalis after GO of 50 µg/mL concentration was added showing an increased height profile up to 571.2 nm. (c) Images of E. faecalis after GO of 100 µg/mL concentration was added showing an increased height profile up to 711.2 nm. (d) A concentration of 150µg/mL of GO was added to images E. faecalis after GO of 150 µg/mL concentration was added showing an increased height profile up to 727.7 nm. (e) Plot of bacterial height profile (y-axis) vs. GO concentration (x-axis); as the concentration of GO increased the E. faecalis, height profile increased.

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CHAPTER-3 Herewith, by using morphological and

spectroscopic data we have confirmed experimentally and theoretically that GO can induce the degradation of the outer and inner cell membranes of E. coli and E. faecalis bacteria. The work presented here demonstrates the great antibacterial action of GO is due to the release of Adenine and protein from Bacteria.

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Cystamine has been successfully conjugated with graphene oxide (GO) as a drug carrier.

The current study used the microdilu tion method to determine the minimum inhibitory concentrations of cystamine-conjugated GO against four types of pathogenic bacteria. Minimum inhibitory concentrations values were 1 μg/mL against Escherichia coli and salmonella typhimurium, 6 μg/mL against Enterococcus faecalis, and 4 μg/mL against Bacillus subtilis.

Toxicity of the conjugate against squamous cell carcinoma 7 cells was minimal at low concentrations, but increased in a dose-dependent manner.

Chapter.4. Oxidative stress and antibacterial properties

of a graphene oxide-cystamine nanohybrid

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CHAPTER-4

Magnified AFM images of GO showed its height 0.8 nm whereas cystamine-conjugated GO shows its height 1.2 nm.

SEM image showed conjugation of cystamine with GO which is confirmed by the reduction of the size of cystamine-conjugated GO. 2

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CHAPTER-4

Cytotoxicity and ROS studies of cystamine conjugated GO UV–Vis spectrum and FT-IR of cystamine, GO, and cystamine-conjugated GO. 2

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CHAPTER-4 XPS measurements provided additional

information about the nature of cystamine-conjugated GO. Both conjugated and unconjugated GO exhibited C=C (sp2), C-C (sp3), C=O (carbonyl), O-C=O (carboxyl), and C-O/C-O-C (hydroxyl and epoxy) groups).

Figure shows the C1S peaks of GO including C-O (hydroxyl and epoxy, 288.1 eV), C=O (carbonyl, 291.4 eV), C=C/C-C (284.7 eV), and O=C-O (carboxyl, 294.8 eV) species. The C=C/C-C peak was 45.36% of the total GO. C-O (hydroxyl and epoxy, 288.5 eV), C=O (carbonyl, 291.9 eV), and O=C-O (carboxyl, 294.5 eV) peaks are shown in Figure B and indicate cystamine conjugation with GO. The major species, C=C/C-C (284.7 eV), was reduced to 36.53% of the total.

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CHAPTER-4 In our study, the differential toxicity of

cystamine-conjugated GO toward Gram-negative bacteria compared to gram-positive bacteria may be related to differences in the natures of their cell walls.

A thin peptidoglycan layer (7−8 nm thickness) is present in gram-negative bacteria whereas a thick peptidoglycan layer (20−80 nm thickness) is present in gram-positive bacteria. The thicker peptidoglycan layer in gram-positive bacteria may explain why these bacteria are more resistant to the antibacterial effects of cystamine-conjugated GO. 2

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CHAPTER.5. FUTURE WORK & PERSPECTIVE

Photothermal therapy employs photosensitizing agents taken up by cells, which generate heat from light absorption, leading to photo-ablation of the cancer cell and subsequent death.

Deep penetration and little non specific photothermal heating in the NIR window are due to the low absorption of light by tissues, which are largely transparent in NIR wavelength range.

CHAPTER-5 Recently, photothermal therapy employing

graphene-oxide as the photo-absorbing agent has emerged as an alternative and promising noninvasive treatment for cancer cells as well as not-cancer related diseases, such as Alzheimer’s disease (AD). It can generate heat from optical energy, leading to the “burning” of cancer cells or AD amyloid- b peptides (A b).

Our current ability to synthesize, manipulate, modify, and functionalize GO in the form of nanohybrids opens a wide vista in the area of biomedical applications. As the living cell is of micrometer-size dimension, nanometer-size entities can be made to address various membrane cell receptors or enter the cell to deliver pharmaceuticals, genes, or heat.

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CHAPTER-5

In this figure, PLGA showed (C-O-C) bending vibration band at 1250 cm -1 ,(CH3) stretching vibration band at 1385 cm-1 and carbonyl band 690 cm-1.

Cystamine conjugated GO showed a temperature up to 650C at 633 nm laser irradiation.

In future, we will make GO composite with PLGA and check it for photothermal activity.

Moreover, PLGA has different intensity in finger print region. So, it can be used for surface enhanced Raman scattering (SERS).

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AMARNATH CA, NANDA SS, PAPAEFTHYMIOU GC, YI DK & PAIK U, (2013) NANOHYBRIDIZATION OF LOW-DIMENSIONAL NANOMATERIALS: SYNTHESIS, CLASSIFICATION, AND APPLICATION, CRITICAL REVIEWS IN SOLID STATE AND MATERIALS SCIENCES (IF:2.714 - PUBLISHED)

NANDA SS, AN SSA, YI DK, (2015) OXIDATIVE STRESS AND ANTIBACTERIAL PROPERTIES OF A GRAPHENE OXIDE-CYSTAMINE NANOHYBRID, INTERNATIONAL JOURNAL OF NANOMEDICINE (IF:4. 195 - PUBLISHED).

NANDA SS, PAPAEFTHYMIOU GC, YI DK, (2015) FUNCTIONALIZATION OF GRAPHENE OXIDE AND ITS BIOMEDICAL APPLICATIONS, CRITICAL REVIEWS IN SOLID STATE AND MATERIALS SCIENCES (IF:2.714 - PUBLISHED).

NANDA SS, KIM K, YI DK, (2015) GRAPHENE OXIDE BASED FLUOROMETRIC DETECTION OF HYDROGEN PEROXIDE IN MILK, JOURNAL OF NANOSCIENCE AND NANOTECHNOLOGY (IF: 1.339 - ACCEPTED).

NANDA SS, WANG T, KIM K, YI DK, (2015) STUDY OF ANTIBACTERIAL MECHANISM OF GRAPHENE OXIDE USING RAMAN SPECTROSCOPY, CURRENT APPLIED PHYSICS (IF: 2.1 - SUBMITTED)

NANDA SS, AN SSA, YI DK, (2015) MEASUREMENT OF CREATININE IN HUMAN PLASMA USING A FUNCTIONAL POROUS POLYMER STRUCTURE SENSING MOTIF, INTERNATIONAL JOURNAL OF NANOMEDICINE (IF:4. 195 –UNDER REVISION).

NANDA SS, AN SSA, YI DK, (2015) RAMAN SPECTRUM OF GRAPHENE OXIDE, ITS DERIVATIVES AND GRAPHENE OXIDE HYBRID MATERIALS, TRENDS IN ANALYTICAL CHEMISTRY (IF: 6. 6 – UNDER PREPARATION)

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