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Design and synthesis of organic small molecules for industrial and biomedical technology nanomaterial augmentation

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Design and synthesis of organic small molecules for industrial and biomedical technology nanomaterial augmentation
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Chapman, James Vincent ( author )
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Photothermal spectroscopy ( lcsh )
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Organic chemistry used to augment nanoparticles and nanotubes, as well as more traditional materials, is a subject of great interest across multiple fields of applied chemistry. Herein we present an example of both nanoparticle and nanotube augmentation with organic small molecules to achieve an enhanced or otherwise infeasible application. The first chapter discusses the modification of two different types of Microbial Fuel Cell (MFC) anode brush bristle fibers with positive surface charge increasing moieties to increase quantitative bacterial adhesion to these bristle fibers, and therefore overall MFC electrogenicity. Type-1 brush bristles, comprised of polyacrylonitrile, were modified via the electrostatic attachment of 1-pyrenemethylamine hydrochloride. Type-2 brush bristles, comprised of nylon, were modified via the covalent attachment of ethylenediamine. Both modified brush types were immersed in an E. Coli broth for 1 hour, stained with SYTO® 9 Green Fluorescent Nucleic Acid Stain from ThermoFisher Scientific (SYTO-9), and examined under a Biotek Citation 3 fluorescent microscope to visually assess differences in bacterial adherence. In both trials, a clear increase in amount of bacterial adhesion to the modified bristles was observed over that of the control. The second chapter demonstrates a potential biomedical technology application wherein a polymerizable carbocyanine-type dye was synthesized and bound to a chitosan backbone to produce a water-soluble photothermal nanoparticle. Laser stimulation of both free and NP-conjugated aqueous solutions of the carbocyanine dye with Near-Infrared (NIR) Spectrum Radiation showed an increase in temperature directly correlated with the concentration of the dye which was more pronounced in the free particle solutions.
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Thesis (M.S.)--University of Colorado Denver.
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James Vincent Chapman III.

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Full Text
DESIGN AND SYNTHESIS OF ORGANIC SMALL MOLECULES FOR INDUSTRIAL
AND BIOMEDICAL TECHNOLOGY NANOMATERIAL AUGMENTATION
by
JAMES VINCENT CHAPMAN III B.S., University of Colorado Denver, 2015
A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Chemistry Program
2017


2017
JAMES VINCENT CHAPMAN III ALL RIGHTS RESERVED
11


This thesis for the Master of Science degree by James Vincent Chapman III has been approved for the Chemistry Program by
Jung-Jae Lee, Chair Scott Reed Xiaotai Wang
Date: May 13, 2017


Chapman III, James Vincent (M.S. Chemistry Program)
Design and Synthesis of Organic Small Molecules for Industrial and Biomedical Technology
Nanomaterial Augmentation
Thesis directed by Associate Professor Jung-Jae Lee
ABSTRACT
Organic chemistry used to augment nanoparticles and nanotubes, as well as more traditional materials, is a subject of great interest across multiple fields of applied chemistry. Herein we present an example of both nanoparticle and nanotube augmentation with organic small molecules to achieve an enhanced or otherwise infeasible application. The first chapter discusses the modification of two different types of Microbial Fuel Cell (MFC) anode brush bristle fibers with positive surface charge increasing moieties to increase quantitative bacterial adhesion to these bristle fibers, and therefore overall MFC electrogenicity. Type-1 brush bristles, comprised of polyacrylonitrile, were modified via the electrostatic attachment of 1-pyrenemethylamine hydrochloride. Type-2 brush bristles, comprised of nylon, were modified via the covalent attachment of ethylenediamine. Both modified brush types were immersed in an E. Coli broth for 1 hour, stained with SYTO 9 Green Fluorescent Nucleic Acid Stain from ThermoFisher Scientific (SYTO-9), and examined under a Biotek Citation 3 fluorescent microscope to visually assess differences in bacterial adherence. In both trials, a clear increase in amount of bacterial adhesion to the modified bristles was observed over that of the control. The second chapter demonstrates a potential biomedical technology application wherein a polymerizable carbocyanine-type dye was synthesized and bound to a chitosan backbone to produce a water-soluble photothermal nanoparticle. Laser stimulation of both free and NP-conjugated aqueous solutions of the carbocyanine dye with Near-
IV


Infrared (NIR) Spectrum Radiation showed an increase in temperature directly correlated with the concentration of the dye which was more pronounced in the free particle solutions.
The form and content of this abstract are approved. I recommend its publication
Approved: Jung-Jae Lee
v


ACKNOWLEDGEMENTS
This work acknowledges the contributions of fellow Lee Lab members Venkatesan Perumal, Hunter Sauerland, Selina Vong, and Rupinder Kaur as well as principle investigator Jung-Jae Lee and collaborators Jae-Do Park of the University of Colorado at Denver Engineering Department and Timberly Roane of the University of Colorado at Denver Biology
Department.
vi


TABLE OF CONTENTS
I. SMALL-MOLECULE MODIFICATION OF MICROBIAL FUEL CELL BRUSH
FIBERS.............................................................1
Introduction....................................................1
Materials and Methods...........................................3
Results.........................................................8
Conclusion......................................................10
II. SYNTHESIS OF WATER-SOLUBLE PHOTOTHERMAL DYES EXCITABLE BY
NEAR-INFRARED SPECTRUM RADIATION...................................11
Introduction....................................................11
Materials and Methods...........................................12
Results.........................................................14
Conclusion......................................................18
REFERENCES............................................................20
APPENDIX
A. Supplemental Data
22


LIST OF FIGURES
Figure 1. Basic repeating structure of polyacrylonitrile, which comprises the bristle component of Mil-Rose bushes under the brand name PX35...............................3
Figure 2. Illustration of the cell membrane-1-pyrenemethylamine hydrochloride-polyacrylonitrile electrostatic
coordination.........................................................................4
Figure 3. Nanotube brush comparison modified vs unmodified...........................8
Figure 4. Nylon brush comparison modified vs unmodified..............................9
Figure 5. ICG fluorescence demonstrating bacterial adhesion to modified nanotube bristles.............................................................................9
Figure 6. ICG fluorescence demonstrating bacterial adhesion to modified nylon bristles... 10
Figure 1A. Side-by-side fluorescent microscopy of modified versus unmodified polyacrylonitrile under DAPI spectrum stimulation...................................22
Figure 2A. Proton NMR of dicarboxlic acid carbocyanine dye precursor................23
Figure 3A. Proton NMR of dicarboxlic acid carbocyanine dye product, with literature spectrum/integration overlay for comparison.........................................24
viii


LIST OF GRAPHS
Graph 1. Change in temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm"2, and laser intensity of 1207 mA......................15
Graph 2. Change in temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm"2, and laser intensity of 2500 mA......................16
Graph 3. Change in temperature with respect to time for varying concentrations of chitosan-carbocyanine nanoparticle conjugate aqueous solutions using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm"2, and laser intensity of 1207 mA... 17
Graph 4. Change in temperature with respect to time for free versus conjugated aqueous solutions of carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm"2, and laser intensity of 1207 mA..........................18
Graph 1A. Temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm"2, and laser intensity of 1207 mA........................................25
Graph 2A. Temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm"2, and laser intensity of 2500 mA........................................26
IX


LIST OF SCHEMES
Scheme 1. Proposed mechanism for attachment of linking agent to nylon polymer subunit ........................................................................................5
Scheme 2. Proposed mechanism for attachment of fluorescent marker molecules........6
Scheme 3. Proposed mechanism for attachment of ethylenediamine for functionalized augmented bristles......................................................................7
Scheme 4. Synthetic pathway for dicarboxylic acid carbocyanine part 1 of 3.12
Scheme 5. Synthetic pathway for dicarboxylic acid carbocyanine part 2 of 3.13
Scheme 6. Synthetic pathway for dicarboxylic acid carbocyanine part 3 of 3.13
Scheme 7. Dr. Perumals reaction of carbocyanine dye with chitosan polymer to form the
conjugate nanoparticle.............................................................14
Scheme 1A. Proposed chemical mechanism for carbocyanine dye synthesis part 1 of 9... .27
Scheme 2A. Proposed chemical mechanism for carbocyanine dye synthesis part 2 of 9... .27
Scheme 3A. Proposed chemical mechanism for carbocyanine dye synthesis part 3 of 9... .28
Scheme 4A. Proposed chemical mechanism for carbocyanine dye synthesis part 4 of 9... .28
Scheme 5A. Proposed chemical mechanism for carbocyanine dye synthesis part 5 of 9... .29
Scheme 6A. Proposed chemical mechanism for carbocyanine dye synthesis part 6 of 9... .29
Scheme 7A. Proposed chemical mechanism for carbocyanine dye synthesis part 7 of 9... .29
Scheme 8A. Proposed chemical mechanism for carbocyanine dye synthesis part 8 of 9... .30
x


Scheme 9A. Proposed chemical mechanism for carbocyanine dye synthesis part 9 of 9... .30
xi


LIST OF ABBREVIATIONS
CC: Cyanuric Chloride
DAPI: 4',6-Diamidino-2-Phenylindole, Dihydrochloride
DIEA: N,N-Diisopropylethylamine
EDC: N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide
FDA: United States Food and Drug Administration
ICG: Indocyanine Green
MFC: Microbial Fuel Cell
NHS: N-Hydroxysuccinimide
NP: Nanoparticle
NT: Nanotube
RMP: Resting Membrane Potential
SYTO-9: SYTO 9 Green Fluorescent Nucleic Acid Stain from ThermoFisher Scientific
TEA: Triethylamine
UCD: University of Colorado at Denver
Xll


CHAPTER I
SMALL-MOLECULE MODIFICATION OF MICROBIAL FUEL CELL BRUSH
FIBERS
Introduction
Microbial Fuel Cells (MFC)s function on the same principle as electrochemical batteries using the transfer of electrons along a conductive material from anode to cathode to produce electrical current. The difference is that in MFCs the electron source consists of living bacteria which transfer electrons onto an external electron acceptor as a natural path of their electron transport chain in oxidative respiration.1 MFCs are attractive as a renewable source of energy because bacteria can be sustained on wastewater nutrients such as acetate. Furthermore, because the bacterial cultures are permanent, so long as their environment remains constant they are suitable for applications where replenishing fuel might otherwise present logistical difficulties, an example being the power source for batteries in deep-sea sensors. Because this process requires physical contact between the bacteria and the anode electron acceptor the electrogenicity of MFCs is directly correlated with anode surface area. Under current methods, MFC anodes are cylindrical brushes where the brush bristle fibers serve as the main site of bacterial adhesion. Modification of these fibers to increase bacterial adhesion is the main focus of improving MFC efficacy.3,45
Biological cells have a net negative surface charge typically in the range of -10 to -120 mV as a result of the voltage gradients generated by membrane-embedded ion pumps which is commonly referred to as Resting Membrane Potential (RMP). Because of this RMP, studies have shown that microbial adhesion to an anode can be increased through electrostatic attraction by attaching a slight positive surface charge to the surface of the
1


anode.6,7,8 The augmentation of MFC brush bristle fibers herein was an expansion on that concept. Chemical modifiers were used to coat the MFC bristle surface in a positive charge to increase microbial surface adhesion.
Nanotube (NT) macromolecules are becoming increasingly common in engineering applications due to their high mechanical strength, surface area, and resistance to corrosion. However, this same inertness can be a hindrance in that nanotube structures are difficult to covalently modify. Therefore the procedure for modification of Type-1 brushes utilized pi-pi stacking of 1-pyrenemethylamine hydrochloride onto the polyaromatic NT as a simple method by which the surface-charge properties of nanotubes could be modified via the application of a charged coating around the nanotube rather than by direct covalent modification, an otherwise expensive and mechanically-demanding task.
Materials and Methods
Type-2 brushes were of a nylon-Fe 2+make. This selection was made because although NTs have superior tensile strength and corrosion resistance, their relatively high cost in comparison to other materials often makes them economically nonviable in large-scale implementation. Most currently-existing MFCs use nylon bristle fibers. However, because nylon is less inert than nanomaterials, covalent modification of the brush was possible. Using cyanuryl chloride as a linking agent, these brush bristle were covalently bound to ethylenediamine.
Post-modification, both brush types (and an unmodified control for each) were immersed in the same E. Coli broth for 1 hour. Microbes were then stained with SYTO-9 and
2


examined under a Citation 3 fluorescent microscope so that differences in adherence could be observed visually.
For the modification of polyacrylonitrile nanotube brush fibers, Mill-Rose brush fibers were graciously provided by Dr. Jae Do Park of the University of Colorado engineering department. 1-pyrenemethylamine was purchased from Sigma-Aldrich. Mill-rose brushes (purchased from Zoltek) use bristles comprised of a substance brand named PX35 which is chemically polyacrylonitrile a nanotube structure shown in Figure 1.
n
Figure 1. Basic repeating structure of polyacrylonitrile, which comprises the bristle component of Mil-Rose
bushes under the brand name PX35.
Because direct chemical modification of polyaromatic systems is synthetically difficult, pi-stacking the naturally-occurring quadrupole attraction between conjugated pi-systems -was used to orient the modifying ligands to the surface of the nanotube. The desired noncovalent coordination between the modified bristles and microbial cell membrane is modelled in Figure 2.
3


Figure 2. Illustration of the cell membrane-l-pyrenemethylamine hydrochloride-polyacrylonitrile noncovalent coordination in T-shaped stacked configuration. Not pictured are parallel-displaced and sandwich oriented pi
stacking.
Adherence of the 1-pyrenemethylamine hydrochloride modifying groups to the polyacrylonitrile brush fibers91011 was achieved by mixing a 373 pM solution of 1-pyrenemethylamine in Milli-Q water and combining ~10 mL of this solution with strands of the Mill-Rose brush fibers in a sealed vortex tube. Mixing was performed with a Branson Sonifter 450 sonicator. Energy was applied to the system under a high-power setting for 5 minutes. After sonication, bristles were washed with Milli-Q water, drained, and freeze-dried overnight. We analyzed the dried bristles under fluorescent microscopy using a Biotek Cytation 3 Imaging Reader in the DAPI spectrum (excitation/emission peaks centered on 358 nm / 461 nm).
4


For the second brush type, a nylon-Fe2+ brush was provided by Dr. Timberly Roane of the UCD Biology Department. In this augmentation procedure,12 initially a mixture of triethylamine, nylon bristles, and cyanuryl chloride was vortexed for 24 hours in a solution at room temperature CH3CI. The proposed chemical mechanism is viewable in Scheme 1.
Scheme 1. Proposed mechanism for attachment of linking agent to nylon polymer subunit. Coordination of the TEA base activates the amidal nitrogen lone pair to prepare it for nucleophilic attack on the cyanuric chloride. Note this step is the same in both test and practical procedures.
For the test procedure, this solution was drained, the bristles were washed three times with CH3CI and then re-immersed in a 373 pM solution of 1-pyrenemethylamine in CH3CI. (cite dudes) To this was added excess triethylamine. This mixture was vortexed for 24 hours, then drained, washed three times with acetone, and analyzed using a Biotek Cytation 3 Imaging Reader in the DAPI spectrum. The proposed chemical mechanism is viewable in Scheme 2.
5


Scheme 2. Proposed mechanism for attachment of fluorescent marker molecules. Only one attachment is depicted for the sake of clarity but note that substitution occurs on both meta-position chlorides.
After confirming ligand attachment efficacy via fluorescent microscopy in the same manner as the polyacrylonitrile bristle trials, the procedure was modified to use ethylenediamine as the source of charge. Although 1-pyrenemethylamine hydrochloride-CC-nylon complex displayed desired electrostatic properties, we desired a demonstration of a cost-effective procedure and 1-pyrenemethylamine hydrochloride is relatively expensive compared to ethylenediamine. So for the practical whole-brush modification procedure, the scheme was altered to use ethylenediamine as the source of charge. The proposed chemical mechanism for the practical procedure is viewable in Scheme 3.
6


4.
Scheme 3. Proposed mechanism for attachment of ethylenediamine for functionalized augmented bristles. Using this modified procedure, an entire brush was treated and from this sample bristles were
removed to be assessed based on microbial binding efficiency.
To demonstrate bacterial adhesion, four sets of bristles (modified polyacrylonitrile, unmodified polyacrylonitrile, modified nylon, and unmodified nylon) were cut from respective brushes and immersed in a both of E. Coli for 1 hour, then removed and rinsed. E. Coli were stained with SYTO-9. These bristles were analyzed via fluorescent microscope to assess microbial adherence.
7


Results
After washing to remove unattached residue, the both the polyacrylonitrile as well as the nylon brush bristles showed the desired fluorescent hotspots, which were absent from the unmodified control bristles. A comparison between the modified and unmodified polyacrylonitrile bristle fibers under fluorescent microscopy may be viewed in Figure 3.
Figure 3. An unmodified polyacrylonitrile brush bristle (left) and an experimentally modified bristle (right) viewed under DAPI-spectrum fluorescent microscopy.
These data showed the desired behavior, since unmodified polyacrylonitrile does not fluoresce in the examined spectra, appearance of fluorescent hotspots meant that the 1-pyrenemethylamine hydrochloride had successfully coordinated to the bristle surface. Augmented vs unmodified nylon fibers displayed similar behavior under fluorescent microscopy and may be viewed in Figure 4.
8


Figure 4. An unmodified nylon brush bristle (left) and an experimentally modified bristle (right) viewed under
DAPI-spectrum fluorescent microscopy.
In this manner, the adherence of 1-pyrenemethylamine hydrochloride to both polyacylonitrile
and nylon surfaces through respective procedures was confirmed.
In the microbial adherence trials, stained E. Coli were visible in much greater quantities on the augmented strands than the unmodified. A side-by-side comparison of microbes adhered to the augmented polyacrylonitrile bristle versus the unmodified bristle may be viewed in Figure 5.
Figure 5. ICG fluorescence demonstrating bacterial adhesion to modified nanotube bristles. Green Dots are
stained A. Coli.
A similar trend was observed in the nylon bristle trials, a side-by-side comparison of which may be viewed in Figure 6.
9


Figure 6. ICG fluorescence demonstrating bacterial adhesion to modified nylon bristles. Green Dots are stained
E. Coli.
In both trials limited amounts of microbial growth was observed on the unmodified bristles,
but in comparatively smaller quantities than were observed on the augmented bristles.
Conclusion
In both trials, treated brush fibers were shown to have higher levels of bacterial adhesion than untreated fibers. Effect on long-term MFC power generation is still under investigation, but the chemical modification procedure has been proven. Future research could investigate polymerization to construct a scaffold around the nanotube brushes, as well as different modifying groups. This is especially true in the case of the nylon brushes, where it could be investigated if other polymers could be attached using the same linking agent. As a proof of concept, this study demonstrates that bristles modified by small molecule attachment can be used to improve bacterial adhesion over unmodified bristles.
10


CHAPTER II
WATER-SOLUBLE PHOTOTHERMAL DYE-NANOPARTICLE CONJUGATES EXCITABLE BY NEAR-INFRARED SPECTRUM RADIATION_FOR USE IN BIOMEDICAL NANOTECHNOLOGY
Introduction
Conjugate dyes which absorb electromagnetic radiation in the MR spectrum (-680-900 nm) are of particular interest to biomedical technology applications because they provide a tissue-penetrating method for the targeted application of heat with minimal energy loss to interference from ambient water.13 In particular, this is a subject of research in anti-cancer therapies because at temperatures of 42 C and higher, tumor cell membranes begin to lyse from heat shock. Photothermal dyes, therefore, can potentially provide a non-invasive method of killing tumor cells. Small-molecule medical biotechnology has received increased interest in recent years for this reason. In this experiment we synthesized a dicarboxylic-acid-substituted carbocyanine dye which incorporates weakly acidic moieties that ionize under physiological pH to generate charged structures which increase water solubility. Moreover, these function as the carboxylate moiety necessary to initiate EDC coupling and can therefore be conjugated onto a suitable polymer backbone through amide bonds. This property allows the molecule to serve as a nanoparticle augment for targeting applications. For this reason the carbocyanine dye synthesized in this experiment is suitable for use in both free-particle aqueous suspensions as well as nanoparticle conjugates. The free particles were subjected to laser stimulation in varying concentrations to assess efficacy in achieving the desired 5 C temperature increase (from physiological temperature of 37 C to tumor-lysing temperature of 42 C). Furthermore, since NPs are crucial in a targeted drug-delivery system,
11


a trial of these carbocyanine dyes was conjugated to chitosan nanoparticles to assess the efficacy of chitosan as a nanoparticle delivery system.
Materials and Methods1415
A mixture of l,l,2-trimethyl-[lH]-benz[e]indole and 3-bromopropanoic acid (1:1 ratio) in 1,2-dichlorobenzene (5 mL per gram of l,l,2-trimethyl-[lH]-benz[e]indole) was heated with stirring at 110 C for 18 hrs. Product was cooled and filtered over vacuum, titurating with DCM to yield a light tan powder. (74.1 % yield) The scheme of this first synthetic step may be viewed in Scheme 4.
Scheme 4. Synthetic pathway for dicarboxylic acid carbocyanine part 1 of 3.
This precipitate was re-dissolved in a 9::1 mixture of MeCN::Water and 1 equivalent of sodium acetate was added. This was called Part A. In a separate flask, cooled to 0 C, glutaconaldehyde dianil monohydrochloride, acetic anhydride, and N,N-diisopropylethylamine (DIEA) were mixed in equimolar amounts (~0.6 equivalents of step 1 product) in DCM in a ratio of 5 mL per gram glutaconaldehyde dianil monohydrochloride This was called Part B. The scheme of this first synthetic step may be viewed in Scheme 5.
12


9 3
Scheme 5. Synthetic pathway for dicarboxylic acid carbocyanine part 2 of 3.
After 1 hour of stirring, Part A was set to reflux and to this, Part B was added dropwise. This solution was then allowed to reflux for 24 hrs. To purify, this solution was cooled to room temperature and filtered over vacuum, triturating with ethyl ether, MeCN, and 3.5 % HC1. The final product was a khaki-green powder. (42.6 % yield.) The scheme of this first synthetic step may be viewed in Scheme 6.
Scheme 6. Synthetic pathway for dicarboxylic acid carbocyanine part 3 of 3.
Final product was purified by filtration over vacuum, titurating with MeCN, ethyl ether, and
3.5% HC1. This pure product was then redissolved in water and using to prepare sample
solutions of varying concentration. Each sample was irradiated using an Arroyo Instruments
6300 Series Laser Diode Controller and temperature of the irradiated solution was recorded
every 30 seconds to assess the relationship between solute concentration and temperature
over time.
13


For the final carbocyanine dye trial, the synthesized dye molecules were attached via EDC coupling onto a chitosan backbone by Dr. Venkatesan Perumal of Lee Lab to form a nanoparticle conjugate. A representation of this polymerization step may be viewed in Scheme 7.
EDC (4 equivalents) NHS (10 equivalents)
Scheme 7. Reaction of carbocyanine dye with chitosan polymer to form the conjugate nanoparticle. In this depiction, chitosan appears simplified to its constituent D-glucosamine monomeric subunit for clarity.
This conjugate was then subject to the same dissolution in water and stimulation by laser as the free-particle trials in the same concentrations (calculated by mass of the carbocyanine used in synthesis) to assess efficacy of the photothermal effect of the free-floating carbocyanine versus the NP conjugate.
Results
Laser-stimulation trials of different concentrations of the dye dissolved in water
showed a quantitative increase in solution temperature positively correlated with the concentration of carbocyanine dye in solution. These data, laid out in graph format, may be viewed in Graph 1.
14


Change in Temperature with Respect to Time for Varying Concentrations of Low-Intensity Laser-Stimulated Carbocyanine
Dye Solutions
-water --50 pM --25 pM 5 pM --2.5 pM --0.5 pM ----Lysis Threshold
Graph 1. Change in temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 mn, power density of 200 mWcnr2, and laser intensity of 1207 inA. Laser analysis procedure performed by Selina Vong at Lee Lab.
The optimal concentration found to elicit the desired 5 C increase in temperature for free
particles at low stimulation intensity was found to be 5 pM. Of note is that the 2.5 pM
concentration solution came close (AT = 4.8 C) to the target threshold, so a solution
concentration between 2.5 and 5 pM might be a good target concentration range for in vivo applications. Solutions excited at a higher intensity showed a faster rise in initial temperature but reached the same asymptotic plateau as those excited at the lower intensity. These data, laid out in graph format, may be viewed in Graph 2.
15


Change in Temperature with Respect to Time for Varying Concentrations of High-Intensity Laser-Stimulated Carbocyanine
Dye Solutions
water 50 pM 25 pM -A-5pM 2.5 pM 0.5 pM
---Lysis Threshold
Graph 2. Change in temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine aqueous solutions using a radiant excitation source of wavelength of 785 mn, power density of 200 mW cnr2, and laser intensity of 2500 inA. Laser analysis procedure performed by Selina Vong at Lee Lab.
The optimal concentration found to elicit the desired 5 C increase in temperature for free particles at high stimulation intensity was found to be 5 pM identical to the low-intensity
trials. From this is it shown that the final change in temperature is independent of laser
intensity and dependent on solution concentration of photothermal dyes.
Chitosan-Carbocyanine NPs displayed water solubility as did the free particles, however under laser stimulation the ratio of change in temperature to carbocyanine concentration was consistently lower than was observed for analogous concentrations in the free particle trials. These data, laid out in graph format, may be viewed in Graph 3.
16


Graph 3. Change in temperature with respect to time for varying concentrations of chitosan-carbocyanine nanoparticle conjugate aqueous solutions using a radiant excitation source of wavelength of 785 mn, power density of 200 mWcnr2, and laser intensity of 1207 inA. Laser analysis procedure performed by Selina Vong at
Lee Lab.
In the case of the chitosan-carbocyanine NPs, the photothermal effect was less pronounced and displayed less consistent behavior than was observed in free-particle trials with the same concentration and laser settings. A comparison between the two types of photothermal solutions with all other factors held constant may be viewed in Graph 4.
17


Change in Temperature with Respect to Time for Similar Concentrations of Free Carbocyanine and Nanoparticle Conjugate
Graph 4. Change in temperature with respect to time for free versus conjugated aqueous solutions of carbocyanine dye using a radiant excitation source of wavelength of 785 mu, power density of 200 mWcnr2,
and laser intensity of 1207 inA.
Chitosan-carbocyanine NP solutions achieved the desired temperature increase but only in concentrations an order of magnitude higher than was necessary for free particle carbocyanine solutions.
Conclusion
The synthesized carbocyanine dye molecules were soluble in water and a photothermal effect in the NIR spectrum was observed. Solution potential to raise temperature was found to be independent of laser intensity and dependent on carbocyanine solute concentration. Ideal dye concentration to achieve the target temperature increase of 5 C was found to be ~5 pM in free particles but required a tenfold increase in concentration to cross that threshold in conjugated NP solutions. While the maximum safe dosage of the carbocyanine dye molecules used in this experiment has not yet been established in humans,
18


mouse trials have undergone 8.5 mg-kg'1 (13.6 pM) injections in experimental settings16 and the FDA-approved chemically-similar carbocyanine dye ICG has a maximum clinical dose of 2 mg-kg"1 (2.6 pM).17 Therefore, although it cannot be stated definitively without further study, the synthesized carbocyanine dyes have good potential suitability for use in biomedical applications. However, it is likely that a different delivery system than chitosan nanoparticles will need to be investigated. Future research may investigate alternative NP conjugates or whether chitosan can be modified to attenuate the observed dampening effect.
19


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13. Ye, Yunpeng; Bloch, Sharon; Kao, Jeffery; and Achilefu, Samuel. Multivalent Carbocyanine Molecular Probes: Synthesis and Applications. Bioconjugate Chem. 2005, 16, 51-61
14. Winstead, Angela J.; Nyambura, Grace; Matthews, Rachael; Toney, Deveine; Oyaghire, Stanley. Synthesis of Quaternary Heterocyclic Salts. Molecules; 18(11): 14306-14319. doi:10.3390/moleculesl81114306. 2014
15. Suganami, Akiko; Toyota, Taro; Okazaki, Shigetoshi; Saito, Kengo; Miyamoto, Katrsuhiko; Akutsu, Yasunori; Kawahira, Hiroshi; Aoki, Akira; Muraki, Yutaka; Madono, Tomoyuki; Hayashi, Hideki; Matsubara, Hisahiro; Omatsu, Takashige; Shirasawa, Hiroshi, Tamura, Yutaka. Preparation and characterization of phospholipid-conjugated indocyanine green as a near-infrared probe. Bioorganic & Medicinal Chemistry Letters 22 (2012) 7481-7485
16. Deng, Yibin; Huang, Li; Yang, Hong; Ke, Hengte; He, Hui; Guo, Zhengqing; Yang, Tao; Zhu, Aijun; Wu, Hong; Chen, Huabing. Cyanine-Anchored Silica Nanochannels for Light-Driven Synergistic Thermo-Chemotherapy. Small 2017, 13, 1602747
17. https://www.drugs.com/pro/indocyanine-green.html (Accessed April 13, 2017)
21


APPENDIX A
SUPPLEMENTAL DATA
Figure 1A. Side-by-side fluorescent microscopy of modified versus unmodified polyacrylonitrile under DAPI
spectrum stimulation.
22


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1000
900
800
700
600
500
400
300
200
100
0
-100
13.0 125 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 25 2.0 1.5 1.0 0.5
fl (ppm)
Figure 2A. Proton NMR of dicarboxlic acid carbocyanine dye precursor.
23


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8.4 8.2 8.0 7.8 7.6 7.4 7.2
6.6
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1700
1600
1500
1400
-1300
1200
-1100
-1000
-900
SOD
700
600
500
-400
-300
-aoo -100 -o -100
B.5 8.4 8.3 8.2 8.1 80 7.9 7.B 7.7 7.6 7.5 7.4 73 7.2 7.1 7.0 6.9 6.8 6.7 6j6 6.5 6.4 6.3 62 6.1
(ppffl) n
Figure 3A. Proton NMR of dicarboxlic acid carbocyanine dye product, with literature spectrum/integration
overlay for comparison.
24


Graph 1A. Temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 mn, power density of 200 mWcm2, and laser intensity
of 1207 mA.
25


Graph 2A. Temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 mn, power density of 200 mWcm2, and laser intensity
of 2500 mA.
26


1.
Scheme 1A. Proposed chemical mechanism for carbocyanine dye synthesis part 1 of 9. Waste products omitted
for clarity.
Scheme 2A. Proposed chemical mechanism for carbocyanine dye synthesis part 2 of 9. Waste products omitted
for clarity.
27


3.
Scheme 3A. Proposed chemical mechanism for carbocyanine dye synthesis part 3 of 9. Waste products omitted
for clarity.
Scheme 4A. Proposed chemical mechanism for carbocyanine dye synthesis part 4 of 9. Waste products omitted
for clarity.
28


5.
Scheme 5A. Proposed chemical mechanism for carbocyanine dye synthesis part 5 of 9. Waste products omitted
for clarity.
Scheme 6A. Proposed chemical mechanism for carbocyanine dye synthesis part 6 of 9. Waste products omitted
for clarity.
Scheme 7A. Proposed chemical mechanism for carbocyanine dye synthesis part 7 of 9. Waste products omitted
for clarity.
29


8.
Scheme 8A. Proposed chemical mechanism for carbocyanine dye synthesis part 8 of 9. Waste products omitted
for clarity.
9.
Scheme 9A. Proposed chemical mechanism for carbocyanine dye synthesis part 9 of 9. Waste products omitted
for clarity.
30


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DISS_title DESIGN AND SYNTHESIS OF ORGANIC SMALL MOLECULES FOR INDUSTRIAL AND BIOMEDICAL TECHNOLOGY NANOMATERIAL AUGMENTATION
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DISS_para Organic chemistry used to augment nanoparticles and nanotubes, as well as more traditional materials, is a subject of great interest across multiple fields of applied chemistry. Herein we present an example of both nanoparticle and nanotube augmentation with organic small molecules to achieve an enhanced or otherwise infeasible application. The first chapter discusses the modification of two different types of Microbial Fuel Cell (MFC) anode brush bristle fibers with positive surface charge increasing moieties to increase quantitative bacterial adhesion to these bristle fibers, and therefore overall MFC electrogenicity. Type-1 brush bristles, comprised of polyacrylonitrile, were modified via the electrostatic attachment of 1-pyrenemethylamine hydrochloride. Type-2 brush bristles, comprised of nylon, were modified via the covalent attachment of ethylenediamine. Both modified brush types were immersed in an E. Coli broth for 1 hour, stained with SYTO 9 Green Fluorescent Nucleic Acid Stain from ThermoFisher Scientific (SYTO-9), and examined under a Biotek Citation 3 fluorescent microscope to visually assess differences in bacterial adherence. In both trials, a clear increase in amount of bacterial adhesion to the modified bristles was observed over that of the control. The second chapter demonstrates a potential biomedical technology application wherein a polymerizable carbocyanine-type dye was synthesized and bound to a chitosan backbone to produce a water-soluble photothermal nanoparticle. Laser stimulation of both free and NP-conjugated aqueous solutions of the carbocyanine dye with Near-Infrared (NIR) Spectrum Radiation showed an increase in temperature directly correlated with the concentration of the dye which was more pronounced in the free particle solutions.
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PAGE 1

DESIGN AND SYNTHESIS OF ORGANIC SMALL MOLECULES FOR INDUSTRIAL AND BIOMEDICAL TECHNOLOGY NANOMATERIAL AUGMENTATION by JAMES VINCENT CHAPMAN III B.S., University of Colorado Denver, 2015 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Chemistry Program 2017

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ii 2017 JAMES VINCENT CHAPMAN III ALL RIGHTS RESERVED

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iii This thesis for the Master of Science degree by James Vincent Chapman III has been approved for the Chemistry Program by Jung Jae Lee Chair Scott Reed Xiaotai Wang Date: May 13, 2017

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iv Chapman III James Vincent (M.S. Chemistry Program ) Design and Synthesis of Organic Small Molecules for Industrial and Biome dical Technology Nanomaterial Augmentation Thesis directed by Associate Professor Jung Jae Lee ABSTRACT Organic chemistry used to augment nanoparticle s and n anotubes as well as more traditional materials, is a subject of great interest across multiple fields of applied chemistry. Herein we present an example of both nanoparticle and nanotube augmentation with organic small molecules to achieve an enhanced or otherwise infeasible application The first cha pter discusses the modification of two different types of Microbial Fuel Cell (MFC) anode brush bristle fibers with positive surface charge increasing moieties to increase quantitative bacterial adhesion to these bristle fibers, and therefore overall M FC e lectrogenicity Type 1 brush bristles, comprise d of polyacrylonitrile, were modified via the electrostatic att achment of 1 pyrenemethylamine hydrochloride Type 2 brush bristles, comprised of nylon, were modified via the cov alent attachment of ethylenediamine Both modified brush types were immersed in an E. Coli bro th for 1 hour, stained with SYTO 9 Green Fluorescent Nucleic Acid St ain from ThermoFisher Scientific ( SYTO 9 ) and examined under a Biotek Citation 3 fluorescent microscope to visually assess differences in bacterial adherence. In both trials, a clear increase in amount of bacterial adhesion to the modified bristles was observed over that of the control. The second chapter demonstrates a potential biomedical techn ology application wherein a polymeri zable carbocyanine type dye was synthesized and bound to a chitosan backbone to produce a water soluble photothermal nanoparticle Laser stimulatio n of both free and NP conjugated aqueous solutions of the carbocyanine dye with Near

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v Infrare d (NIR) Spectrum Radiation showed an increase in temperature directly correlated with the concentrati on of the dye which was more pronounced in the free particle so lutions. The form and content of this abstract are approved. I recommend its publication Approved: Jung Jae Lee

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vi ACKNOWLEDGEMENTS This work acknowledges the contributions of fellow Lee Lab members Venkatesa n Perumal, Hunter Sauerland, Selina Vong and Rupinder Kaur as well as principle investigator Jung Jae Lee and collaborators Jae Do Park of the University of Colorado at Denver Engineering Department and Timberly Roane of the University of Colorado at Denver Biology Department.

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vii TABLE OF CONTENT S I. SMALL MOLECULE MODIFICATION OF MICROBIAL FUEL CELL BRUSH FIBER S 1 Introduction Materials and Methods Results Conclusion 10 II. SYNTHESIS OF WATER SOLUBLE PHOTOTHERMAL DYES EXCITABLE BY NEAR INFRARED SPECTRUM RADIATION 1 1 Introduction 1 1 Materials and Methods 1 2 Results 1 4 Conclusion 1 8 REFERENCES 20 APPENDIX A. Supplemental Data 22

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viii LIST OF FIGURES Figure 1. Basic repeating structure of polyacrylonitrile, wh ich comprises the bristle component of Mil Rose bushes under the brand name PX35 3 Figure 2 Illustration of the cell membrane 1 pyrenemethylamine hydrochloride polyacrylonitrile electrostatic coordination 4 Figu re 3 Nanotube brush comparison modified vs unmodified 8 Figure 4 Nylon brush comparison modified vs unmodified 9 Figure 5 ICG fluorescence demonstrating bacterial adhesio n to modified nanotube bristles 9 Figure 6 I CG fluorescence demonstrating bacter ial adhesion to modified nylon bristles 10 Figure 1A Side by side fluorescent microscopy of modified versus unmodified polyacrylonitrile under DAPI spectrum stimulation 22 Figure 2 A. Proton NMR of dicarboxlic acid carbocyanine dye precursor 23 Figure 3A Proton NMR of dicarboxlic acid carbocyanine dye product, with literature spectrum/int egration overlay for comparison 24

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ix LIST OF GRAPHS Graph 1. Change in temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm 2 and laser intensity of 1207 mA 1 5 Graph 2 Change in temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm 2 and laser intensity of 2500 mA 16 Graph 3 Change in temperature with respect to time for varying concentrations of chitosan carbocyanine nanoparticle conjugate aqueous solutions using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm 2 and laser intensity of 1207 mA 17 Graph 4 Change in temperature with respect to time for free versus conjugated aqueous solutions of carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm 2 and laser intensity of 1207 mA 18 Gra ph 1 A Temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mW cm 2 and laser intensity of 1207 mA 25 Graph 2 A Temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mW cm 2 and laser intensity of 2500 mA 2 6

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x LIST OF SCHEMES Scheme 1. Proposed mechanism for attachment of linking agent to nylon polymer subunit .. 5 Scheme 2 Proposed mechanism for attachment of fluorescent marker molecules. .. 6 Scheme 3 Proposed mechanism for attachment o f ethylenediamine for functionalized augmented bristles. 7 Scheme 4 Synthetic pathway for dicarboxylic acid carbocyanine part 1 of 3. 12 Scheme 5 Synthetic pathway for dicarboxylic acid carbocyanine p art 2 of 3. 13 Scheme 6. Synthetic pathway for dicarboxylic acid carbocyanine p art 3 of 3 13 Scheme 7. dye with chitosan polymer to form the conjugate nanoparticle 1 4 Scheme 1A Proposed chemical mechanism for carbocyanine dye synthesis part Scheme 2A Proposed chemical mechanism for carbocyanine dye synthesis part Scheme 3A Proposed chemical mechanism for carbocyanine dye synthesis part 28 Scheme 4A Proposed chemical mechanism for carbocyanine dye synthesis part 28 Scheme 5A Proposed chemical mechanism for carbocyanine dye synthesis part 29 Scheme 6A Proposed chemical mechanism for carbocyanine dye synthesis part 29 Scheme 7A Proposed chemical mechanism for carbocyanine dye synthesis part 29 Scheme 8A Proposed chemical mechanism for carbocyanine dye synthesis part 30

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xi Scheme 9A Proposed chemical mechanism for carbocyanine dye synthesis part 30

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xii LIST OF ABBREVIATIONS CC: Cyanuric Chloride DAPI: 4',6 Diamidino 2 Phenylindole, Dihydrochloride DIEA: N,N Diisopropylethylamine EDC: N (3 Dimethylaminopropyl) N ethylcarbodiimide FDA: United States Food and Drug Administration ICG: Indocyanine Green MFC: Microbial Fuel Cell NHS: N Hydroxysuccinimide NP: Nanoparticle NT: Nanotube RMP: Resting Membrane Potential SYTO 9: SYTO 9 Green Fluorescent Nucleic Acid St ain from ThermoFisher Scientific TEA: Triethylamine UCD: University of Colorado at Denver

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1 CHAPTER I SMALL MOLECULE MODIFICATION OF M ICROBIAL FUEL CELL BRUSH FIBERS Introduction Microbial Fuel Cells ( MFC ) s function on the same principle as electrochemical batteries using the transfer of electrons along a conductive material from anode to cathode to produce electrical current. The difference is that in MFCs the electron source consists of living bacteria which transfer electrons onto an external electron acceptor as a natural path of their electron transport chain in oxidative respiration. 1 MFCs are attractive as a renewable source of energy because bacteria can be sustained on wastewater nutrients such as acetate. Furthermore, because the bacterial cultures are permanent so long as their environment remains constant they are suitable for applications where replenishing fuel might otherwise present logistical difficulties, an ex ample being the power source for batteries in deep sea sensors. Because this process requires physical contact between the bacteria and the anode electron acceptor the electrogenicity of MFCs is direct ly correlated with anode surface area. Under current me thods MFC anodes are cylindrical brushes wh ere the brush bristle fibers serve as the main site of bacterial adhesion. Modification of these fibers to increase bacterial adhesion is the main f ocus of improving MFC efficacy. 3, 4, 5 Biological cells have a net negative surface charge typically in the range of 10 to 12 0 mV as a result of the voltage gradients generated by membrane embedded ion pumps which is commonly referred to as Resting Membrane Potential (RMP) Because of this RMP s tudies have s hown that microbial adhesion to an anode can be increased through electrostatic attraction by attaching a slight positive surface charge to the surface of the

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2 anode. 6,7,8 The augmentation of MFC brush bristle fibers herein was an expansion on that concept. C hemical modifiers were used to coat the MFC bristle surface in a positive charge to increase microbial surface adhesion. Nanotube (NT) macromolecules are becoming increasing ly common in engineering applications due to their high mechanical strength surface area, and resistance to corrosion However, this same inertness can be a hindrance in that nanotube structures are difficult to covalently modify. The refore the procedure for mod ification of Type 1 brushes utilized pi pi stacking of 1 pyrenemethylamine hydrochloride onto the polyaromatic NT as a simple met hod by which the surface charge properties of nanotubes could be modified via the application of a charged coating around the nanotube rather than b y direct covalent modification an otherwise expensive and mechanically demanding task Materials and Methods Type 2 brushes were of a nylon Fe 2+ make. This selection was made because although NTs have superior tensile st rength and corrosion resistance, their relatively high cost in comparison to other materials often makes them economically nonviable in large scale implementation. Most currently existing MFCs use nylon bristle fibers. However, because nylon is less inert than nanomaterials, covalent modification of the brush was possible. Using cyanuryl chloride as a linking agent, these brush bristle were coval ently bound to ethylenediamine. Post modification, both brush types (and an unmodified control for each) were imm ersed in the same E. Coli broth for 1 hour. Microbes were then stained with SYTO 9 and

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3 examined under a Citation 3 fluorescent microscope so that differences in adherence could be observed visually. For the modification of polyacrylonitrile nanotube brush fibers, Mill Rose brush fibers were graciously provided by Dr. Jae Do Park of the University of Colorado engineering department. 1 pyrenemethylamine was purchased from Sigma Aldri ch. Mill rose brushes ( purchased from Zoltek ) use bristles comprised of a substance brand name d PX35 which is chemically polyacrylonitrile a nanotube structure shown in Figure 1. Figure 1 Basic repeating structure of polyacrylonitrile, which comprises the bristle component of Mil Rose bushes under the brand name PX35 Because direct chemical modification of polyaromatic systems is synthetically difficult, p i stacking the naturally occ urring quadrupole attraction between conjugated pi systems was used to orient the modifying ligands to the surface of the nanotube. The desired noncovalent coordination between the modified bristles and microbial cell membrane is modelled in Figure 2.

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4 Figure 2 Illustration of the cell membrane 1 pyrenemethylamine hydrochloride polyacrylonitrile noncovalent coordinatio n in T shaped stacked configuration. Not pictured are parallel displaced and sandwich oriented pi stacking. Adherence of the 1 pyrenemethylamine hydrochloride modifying groups to the polyacrylonitrile brush fibers 9,10,11 was achieved tion of 1 pyrenemethylamine in Milli Q water and combining ~10 mL of this solution with strands of the Mill Rose brush fibers in a sealed vortex tube Mixing was performed with a Branson Sonifier 450 sonicator. Energy was applied to the system under a high power setting for 5 minutes. After sonication, bristles were washed with Milli Q water drained, and freeze dried overnight. W e analyzed the dried bristles under fluorescent microscopy using a Biotek Cytation 3 Imaging Re ader in the DAPI spectrum ( excitation/emission peaks centered on 358 nm / 461 nm )

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5 For the second brush type, a nylon Fe 2+ brush was provided by Dr. Timberly Roane of the UCD Biology Department. In this augmentation procedure 12 initially a mixture of triethylamine, nylon bristles, and cyanuryl chloride was vortexed for 24 hours in a solution at room temperature CH 3 Cl. The proposed chemical mechanism is viewable in Scheme 1. Scheme 1. Proposed mechanism for att achment of linking agent to nylon polymer subunit Coordination of the TEA base activates the amidal nitrogen lone pair to prepare it for nucleophilic attack on the cyanuric chloride. Note this step is the same in both test and practical procedures. For the test procedure, this solution was drained, the bristles were washed three times with CH 3 Cl and then re pyrenemethylamine in CH 3 Cl. (cite dudes) To this was added excess triethylamine. This mixture was vortexed for 24 h ou rs, then drained, washed three times with acetone, and analyzed using a Biotek Cytation 3 Imaging Reader in the DAPI spectrum. The proposed chemical mechanism is viewable in Schem e 2.

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6 Scheme 2 Proposed mechanism for attachment of fluorescent marker molecules. O n l y one attachment is depicted for the sake of clarity but note that substitution occurs on both meta position chlorides. After confirming ligand attachment efficacy via fluorescent microscopy in the same manner as the polyacrylonitrile bristle trials the procedure was modified to use ethylenediamine as the source of charge. Although 1 pyrenemethylamine hydrochloride CC nylon complex displayed desired electrostatic properties we desired a demonstration of a cost effective procedure and 1 pyrenemethylamine hydrochlo ride is relatively expensive compared to ethylenediamine. So for the practical whole brush modification procedure, the scheme was altered to use ethylenediamine as the source of charge. The proposed chemical mechanism for the practical procedure is viewabl e in Scheme 3.

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7 Scheme 3 Proposed mechanism for attachment of ethylenediamine for functionalized augmented bristles. Using this modified procedure, an entire brush was treated and from this sample bristles were removed to be assessed based on microbial binding efficiency. To demonstrate bacterial adhesion, f our sets of bristles (modified polyacrylonitrile, unmodified polyacrylonitrile, modified nylon, and unmodified nylon) were cut from respective brushes and immersed in a both of E. Coli for 1 hour, then removed and rinsed. E. Coli were stained with SYTO 9 These bristles were analyzed via fluorescent microscope to assess microbial adherence.

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8 Results After washing to remove unattached residue, the both the polyacrylonitrile as well as the nylon brush bristles showed the desired fluorescent hotspots, which were absent from the unmodified control bristles. A comparison between the modified and un modified polyacrylonitrile bristle fibers under fluorescent microscopy may be viewed in Figure 3. Figure 3 An unmodified polyacrylonitrile brush bristle (left) and an experiment ally modified bristle (right) viewed under DAPI spectrum fluorescent microscopy. These data showed the desired behavior, since unmodified polyacrylonitrile does not fl uoresce in the examined spectra appearance of fluorescent hotspots meant that the 1 pyrenemethylamine hydrochloride had successfully coordinated to the bristle surface. Augmented vs unmodified nylon fibers displayed similar behavior under fluorescent microscopy and may be viewed in Figure 4

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9 Figure 4 An unmodified nylon brush bristle (left) and an experimentally modified bristle (right) viewed under DAPI spectrum fluorescent microscopy. In this manner, the adherence of 1 pyrenemethylamine hydrochloride to both polyacylonitrile and nylon surfaces through respective procedures was confirmed. In the microbial adherence trials, stained E. Coli were visible in much greater quantities on the augmented strands than the unmodified. A side by side comparison of microbes adhered to the augmented polyacrylonitrile bristle versus the unmodified bristle may be viewed in Figure 5. Figure 5 ICG fluorescence demonstrating bacterial adhesion to modified nanotube bristles. Green Dots are stained E. Coli A similar trend was observed in the nylon bristle trials, a side by side comparison of which may be viewed in Figure 6

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10 Figure 6 ICG fluorescence dem onstrating bacterial adhesion to modified nylon bristles. Green Dots are stained E. Coli. In both trials l imited amounts of microbial growth was observed on the unmodified bristles, but in comparati vely smaller quantities than were observed on the au gmente d bristles. Conclusion In both trials, t reated brush fibers were shown to have higher levels of bacterial adhesion than untreated fibers. Effect on long term MFC power generation is still under investigation, but the chemical modification procedure has bee n proven. Future research could investigate polymerization to construct a scaff old around the nanotube brushes, as well as different modifying groups. This is especially true in th e case of the nylon brushes, where it could be investigated if other polymer s could be attached using the same linking agent. As a proof of concept, this study demonstrates that bristles modified by small molecule attachment can be used to improve ba cterial adhesion over unmodified bristles.

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11 CHAPTER II WATER SOLUBLE PHOTOTHERMAL DYE NANOPARTICLE CONJUGATES EXCITABLE BY NEAR INFRARED SPECTRUM RADIATION FOR USE IN BIOMEDICAL NANOTECHNOLOGY Introduction Conjugate dyes which absorb electromagnetic radiation in the NIR spectrum (~680 900 nm) are of particul ar interest to biomedical technology applications because they provide a tissue penetrating method for the targeted application of heat wi th minimal energy loss to interference from ambient water 13 In particular, this is a subject of research in anti cancer therapies because at temperatures of 42 C and higher, tumor cell membranes begin to lyse from heat shock. Photothermal dyes, th erefore, can potentially provide a non invasive method of killing t umor cells Small molec ule medical biotechnology has received increased interest in recent years for this reason. I n this experiment we synthesized a dicarboxylic acid substituted carbocyanine dye which incorporates weakly acidic moieties that ionize under physiological pH to generate charged structures wh ich increase water solubility. M oreover, these function as the carboxylate m oiety necessary to initiate EDC coupling and can therefore be conjugated onto a suitable polymer backbone through amide bonds. This property allows the molecule to serve as a nanoparticle augment for targeting applications. F or this reason the carbocyanine dye s ynthesized in this experiment is suitable for use in both free particle aqueous suspens ions as well as nanoparticle conjugates The free particles were subjected to laser stimulation in varying concentrations to assess efficacy in achieving the desired 5 C temperature increase (from physiological temperature of 37 C to tu mor lysing temperature of 42 C ) Furthermore, since NPs are crucial in a tar geted drug delivery system,

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12 a trial of these carbocyanine dyes was conju gated to chitosan nanoparticles to assess the efficacy of chitosan as a nanoparticle delivery system. Materials and Methods 14,15 A mixture of 1,1,2 trimethyl [1H] benz[e]indole and 3 b romopropanoic acid (1:1 ratio) in 1,2 dichlorobenzene (5 mL per gram of 1,1,2 trimethyl [1H] benz[e]indole) was heated with stirring at 110 C for 18 hrs. Product was cooled and filtered over vacuum, titurating with DCM to yield a light tan powder. (74.1 % yield) The scheme of this first synthetic step may be viewed in Scheme 4 Scheme 4 Synthetic p athway for dicarboxylic acid carbocyanine p art 1 of 3 This precipitate was re dissolved in a 9::1 mixture of Me CN ::Water and 1 equivalent of s odium acetate was added. This was called Part A. In a separate flask, cooled to 0 C, glutaconaldehyde dianil monohydrochloride, acetic anhydride, and N,N diisopropylet hylamine (DIEA) were mixed in equimolar amounts (~0.6 equival ents of step 1 product) in DCM in a ratio of 5 mL per gram glutaconaldehyde dianil mon ohydrochloride This was called Part B. The scheme of this first synthetic step may be viewed in Scheme 5.

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13 Scheme 5 Synthetic p athway for dicarboxylic acid carbocyanine part 2 of 3 After 1 hour of stirring, Part A was set to reflux and to this, Part B was added dropwise. This solutio n was then allowed to reflux for 24 hrs. To purify, this solution was cooled to room temperature and filtered over vacuum, triturating with ethyl ether, Me CN, and 3.5 % HCl. The final product was a khaki green powder. (42.6 % yield.) The scheme of this first synthetic step may be viewed in Scheme 6. Scheme 6 Synthetic p athway for dicarboxylic acid carbocyanine part 3 of 3 Final product was purified by filtr ation over vacuum, titurating with MeCN, ethyl ether, and 3.5% HCl. This pure product was then redissolved in water and using to prepare sample solutions of varying concentration. Each sample was irradiated using an Arroyo Instruments 6300 Series Laser Dio de Controller and temperature of the irradiated solution was recorded every 30 seconds to assess the relationship between solute concentration and temperature over time.

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14 For the final carbocyanine dye trial, the synthesized dye molecules were attached via EDC coupling onto a chitosan backbone by Dr. Venkatesan Perumal of Lee Lab to form a nanoparticle conjugate. A representation of this polymerization step may be viewed in Scheme 7. Scheme 7 R eaction of carbocyanine dye with chitosan polymer to form the conjugate nanoparticle. I n this depiction, c hitosan appears simplified to its constituent D glucosamine monomeric subunit for clarity. This conjugate was then subject to the same dissolution in water and stimulation by laser as the free particle trials in the same concentrations (calculated by mass of the carbocyanine used in synthesis ) to assess efficacy of the photothermal effect of the free floating carb ocyanine versus the NP conjugate. Results Laser stimulation trials of different concentrations of the dye dissolved in water showed a quantitative increase in solution temperature positively correlated with the concentration of carbocyan ine dye in solution. These data, laid out in graph format, may be viewed in Graph 1.

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15 Graph 1 Change in temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm 2 and laser inte nsity of 1207 mA. Laser analysis procedure performed by Selina Vong at Lee Lab. The optimal concentration found to elicit the desired 5 C increase in temperature for free particles at low stimulation intensity was found to be concentration solution came close ( T 4.8 C) to the target threshold so a solution in vivo applications Solutions excited at a higher intensity showed a faster rise in i nitial temperature but reached the same asymptotic plateau as those excited at the lower intensity. These data, laid out in graph format, may be viewed in Graph 2. 0 5 10 15 20 25 0 50 100 150 200 250 300 Time (s) Change in Temperature with Respect to Time for Varying Concentrations of Low Intensity Laser Stimulated Carbocyanine Dye Solutions water M M M M M Lysis Threshold

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16 Graph 2 Change in temperature with respect to time for varying conce ntrations of dicarboxylic acid carbocyanine aqueous solutions using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm 2 and laser intensity of 2500 mA. Laser analysis procedure performed by Selina Vong at Lee Lab. The optimal concentration found to elicit the desired 5 C increase in temperature for free particles at high stimulation intensity was found to be identical to the low intensity trials. From this is it shown that the final change in temperature is independent of laser intensity and dependent on solution concentration of photothermal dyes. Chitosan Carbocyanine NPs displayed water solubility as did the free particles, however under laser stimulati on the ratio of change in temperature to carbocyanine concentration was consistently lower than was observed for analogous concentrations in the free particle trials. These data, laid out in graph format, may be viewed in Graph 3 0 5 10 15 20 25 30 0 50 100 150 Time (s) Change in Temperature with Respect to Time for Varying Concentrations of High Intensity Laser Stimulated Carbocyanine Dye Solutions water M M M M M Lysis Threshold

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17 Graph 3 Change in temperature with respect to time for varying concentrations of chitosan carbocyanine nanoparticle conjugate aqueous solutions using a radiant excitation source of wavele ngth of 785 nm, power density of 200 mWcm 2 and laser intensity of 1207 mA. Laser analysis procedure performed by Selina Vong at Lee Lab. In the case of the chitosan carbocyanine NPs, the photothermal effect was less pronounced and displayed less consistent behav ior than was observed in free particle trials with the same concentration and laser settings. A comparison between the two types of photothermal solutions with all other factors held constant may be viewed in Graph 4. 0 2 4 6 8 0 50 100 150 200 250 300 350 Time (s) Change in Temperature with Respect to Time for Varying Concentrations of Carbocyanine Nanoparticle Conjugate 5 M 25 M 50 M water Lysis Threshold

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18 Graph 4 Change in temperature with respect to time for free versus conjugate d aqueous solutions of carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm 2 and laser intensity of 1207 mA. Chi tosan carbocyanine NP solutions achieved the desired temperature increase but only in concentrations an order of magnitude higher than was necessary for free particle carbocyanine solutions. Conclusion The synthesized carbocyanine dye molecules were soluble in water and a photothermal effect in the NIR spectrum was ob served Solution potential to raise temperature was found to be independent of laser intensity and dependent on carbocyanine solute concent ration. Ideal dye concentration to achieve the target temperature increase of 5 C was found to be ~5 cross that threshold in conjugated NP solutions While t he maximum safe dosage of the carbocyanine dye molecules used in this experiment has not yet been established in humans, 0 5 10 15 20 25 0 50 100 150 200 250 300 Time (s) Change in Temperature with Respect to Time for Similar Concentrations of Free Carbocyanine and Nanoparticle Conjugate M Chitosan Carbocyanine M Free Carbocyanine M Chitosan Carbocyanine M Free Carbocyanine Lysis Threshold

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19 mouse trials have undergone 8.5 mg kg 1 16 and the FDA approved chemically similar carbocyanine dye ICG has a maxi mum clinical dose of 2 mg kg 1 ( ) 17 Therefore, a lthough it cannot be stated definitively without further study the synthesized carbocyanine dyes have good potential suitability for use in biomedical applications. However, it is likely tha t a different delivery syste m than chitosan nanoparticles will need to be investigated. Future research may investigate alternative NP conjugates or whether chitosan can be modified to attenuate the observed dampening effect.

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20 REFERENCES 1. Logan, Bruce E.; Hamelers, Bert; Rozendal, Rene; Schroder, Uwe; Keller, Jurg ; Freguia, Stefano; Aelterman, Peter; Verstate, Willy; Rabaey, Korneel Microbial Fuel Cells: Methodology and Technology. VOL. 40, NO. 17, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 518 1 5192 2. Picota, Matthieu; Lapinsonnirea, Laure; Rothballer,Michael; Barrirea, Frdric. Graphite anode surface modification with controlled reduction of specific aryl diazonium salts for improved microbial fuel cells power output Biosensors and Bioel ectronics 28 ( 2011 ) 181 188 3. Elimelech, Menachem; Nagai, Masahiko; Ko, Chun Han; Ryan, Joseph N. Relative Insignificance of Mineral Grain Zeta Potential to Colloid Transport in Geochemically Heterogeneous Porous Media Environ. Sci. Technol. 2000 34, 2143 2148 4. Li, Dan; Babel, Amit; Jenekhe, Samson A.; Xia, Younan. Nanofibers of Conjugated Polymers Prepared by Electrospinning with a Two Capillary Spinneret Adv. Materials, 16 22, November 2004 5. Xu, Yunhua; Zhu, Yujie; Han, Fudong; Luo, Chao ; and Wang, Chunsheng. 3D Si/C Fiber Paper Electrodes Fabricated Using a Combined Electrospray/Electrospinning Technique for Li Ion Batteries Adv. Energy Mater. 2015 5, 1400753 6. Zhongqian Song, Yuanhong Xu, Wenrong Yang, Liang Cui, Jizhen Zhang Jingquan Liu. Graphene/tri block copolymer composites prepared via RAFT polymerizations for dual controlled drug delivery via pH stimulation and biodegradation European Polymer Journal 69 ( 2015 ) 559 572 7. Baikun, Li; Logan, Bruce E Bacterial adhesion to glass and metal oxide surfaces. Colloids and Surfaces B: Biointerfaces 36 ( 2004 ) 81 90 8. Weifang Chen, Fred S. Cannon. Thermal reactivation of ammonia tailored granular activated carbon exhausted with perchlorate Carbon 43 ( 2005 ) 2742 2749 9. Cheng, Shaoan; Logan, Bruce E. Ammonia treatment of carbon cloth anodes to enhance power generation of microbial fuel cells. Electrochemistry Communications 9 ( 2007 ) 492 496 10. Anion recognition using dim etallic coordination complexes. Coordination Chemistry Reviews 250 ( 2006 ) 3068 3080 11. Tan, Ming hui; Li, Peng; Zheng, Jing tang; Noritatsu, Tsubaki; Wu, Ming boo. Preparation and modification of high performance porous carbons from petroleum

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21 coke for use as supercapacitor electrodes New Carbon Materials, 2016 31(3): 343 351 12. Bunge, Meagan A.; Ruckart, K. Niel; Leavesley, Silas; Peterson, Gregory W.; Nguyen, Ni en; West, Kevin N.; Glover, T. Grant. Modification of Fibers with Nanostructures Using Reactive Dye Chemistry. Ind. Eng. Chem. Res. 2015 54, 3821 3827 13. Ye, Yunpeng; Bloch, Sharon; Kao, Jeffery; and Achilefu, Samuel Multivalent Carbocyanine Molecular Probes: Synthesis and Applications. Bioconjugate Chem 2005 16, 51 61 14. Winstead, Angela J.; Nyambura, Grace; Matthews, Rachael; Toney, Deveine; Oyaghire, Stanley. Synthesis of Quaternary Heterocyclic Salts. Molecules; 18(11): 14306 14319. doi:10.3390/molecules181114306. 2014 15. Suganami, Akiko; Toyota, Taro; Okazaki, Shigetoshi; Saito, Kengo; Miyamoto, Katrsuhiko; Akutsu, Yasunori; Kawahira, Hiroshi; Aoki, Akira; Muraki, Yutaka; Madono, Tomoyuki; Hayashi, Hideki; Matsubara, Hisahir o; Omatsu, Takashige; Shirasawa, Hiroshi, Tamura, Yutaka. Preparation and characterization of phospholipid conjugated indocyanine green as a near infrared probe. Bioorganic & Medicinal Chemistry Letters 22 ( 2012 ) 7481 7485 16. Deng, Yibin; Huang, Li; Yang Hong; Ke, Hengte; He, Hui; Guo, Zhengqing; Yang, Tao; Zhu, Aijun; Wu, Hong; Chen, Huabing. Cyanine Anchored Silica Nanochannels for Light Driven Synergistic Thermo Chemotherapy. Small 2017, 13, 1602747 17. https://www.drugs.com/pro/indocyanine green.html (Accessed April 13, 2017)

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22 APPENDIX A SUPPLEMENTAL DATA Figure 1A. Side by side fluorescent microscopy of modified versus unmodified polyacrylonitrile under DAPI spectrum stimulation.

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23 Figure 2 A. Proton NMR of d icarboxlic acid carbocyanine dye precursor.

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24 Figure 3 A. Proton NMR of d icarboxlic acid carbocyanine dye product, with literature spectrum/integration overlay for comparison.

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25 Graph 1 A T emperature with respect to time for varying concentrations of dic arboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm 2 and laser intensity of 1207 mA. 21 26 31 36 41 0 50 100 150 200 250 300 Time (s) Temperature with Respect to Time for Varying Concentrations of Low Intensity Laser Stimulated Carbocyanine Dye Solutions water M M M M M M

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26 Graph 2 A Temperature with respect to time for varying concentrations of dicarboxylic acid carbocyanine dye using a radiant excitation source of wavelength of 785 nm, power density of 200 mWcm 2 and laser intensity of 2500 mA. 19 24 29 34 39 44 49 0 50 100 150 200 Time (s) Change in Temperature with Respect to Time for Varying Concentrations of High Intensity Laser Stimulated Carbocyanine Dye Solutions water M M M M M M

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27 Scheme 1A Proposed chemical mechanism for carbocyanine dye synthesis part 1 of 9. Waste products omitted for clarity. Scheme 2 A Proposed chemical mechanism for carbocyanine dye synthesis part 2 of 9. Waste products omitted for clarity.

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28 Scheme 3 A Proposed chemical mechanism for carbocyanine dye synthesis part 3 of 9. Waste products omitted for clarity. Scheme 4 A Proposed chemical mechanism for carbocyanine dye synthesis part 4 of 9. Waste products omitted for clarity.

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29 Scheme 5 A Proposed chemical mechanism for carbocyanine dye synthesis part 5 of 9. Waste products omitted for clarity. Scheme 6 A Proposed chemical mechanism for carbocyanine dye synthesis part 6 of 9. Waste products omitted for clarity. Scheme 7A Proposed chemical mechanism for carbocyanine dye synthesis part 7 of 9. Waste products omitted for clarity.

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30 Scheme 8A Proposed chemical mechanism for carbocyanine dye synthesis part 8 of 9. Waste products omitted for clarity. Scheme 9A Proposed chemical mechanism for carbocyanine dye synthesis part 9 of 9. Waste products omitted for clarity.