tailorable and deployable trans-corneal drainage...
TRANSCRIPT
TAILORABLE AND DEPLOYABLE TRANS-CORNEAL DRAINAGE DEVICE
FABRICATED WITH NANOPOROUS LIQUID CRYSTAL ELASTOMER
by
ROSS VOLPE
B.S. SUNY Environmental Science and Forestry, 2014
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
Of the requirements of the degree of
Masters of Science
Bioengineering
2016
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© 2016
ROSS VOLPE
ALL RIGHTS RESERVED
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This thesis for the degree of Master of Science by
Ross Volpe
Has been approved for the
Bioengineering Program
By
Kendall S. Hunter, Chair
Christopher M. Yakacki, Advisor
David Ammar
Amir Torbati
April 29, 2016
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Volpe, Ross (M.S. Bioengineering)
Tailorable and Deployable Trans-Corneal Drainage Device Using Nanoporous Liquid
Crystal Elastomer
Thesis directed by Assistant Professor Christopher M Yakacki
ABSTRACT
While the pathophysiology of glaucoma is widely unknown, current treatment relies
on lowering intraocular pressure (IOP) in order to delay vision loss. The gold standard in the surgical
treatment of glaucoma is the Ahmed valve. This device offers unpredictable intraocular pressure
(IOP) lowering efficacy as well as a milieu of acute and chronic complications. On the forefront of
glaucoma device interest is trans-corneal drainage, which offers a predictable IOP lowering
performance while eliminating procedural outcomes that lead to many of the current complications
seen (e.g. blebs). An investigation into a novel manufacturing process of a trans-corneal drainage
device constructed from a porous liquid crystal elastomer (LCE) is proposed herein.
Using a sacrificial template of water soluble nanofibers, several hundred thousand sub-
micron channels are created in a cylindrical LCE with diameter ~200 microns. Tailorable sacrificial
templates of nanofibers are formed via electrospinning of poly(vinylalcohol). LCE monomers are
polymerized around these sacrificial templates, and once these fibers are dissolved from a bulk LCE,
the remaining channels allow for constant and predictable flow through the material. Existence of
pores is confirmed with SEM and fluorescent microscopy. Finally, the drainage efficacy is tested
using a water column perfusion test.
Design of the device involves a two piece system: a silicone outer housing which is inserted
into the cornea and a separate LCE filter. A collagenous exterior may be introduced to the outer
housing to promote integration into the cornea. The LCE filter contains channels with controlled
diameters to provide adequate drainage properties, while being small enough to block most corneal
flora. The filter also may be chemically coated with a layer of copper to provide additional
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antimicrobial properties. The chemical and physical barriers to microbes yield an antimicrobial LCE,
or A-LCE.
The shape switching property of LCEs allows this device to overcome several disadvantages
of glaucoma devices which are currently available to patients. The first of which is a limited lifetime
of devices caused by scarring over or migration of a device. The proposed device may be cooled
down to be easily removed and replaced upon inactivation. A second shortcoming of current devices
involves improper IOP control. Several styles of the proposed device could easily be made through
manufacturing process which could provide varying levels of IOP relief. This would make the device
appropriate for patients with mild to severe glaucoma. Finally, the use of heat activated expansion
allows for simple, non-surgical insertion. This advantage may prove to be the most crucial in the
development of the device. Non-surgical insertion and an attractive price point would make the
device accessible in developing and third world countries where ophthalmic surgeons are rare or
otherwise inaccessible. This unique attribute would open up new markets not currently realized with
other glaucoma drainage devices.
The form and content of this abstract are approved. I recommend its publication.
Approved: Chris Yakacki
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ACKNOWLEDGEMENTS
My parents Raymond and Andrea Volpe have always told me to not look to others to
see what is possible, but to look within myself and achieve my dreams. Without advice like
this and a constant backbone of support, I would not be here. My heartfelt thanks for making
this possible.
I would like to thank my mentors for their patience in giving me advice and insight
into the world of academia which few navigate with such grace and levelheadedness. Dr.
Chris Yakacki and Dr. Amir Torbati.
I also thank Dr. Kendall Hunter for his will to see me succeed through unexpected
and sometimes adverse situations.
Dr. Ammar gave invaluable insight into the world of ophthalmology at short notice,
and always made time to give help and advice. Thank you.
Imaging was made possible by the much appreciated work of Dan Merkl (University
of Wyoming) and Melissa Laughter (University of Colorado).
I would be at a loss without the acknowledgement and thanks of the University of
Colorado Denver Calibration and Machine Laboratory, specifically Rich Wojzick, Jack and
Tom and the many others who helped machine testing apparatuses and kept my smiling while
I dug through 30 years of fittings and wiring.
Last but not least, the SMAB Lab has provided me a place to not only complete
meaningful and life changing research, but has become a haven from the daily grind and a
place I will never forget.
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DEDICATION
To my mother.
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TABLE OF CONTENTS
CHAPTER
I. INTRODUCTION………………………………………………………………… 1
Glaucoma………………………………………………………………………….. 3
Existing Treatments…………………………………………………………. 3
Drugs…………………………………………………………………...4
Surgery………………………………………………………………... 5
Drainage Devices…………………………………………………....... 6
Liquid Crystal Elastomers………………………………………………………… 12
Background and Synthesis………………………………………………….. 12
Liquid Crystal Elastomers as Trans-Corneal Filter…………………………. 17
Advantages of Liquid Crystal Elastomers…………………………….. 18
Antimicrobial Properties……………………………………………… 18
Electrospinning……………………………………………………………………. 18
Background…………………………………………………………………. 18
Tailoring Fiber Diameters…………………………………………………... 20
II. MATERIALS AND METHODS…………………………………………………. 21
Poly(vinylalcohol) Solution Preparation………………………………………….. 21
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Electrospinner Fabrication………………………………………………………... 21
Nanofiber Production……………………………………………………………... 22
Polymer Synthesis…………………………………..…………………………….. 23
LCE Synthesis………………………………………………………………. 23
Test Samples………………………………………………………………… 25
Composite Formation…………………………………………………………….. 26
Nanochannel Formation…………………………………………………………... 27
Imaging…………………………………………………………………………… 28
Perfusion Testing…………………………………………………………………. 29
III. RESULTS………………………………………………………………………… 34
Electrospinning…………………………………………………………………… 32
Nanofiber/Polymer Composite…………………………………………………… 41
Porous LCE………………………………………………………………………. 41
Fluorescent Imaging……………………………………………………………… 44
Perfusion Testing…………………………………………………………………. 46
IV. DISCUSSION………………………………………………………………… 50
V. CONCLUSION……………………………………………………………….. 56
REFERENCES………………………………………………………………………... 59
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LIST OF FIGURES
Figure
1. Aqueous Humor Drainage Path………………………………………………….. 1
2. Ocular Surface Irritation…………………………………………………………. 4
3. Trabeculectomy Procedure………………………………………………………. 6
4. Acute Blebitis……………………………………………………………………. 7
5. Traditional Drainage Devices……………………………………………………. 7
6. MIGS Device…………………………………………………………………….. 9
7. MicroOptx Trans-Corneal Device ………………………………………………. 10
8. Liquid Crystal Elastomer Configurations……………………………………….. 12
9. TAMAP Reaction Visualization…………………………………………………. 14
10. Liquid Crystal Elastomer Actuation……………………………………………... 16
11. Trans-Corneal Filter ……………………………………………………………... 17
12. Electrospinning Unit Picture and Circuitry……………………………………….21
13. Electrospinning Collectors……………………………………………………….. 22
14. Liquid Crystal Elastomer Monomers…………………………………………….. 23
15. Cylindrical Composite Process Flow……………………………………………. 26
16. Rectangular Composite Fabrication……………………………………………… 27
17. Pipette Perfusion Apparatus……………………………………………………… 30
18. Water Column Perfusion Apparatus……………………………………………... 30
19. Disc Perfusion Fixture……………..…………………………………………….. 32
20. Photo of Perfusion Fixture………………………………………………………. 33
21. SEM Images of Randomly Oriented Fibers……………………………………… 34
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22. Graph of Nanofiber Sizes vs. Solution Concentration…………………………… 36
23. SEM and Scanning Laser Topography Images of Aligned Fibers………………. 39
24. SEM Images of Fibers on a Wire………………………………………………… 40
25. Cylindrical Composite…………………………………………………………… 41
26. Cylindrical LCE Actuation………………………………………………………. 42
27. SEM Image of Nanochannels……………………………………………………. 43
28. Fluorescent Confocal Microscopy Image of Nanochannels……………………... 44
29. Bar Graph Comparing Fluorescent Fibers and Sacrificial Template……………. 46
30. Pipette Perfusion Results………………………………………………………… 47
31. Large Cylinder/Water Column Perfusion Results……………………………….. 48
32. Disc Fixture/Water Column Perfusion Results…………………………………... 49
33. Poly(vinylalcohol) Synthesis…………………………………………………….. 51
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LIST OF TABLES
Table
1. Mowiol 8-88 Fiber Measurements……………………………………………… 37
2. Mowiol 10-98 Fiber Measurements…………………………………………….. 38
3. Fluorescent Fiber Measurements………………………………………………... 45
4. Perfusion Data and Conversions………………………………………………... 49
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LIST OF ABBREVIATIONS
AH - Aqueous humor
IOP - Intraocular pressure
GDD - Glaucoma drainage device
NTG - Normal tension glaucoma
LT - Laser Trabeculoplasty
MIGS - Micro invasive glaucoma surgery
LCE - Liquid crystal elastomer
Ti - Initiation temperature
TAMAP - Two-stage thiol acrylate Micheal addition reaction
SEM - Scanning electron microscopy
MMA - Methyl methacrylate
DEGDMA - Di(ethyleneglycol) dimethacrylate
PEGDMA - Poly(ethyleneglycol) dimethacrylate
DMF - Dimethylformamide
fBA - tert(butyl)acrylate
PVA - Poly(vinylalcohol)
AC - Alternating current
DC - Direct current
kV - Kilovolt
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CHAPTER I
INTRODUCTION
There is a dire need to develop an effective and consistent treatment to glaucoma, a
disease that is predicted to effect 80 million people by 2020(3). Glaucoma is the second
leading cause of blindness worldwide, with one in six cases leading to bilateral blindness(4).
The biological pathophysiology of glaucoma is widely unknown due to the difficulty of
Figure 1 – A) Diagram showing aqueous humor
(green arrow) in a healthy eye flow from the ciliary
body around the iris, through the trabecular
meshwork and out Schlemm’s Canal into an
episcleral vein. B) Open angle glaucoma eye has
limited egress through trabecular meshwork. (2)
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studying ocular tissues in vitro. The only proven factor in decreasing the risk of blindness is
by maintaining a healthy intraocular pressure (IOP) of 15 mmHG or lower(5-7). If pressure
rises above this, ganglion cells rooted in the ocular nerve are damaged and lose function.
Aqueous humor (AH) is the fluid that fills the eye and is constantly secreted from the ciliary
processes posterior to the iris at a rate of 2 to 4 μl/minute(8).
In a healthy eye, the trabecular meshwork provides an egress for the AH in order to
maintain a constant IOP at the interface of the iris and sclera (Figure 1, A). AH is
subsequently shuttled into collection ducts in the anterior wall of Schlemm's canal and finally
into aqueous veins. The pressure of the eye is determined by a balance between the
production of aqueous humor and its outflow through the trabecular meshwork.
This study will focus on open angle glaucoma, the most prevalent form of the disease.
In open angle glaucoma, cells in the trabecular meshwork morphologically or otherwise
change, and AH cannot efficiently escape the eye (2). The most effective means of
maintaining low IOP in patients has been shown to be glaucoma drainage devices
(GDDs)(9). The design of these devices generally utse ab interno placement in order to
facilitate drainage from inside of the eye to into another part of the eye. (10). This type of
drainage device presents risk of various complications and efficacy issues that retract from
patients’ willingness to undergo GDD procedures, especially due to the fact that the disease
usually presents very late in life(5).
A transcorneal GDD would allow for the efficacy of a device based solution, while
avoiding the many limitations of an ab interno device. The purpose of this study is to
confirm the viability of manufacturing a transcorneal GDD from a nanoporous shape
memory polymer created using a sacrificial nanofiber template.
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Glaucoma
Glaucoma is characterized by damage to ganglion cells rooted in the ocular nerve.
There are several types of glaucoma with the most common being open angle, also known as
primary, glaucoma. This is the main type of glaucoma that may be treated with a glaucoma
drainage device. In open angle glaucoma, the trabecular meshwork is still exposed despite
ocular hypertension. It is known that the trabecular meshwork is unable to efficiently drain
fluid, but a drug which corrects the cellular-level function is still unknown.
Another type of glaucoma is closed angle, which has a quick onset and is an acute
disease resulting in fast deterioration of the optic nerve. The IOP spikes so dramatic because
the iris presses against the trabecular meshwork, effectively closing the angle between them
and eliminated the main egress of AH. This type of glaucoma requires quick reversal of angle
closure in order to salvage vision, and a long term treatment such as a GDD is not usually
considered.
A third, and perhaps most intriguing, type of glaucoma is called normal tension
glaucoma (NTG). Patients with NTG experience loss of ganglion cell function despite a
normal IOP. A treatment to NTG would give the community great insight into the disease
(8).
Existing Treatments of Glaucoma
There is no known cure for the disease, and the cellular pathophysiology is largely
unknown. The cells in the trabecular meshwork are believed to reorder their cytoskeleton, but
studying this change presents significant difficulty as the cells do not maintain their character
once taken from the living eye. There are, however, proven ways of managing glaucoma and
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preventing further ocular nerve damage. By maintaining a normal IOP, which is around 10-
20 mmHG, the nerves experience markedly decreased degeneration(4). There are three
strategies of managing glaucoma: drugs, surgery, and drainage devices.
Drugs
Topical drugs are usually the first line of offense used by doctors to combat high IOP.
There are many drugs on the market, most notably are Bimatoprost, Travoprost, Latanoprost,
and Timolol. All of these drugs are β-blockers, which cause vasodilation of the ciliary
arteries leading to less blood flow to the eyes(11). Although this strategy does not target
specific pathophysiology of glaucoma, it is nevertheless effective in reducing IOP –
generally by around 30% (12).
Prostaglandins have been recently been used more commonly because they only
require application once a day, compared to β-receptor antagonists which may be applied
three or more times a day (13, 14). This type of drug increases the outflow of AH from the
eye and have several side effects including eyelash growth and iris color change.
Figure 2 – A patients eye exhibiting ocular
surface irritation
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Investigations on the efficacy of prostaglandins are limited by species-dependent reaction to
these drugs (15).
There are several challenges in using topical drugs to lower IOP. First is the
frequency of application, which may be one to two times a day (14). Patient adherence to
daily topical drugs has been shown to be much less than they report to the clinician, as low as
59%, resulting in failure to lower IOP(16).There is an associated ocular surface irritation in
some patients that makes this type of treatment unbearable (Figure 2). The chemical used in
glaucoma drugs have a body-wide effect, sometimes causing respiratory and/or
cardiovascular complications(17). Finally, the IOP lowering efficacy of drugs for patients
with higher (over 21mmHg) IOP is not consistent (15).
Surgery
Each patient responds differently to medication, and if treatment with drugs alone is
not sufficiently reducing IOP, doctors may suggest a surgical solution. Surgery is a more
aggressive tactic in combatting high IOP, with effective results and many associated
risks(18).
Laser trabeculoplasty (LT) induces a thermal reconstruction of the trabecular
meshwork, leading to higher drainage rate. Although LT is a common procedure due to its
low risk of infection, it is ineffective in late stage glaucoma and subsequent treatments are
generally minimally effective at further lowering IOP(19, 20). Furthermore, this technique
requires a high degree of skill for the surgery.
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Trabeculectomy is the gold standard invasive glaucoma procedure that aims to bypass
the trabecular meshwork by dissection of the sclera, creating a scleral flap and opening a hole
to the anterior chamber (Figure 3). Approximately 24,000 Medicare patients receive this
treatment each year(21).This technique results in a bleb, or blister like pouch on the surface
of the sclera which AH is allowed to drain into. Blebs have a tendency for long term
complications including acute blebitis (shown in Figure 4) and leaking(10). The high
morbidity of a trabeculecomy limits its use. The acute post-operative complications include
choroidal effusion (13%), wound leaks (11%), shallow anterior chamber (10%), and anterior
chamber bleeding (8%) (22). There are also many long term complications which include
corneal edema (9%), dysesthesia (ocular discomfort) in 8%, fluid leaks (6%) and
endophthalmitis (serious eye infection) in 5% of patients (22). These complications, along
with scarring over of the anterior chamber shunt, lead to a 46.9% failure rate over five years
(23).
Figure 3 – Diagram of trabeculectomy
procedure. An alternative egress for AH
is introduced underneath the sclera.
Green arrow shows new path of AH.
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Drainage Devices
An alternative to traditional surgery is a glaucoma drainage device. These devices
come in many varieties that act in several ways. The most common device is called an
Ahmed Valve or tube shunt, several of which are shown in Figure 5. This device acts in a
Figure 4 – Acute blebitis in a patient who
has undergone a trabeculectomy
Figure 5 – (Left) A variety of shapes of tube shunts that may be
placed in an eye to lower IOP. (Left) An animation of an
Ahmed Valve after being sutured into place. In practice, the
pouch would be covered by the sclera.
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similar way to a trabeculectomy. A pouch is surgically attached under the sclera and a tube in
inserted into the anterior chamber to provide an additional egress for AH. There are many
variations of this type of valve shown in Figure 5 (Left). The top row shows Molteno
implants. The middle row shows a Krupin slit valve (left) and an Ahmed valve (right)
implant. The bottom row shows a variety of Baerveldt drainage devices.
Complications of this type of device include improper drainage (hypotony or failure
to lower IOP), bleb infection and scleral tissue erosion. Early stage complications are
experienced by approximately 21% of patients, and late stage complications occur in about
34% of patients (23). Furthermore, the relatively large size of these devices limits the number
of additional devices that can be implanted upon failure. Doctors can generally only implant
two devices into an eye before they deplete the accessible scleral tissue. If additional devices
do not correct the problem, patients will undergo a cyclodestruction therapy, which generally
consists of laser treatment around the ciliary body. This is aimed to decrease AH production
but often results in significant collateral tissue damage resulting in a decline or loss of vision.
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Micro-Invasive Glaucoma Surgery (MIGS) devices are an alternative to traditional
GDDs in mild cases of glaucoma which aim to minimize surgical complications. These
devices are inserted ab interno, meaning they are placed in the interior the eye and drain to
another chamber inside of the eye. As seen in Figure 6, the iStent (Glaukos, USA) is a
titanium tube shaped device that is inserted into the trabecular meshwork to shunt AH
directly into the Canal of Schlemm. Complications for iStent include fibrotic blockage,
temporary IOP spike, corneal edema, stent obstruction by blood clot or iris, anterior chamber
collapse, and vitreous incarceration (24).
A novel approach was achieved by Transcend Medical with their Cypass Microstent.
This device shunts AH into the suprachoroidal space, a physiological drainage site which is
secondary to the trabecular meshwork. It has been shown that the suprachoroidal space has a
great capacity for drainage once an artificial fistula is introduced(25).
Although MIGS devices offer fewer and less severe post-operative complications,
there efficacy is only shown for individuals with mild ocular hypertension (24). Additionally,
due to their small size there is a high rate of scarring over of the devices inside the eye, where
Figure 6 – Size of the iStent Drainage Device (Glaukos)
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repair is fast and largely unpredictable. This leads to uncontrollable drainage rates resulting
in either hypotony or no IOP lowering effect.
A transcorneal drainage method of reducing IOP offers several advantages compared
to GDDs currently on the market. This strategy aims to shunt aqueous humor across the
cornea directly onto the tear film, where it is naturally evacuated through the puncta.
Microoptx (USA) is currently developing a transcorneal drainage device called the BG
Implant (Figure 7, Top) and released some clinical data in the summer of 2015. This data
Figure 7 – (Top) The MicroOptx BG Implant
next to an Ahmed Valve and a dime, for size
reference. (Bottom) A showing in vitro data of
the BG Implant maintaining a constant IOP at
12 mmHg over a 14 day period.
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indicates that the BG Implant achieved a stable IOP at around 12 mmHg while maintaining a
microbe free environment through the device (Figure 7, Bottom) (26). These results show a
promising future in the use of transcorneal devices as they require a low level of surgical
expertise, making them widely available in countries like India where technical expertise is
not readily available, through monetary or geographic reasons.
Although Microoptx offers a viable device for the treatment of glaucoma, they are
missing several key features. The device is coated in a collagen layer which helps the device
adhere to the corneal tissue. This makes the device permanently attached in the eye and will
not allow replacement if the device is clogged by proteins or debris in the AH. Also, the
device is not tailorable to individual IOP reduction needs. This follows the “one-size-fits-all”
approach similar to many devices, which ultimately is the reason for many post-operative
complications.
Sizing of MIGS devises are generally on the millimeter scale. The exterior form
factor usually differs from the actual drainage path, including anchors or features which
allow the device to secure itself into the eye. Drainage paths vary enormously, depending on
the downstream pressure barrier. For example, a device draining into the suprchoroidal space
will provide much less internal resistance than a device draining to the tear film. This is
because the suprachoroidal space contains intrinsic resistance to flow or AH accumulation
whereas the tear film can accept as much AH as physiological flow rate. For comparison, 2-4
microliters per minute of physiological flow equates to about one drop every half hour.
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Liquid Crystal Elastomers
Background and Synthesis
Liquid crystal elastomers (LCEs) are a class of smart material which are defined by
thier combination of rubber elasticity of a lightly crosslinked polymer with self-organizing
liquid crystals. This material system was first proposed by de Gennes et al in 1975(27) and
then realized by Finkleman et al in 1981(28). Such a unique combination of subsystems
gives LCEs the ability to reversibly and repeatedly change its shape and optical properties
due to exposure to a stimulus, such as heat (29).
The stimulus induced phase transition is made possible by the self-organization of the
LCE monomers called mesogens. Mesogens are rigid molecules usually comprised of two or
more aromatic rings connected in shapes like rods, discs, or bent ‘banana’ shapes(30). These
mesogens can be linked together with flexible spacer molecules to form a main chain LCE,
or may be attached to a polymer as a side group to form an end-on or side-on LCE, pictured
in Figure 8. Main chain LCEs have been of particular interest in the past few years because
Figure 8 – a) Grey mesogens in an end-on configuration. b)
Mesogens in a side on configuration. c) A main chain LCE
with mesogens integrated into the polymer chain.
13
of their high strain actuation compared to side-on or end-on LCEs as well as newly
discovered synthesis pathway which allows for highly reproducible and tailorable samples to
be made (31).
The unique properties of mesogens manifest in a phase transition at the isotropic
temperature, or Ti. Below Ti, mesogens will naturally align organize into monodomains
which can form in two general configurations. When mesogens are aligned in only one axis,
it is termed a nematic monodomain. When alignment occurs in two directions it is a smectic
monodomain. The natural alignment of mesogens into a monodomain relies on their ability to
move freely. However, when a crosslinked network is introduced to form an LCE, covalent
bonds disrupt the free movement of the mesogens. This results in a phase of only localized
order, called the polydomain. In all three cases, when the material is taken above Ti, it will
undergo a phase transition as the mesogens completely lose their order and become randomly
oriented. This is now called the isotropic phase. An optical change can be observed while
heating a sample as the opaque crystal structure turns unordered and transparent.
Although naturally forming in a polydomain, LCEs can be programmed into a
monodomain. This is known as a liquid single crystal elastomer. In this state, the mesogens
are aligned uniformly throughout the entire sample. Monodomain can be achieved through
several means, depending on the chemistry used to synthesize the LCE. Traditionally
programming of the monodomain was achieved at the same time as synthesis by three basic
tactics: wiping a glass slide with polyimide in the desired direction of mesogens orientation,
exposure to a strong (>1Tesla) magnetic field, and mechanical stretching during the reaction
(30). These methods of programming monodomain during synthesis can be unreliable, not
easily reproducible and present strong limitations of sample geometry (only thin films are
14
able to be made with polyimide and magnetic orientation). Recently, Yakacki et al has
proposed a two stage thiol acrylate Michal Addition photopolymerization (TAMAP) reaction
to synthesize a reproducible, facile and tailorable nematic main chain LCE(29). Several
recent studies show the effectiveness of the TAMAP methodology (32-34).
The TAMAP reaction represents a dramatic change in the way which researchers are
able to synthesize and explore the mechanics of LCEs. Up to 2012, the only relatively simple
method to create an LCE from a functionalized monomer was a free radical polymerization
(30). This reaction was often hard to carry through in a homogeneous way, leading to
uncontrollable liquid crystal domains.
Another reaction, proposed by Kupfer et al relies on the functionalization of a Si-H
bond in the presence of a Pt catalyst (35). This reaction, deemed hydrosylilation, relies on the
functionalization of a cross linking molecule with both a vinyl and an acrylate group, which
exhibit two different reaction rates. The vinyl groups react quickly in the presence of a Pt
catalyst while the acrylate groups react slower. This allows the researcher to stretch the
(a) (b) (c)
Figure 9 – Schematic of the second stage of a TAMAP reaction of an LCE. Sample
starts after the first stage as a stable polydomain LCE (a). Sample is then stretched
uniaxially to align mesogens within the polymer chains (b). Finally the polymer is
photopolymerized to connect excess acrylates present in the sample (c). The polymer is
now programed into a monodomain phase.
15
polymer into a monodomain once they feel the vinyl groups have reacted, but the acrylate
groups have yet to react. This reaction allowed researchers to create a monodomain bulk
polymer, albeit with quite unpredictable crosslinking densitities and degrees of alignment
within the monodomain(36).
To be successful, these reactions must be carried out under very strict temperature
conditions. Furthermore, only side-chain LCEs were able to be synthesized from these
reaction. This limited not only the application of the reaction, but the interest of researchers
who were searching for truly applicable “hands-free” actuation. A Michael-Addition reaction
offers a new perspective on a traditionally grueling and unpredictable synthesis.
16
Using the two stage Michael-Addition reaction, LCE can first be synthesized in the
polydomain and subsequently programmed into monodomain during the second stage via
photopolymerization-induced crosslinking during mechanical straining (Figure 9). The
mechanical properties and glass transition temperature of the LCE have been shown to be
tailorable through varying the crosslinking density during the first stage of the reaction.
Figure 10 shows a strip of monodomain LCE at room temperature get heated up past its Ti
and lift a small weight. It then cools back to room temperature and returns to its original
shape.
Figure 10 - A monodomain LCE has been programmed
prior to this animation. The sample starts at room
temperature with a small binder clip attached to the end. As
the sample is heated up the polymer transitions to
polydomain. Once heat is removed, the LCE sample cools
back down and expands to its original position.
17
Liquid Crystal Elastomers as a Transcorneal Filter
Using this system, a cylindrical filter synthesized from LCE in the polydomain phase
may be stretched along its axis into monodomain phase, locked in place via UV curing, and
may switch between a longer thin cylinder at cool temperatures to a shorter thick cylinder at
body temperatures. The filter will be designed in a way which expands and locks into an
outer housing in the cornea once it reaches body temperature. Pictured in Figure 11 is the
schematic of a device as the filter is placed into the outer housing, expands and locks in place
and is subsequently removed by cooling of the device. This gives the transcorneal filter a
unique advantage over any existing glaucoma drainage devices. Insertion and removal of the
filter will be performed with an application device, not pictured.
Figure 11 – A schematic describing the two-part device concept and the replacing of a filter. a)
the silicone/collagen outer housing is placed in a pilot hole on the edge of the cornea. b) A
chilled drainage filter is placed inside the outer housing. This schematic shows an
Antimicrobial LCE (A-LCE) which has been chemically coated with a layer of copper. c) As
the drainage filter reaches body temperature, the filter will expand causing it to lock in place
and maintain a microbe-free barrier between the filter and the outer housing. d) Once the
device needs replacing, the device is chilled and can be easily removed from the outer
housing.
Remove and ReplaceChill to
18
Advantages of Using LCE
One key feature of the LCE is the heat activated shape switching ability, which
allows the device to be easily placed into, and taken out of, the outer housing. This gives
surgeons the option to easily switch in different devices which have different drainage
characteristics. This feature also affords patients reliable lifelong glaucoma relief by allowing
replacement if the device is clogged by accumulation of protein in the aqueous humor.
Antimicrobial Properties
Introducing a transcorneal egress for aqueous humor has the potential to put patients
at risk of infection. To combat this risk, the pores in the transcorneal filter are designed to be
less than one micron, the average size of human corneal bacteria (6). This size based barrier,
combined with the constant outflow of aqueous humor through the device, ensure that there
will be no bacterial infection happening through the body of the drainage device. It is
important to note that not all bacteria will be larger than one micron. The constant flow of
AH will prevent these small bacteria from infiltrating the device.
Electrospinning
Background
In order to produce consistent, sub-micron channels in the LCE, a two polymer
system is used that relies on a sacrificial template of nanofibers. A difference of solubility
was taken advantage of in this study by using a water soluble polymer to form the sacrificial
nanofibers. This allows the fibers to remain stable as the LCE monomers are infiltrating the
fibers. Toluene was used as a solvent for the LCE and will not disturb the nanofiber system.
19
A similar study was conducted by Luo et al during work aimed at developing a self-
healing polymer by embedding in it electrospun nanofibers to create a two-polymer
composite(37). This system also relied on the solubility differences between the polymers,
but differed from the system in this study by leaving the water soluble nanofibers intact in the
polymer.
Another study was conducted with yet a more similar system of nanofiber
composites, conducted by Bellan et al (38). In this study, a composite of water soluble
nanofibers inside a bulk hydrophobic polymer (polydimethylsiloxane) was prepared and the
electrospun fibers were subsequently dissolved out. This study aims to test if this technique is
compatible with a shape memory polymer.
In both these studies, electrospinning was used to create the nanofibers. This has been
shown to be a simple and fairly reliable way to produce continuous fibers on the nano- to
micro-scale(39-42). The process of electrospinning is based on a high voltage DC field
dragging a conductive polymer solution across a charge gradient onto a grounded collector
(39). The polymer is typically ejected from a syringe at a low flow rate while the positive
charge is placed directly to the syringe needle.
The collector may have a variety of configurations, including a flat plate, rotating
drum, wire, or pin electrode (pictured in Figure 345y). The type of collector plays a major
part in the morphology of fibers collected as well as several other key parameters: voltage
charge applied, tip-to-collector distance, polymer concentration, molecular weight of
polymer, humidity of the room, and additions to the polymer solution (salts, pH control, etc.)
(41). It is noted in literature that the parameters most influential on the fiber morphology are
20
polymer solution concentration and tip-to-collector distance (40-42). Nanofiber diameters are
increased by increasing solution concentrations and decreasing tip-to-collector distance.
Tailoring Fiber Diameters
Tailoring the nanofiber diameters is an important step in developing a robust solution
to transcorneal drainage because the size of the nanofibers produced will correlate to the
diameter of pores left in the filter after dissolution of the fibers. The diameter of pores in the
sample is perhaps the most impactful parameter of the filter’s drainage ability. If we consider
only one pore which runs through the filter, its capacity to drain fluid at a given pressure is
given by the Hagen-Poiseuille equation:
𝑄 = ∆𝑃𝜋𝑑4
128𝜇𝐿
Where Q is the flow rate, P is the pressure gradient, d is the pore diameter, μ is the
fluid viscosity and L is the length of the tube in question. It is clear that a small change in
diameter will greatly affect flow rate through the tube. Therefore the diameter of the
electrospun fibers is a key parameter is the overall filtration rate of a transcorneal GDD and
will be investigated during this study.
In summary, a transcorneal glaucoma drainage device will be fabricated so as to
maintain an IOP of ~15mmHG under physiologic AH production of 2-4 μl/min. The filter
will be made using a sacrificial template of electrospun water soluble nanofibers embedded
in a bulk LCE. The system will be qualified with SEM and fluorescent microscopy images as
well as perfusion tests.
21
CHAPTER II
MATERIALS AND METHODS
PVA Solution Preparation
Granualted polyvinyl alcohol (Mowiol 8-88, Sigma Aldrich) was dissolved in water
at several concentrations (8%, 10%, 12%, 17%, 20% and 22%), by weight, by vigorously
mixing at 85 oC overnight.
An additional granulated poly(vinylalcohol) (Mowiol 10-98, Sigma Aldrich, USA)
was also prepared for electrospinning by dissolution in pure water. Concentrations of 8%,
10% and 13% were used in this study. Solutions were prepared by stirring at 95 oC over 48
hours.
Electrospinner Fabrication
In order to avoid extreme and unnecessary prices of commercially available
electrospinning machines, one was designed and fabricated in house. Two items are essential
when making such a device – an AC/DC converter (ESCC 0305, Astrodyne, Mansfield MA),
Figure 12 – Image on the left shows the DC electric current output box. The diagram on
the right shows the components and circuit configuration inside the box. a) AC current
input. b) AC/DC power converter c) High voltage converter. d) LCD display e)
Potentiometer f) Switch g) LCD display power input. h) High voltage ground, labeled
“GROUND” on left. i) High voltage DC output, labeled “HIGH VOLTAGE” on left.
22
and a high voltage output (25A12 Series, Ultravolt, Ronkonkoma NY). An acrylic box was
constructed to hold these two components, as well as an additional AC/DC converter to
provide power to a rotating drum collector, an LCD readout screen displaying the current
voltage (PM128, Jameco, Belmont CA), and adjustment knobs provided courtesy of the
University of Colorado machine and calibration lab. In order to avoid the high voltage from
deactivating other components, the high voltage output had to be removed from the main
cabin and placed into a small acrylic box of its own and attached to the top of the main
acrylic box, where it was accessible to wiring. Wiring schematic and photo of the
electrospinning device is shown in Figure 12.
Nanofiber Production
Nanofibers are produced by electrospinning a polymer jet of polyvinyl alcohol onto a
wire collector. The polymer solution is placed in a 20ml syringe and is ejected from the tip of
a 22 gauge blunted needle at a rate of 1 ml/h by a standard syringe pump. The positive side
of the high voltage DC power converter 8is connected to the needle with a charge of 10 kV.
The collector is a grounded 25 um copper wire with a length of around 7 inches. It is placed
Figure 13 – (Left) The electrospinning setup (excluding the power supply and syringe
pump). A polymer jet is dragged across a DC charge gradient from a needle tip onto a
grounded wire in the form of continuous nanofibers. (Right) A pin-gap electrode is
pictured with blue PVA nanofibers collecting across the gap. The two pins are
separated by ~1 in. and are both grounded.
23
perpendicular to the needle at a distance of 8 inches. The electospinning process is allowed to
proceed for approximately 30 minutes, with attention to not allow web formation connecting
the grounded wire to surrounding environment. All electrospinning is conducted at room
temperature and a relative humidity of 14%. The experimental setup is pictured in Figure 13.
Experiments were also done using a house made rotating drum collector and pin electrode
collector in order to produce fibers arrays of aligned morphology.
The production of different shapes and sizes of fibers is able to be achieved by
altering many parameters of the electrospinning process such as voltage charge, tip-to-
collector distance, needle size, solution flow rate, collector geometry, and solution
concentration. In a preliminary study, it was observed that solution concentration had the
most drastic effect on fiber morphology and therefore was the one parameter that was tested
throughout this study.
Polymer Synthesis
LCE Synthesis
LCE was synthesized according to Yakacki et al using a thiol-acrylate Michael
Addition reaction. The mesogenic monomer used was RM 257 (Wilshire Technologies,
Princeton NJ), and the flexible spacer was 2,2-(Ethylenedioxy) diethanethiol (EDDT) (Sigma
a b c
Figure 14 – Chemical structures of the monomers involved in the TAMAP reaction. a) RM
257 b) EDDT and c) pentatetrakis (3-mercaptopropionate)
24
Aldrich, USA). The LCE was syntheized with 15% tetra-thiol crosslinkers, pentaerythritol
tetrakis (3-mercaptopropionate). Figure 14 shows the monomers. Samples were synthesized
with a stoichiometric ratio of functional acrylates to thiol groups, with 15% of the functional
thiol groups belonging to the crosslinking molecule.
LCE was typically made in small batches according to the following: to a 20 ml
scintillation vial, 1 gram of RM257 powder was added. This was dissolved in 0.30 g of
toluene at 80 oC. To the solution, 0.2942 g EDDT spacer and 0.021 g of pentatetrakis (3-
mercaptopropionate) crosslinker was added. After thorough mixing, 0.245 g of a 50:1
toluene/DPA catalyst was added and thoroughly mixed, which initiated the reaction. Within 5
minutes of adding catalyst, LCE was poured into a mold and left to fully polymerize
overnight.
The mold which LCE was polymerized in was generally a pipette, which was a cheap
and readily available method of creating cylindrical samples. For preparing porous LCE, the
tip of the pipette was used. First, a wire coated with electrospun nanofibers was inserted
approximately one inch into the tip. The tip was then dipped in a mixture of LCE monomers
soon after catalyst was added and the mixture was still quite fluid. after a vacuum treatment
to remove air bubbles, the pipette was placed vertically to allow polymerization overnight.
In preparation of samples which showed the shape changing characteristics of the
LCE, the wide end of the pipette was used. To synthesize these samples, the tip of the pipette
was first plugged with wax. Soon after adding catalyst to a mixture of LCE monomers, the
reaction was poured into the back end of the pipette until filled. Again, a vacuum treatment
was applied and the LCE was left to polymerize overnight. After polymerization was
25
complete, the pipette was smashed and LCE was left overnight to evaporate all remaining
solvent.
Test Samples
In order to quickly test composite systems, a shape memory polymer (SMP) which
could be rapidly fabricated was used in place of LCE in some cases. A key attribute of the
SMP used in place of LCE was its low glass transition temperature, which made the polymer
rigid at room temperature. The SMPs were also amorphous which made them optically
transparent, allowing imaging through samples possible.
Two different SMPs were used in this study. The first was a 5:1 tert(butylarylate) tBA
and di(ethyleneglycol) dimethacrylate (DEGDMA) mixture. The second was a 2:3 methyl
methacrylate (MMA) and poly(ethyleneglycol) dimethacrylate (PEGDMA) mixture. Both of
these were free radical driven polymerizations that were cured in 20 minutes under UV light.
The MMA/PEGDMA mixture exhibited less shrinkage when polymerized which made
possible synthesis of very thin, homogeneous samples.
26
Composite Formation
Composites were formed in a variety of ways, depending on their end use. These are
diagramed in Figure 15. The first way was a mat composite, where the PVA nanofibers were
sandwiched between two layers of LCE. To accomplish this, immediately after adding
catalyst to the LCE monomers, the reaction was brushed on a glass slide coated with a
hydrophobic rain repellent (used as a release agent). An electrospun fiber mat was laid on top
of the first layer of LCE and then more LCE was spread on top of the nanofiber mat. Another
glass slide coated in release agent was clamped on top of the composite overnight while the
LCE completed polymerization.
To make prototypes suitable to test perfusion using basic laboratory equipment,
composites are formed in pipettes. After a wire collector has been removed from the
Figure 15 – Picture schematic of a circular composite being formed. A 25
micron copper wire is a) electrospun onto and then b) inserted into a tube,
either capillary tube, pipette or vial depending on the end use. The cross
section of the tube with wire inside is seen on the bottom left. c) The
nanofibers are then infiltrated by LCE polymer and vacuum treated to
evacuate any air. In a later step, d) the the fibers are dissolved out leaving a
nanoporous sample.
27
elctrospinning apparatus by cutting both ends, it is strung through the pipette with the
midpoint of the wire in the widest portion of the pipette. The wire is secured into place at the
tip of the pipette with superglue, while also sealing off the tip of the pipette. A custom made,
3D printed cap is placed at the other end of the pipette which allows for centering of the wire,
as well as supplying an ingress for LCE to be poured. The LCE monomers are prepared and
quickly after adding catalyst, the reaction mixture is injected through the pipette cap opening
with a syringe. The pipette is placed in a 15 mmHG vacuum for five minutes to degas the
composite and is left to cure overnight.
Composite samples using tBA and DEGDMA (5:1) were also synthesized in the front
and back end of glass pipettes. These samples had a larger size (1mm and 5mm diameter) for
the front and back end respectively, which made them easier to handle during perfusion tests.
These samples were also formed in rectangular geometries by sandwiching tBA/DEGDMA
and a flat sheet of nanofibers between two glass slides (Figure 16). The rigidity of the
tBA/DEGDMA polymer at room temperature also aided in testing.
Figure 16 – Picture schematic of creation of mat composites.
Nanofibers are engulfed on top and bottom with LCE polymer. Fibers
are dissolved out in a later step.
28
Nanochannel Formation
Once a composite has been removed from its mold (pipette, glass slides), it is dried
overnight in an 80 oC oven. The composite is then sliced into smaller pieces and placed in a
vial of water. The composite is gently stirred in this solution for two days at 60 oC.
Imaging
Nanopores are confirmed by first freezing samples and then slicing cross sections of
the cylinders near the center. Carbon sputtered SEM imaging was used to obtain high
resolution images of the individual nanopores. Imaging was kindly conducted by Dan Merkl
from the University of Wyoming Mechanical Engineering department.
Fluorescent imaging was also performed in order to see continuous channels. Samples
used in fluorescent imaging were prepared with a MMA and PEGDMA (3:2) mixture instead
of LCE for several reasons. First of all, the MMA/PEGDMA is an amourphous glassy
polymer at room temperature, which in lay terms is clear and hard. This makes handling and
imaging possible at room temperature.
To prepare samples, the 3:2 mixture of MMA/PEGDMA was placed in a 1mm thick
mold until it filled the mold half way. A section of an electrospun sheet, created on a flat
plate collector, was placed on the MMA/PEGDMA and the mold was filled up all the way.
The MMA/PEGDMA was subsequently crosslinked via UV irradiation. Once the composite
was removed from the mold and the nanofibers were dissolved out in water for 2 days in 60
oC water, the samples were dried in a 70 oC vacuum oven. After the channels were
evacuated, the samples were submerged in a 100 μM solution of fluorescein (Sigma Aldrich,
USA). After soaking for 30 minutes, the submerged samples were introduced into a 15 inHg
29
(381 mmHg) vacuum 4 times in order to clear the air from the nanochannels and replace that
space with fluorescein solution. After soaking for two days, the sample surfaces were dried
with a wipe prior to imaging.
Confocal images were collected using a Nikon Eclipse Ti C2 LUN-A microscope
(Nikon, Tokyo) equipped with two C2-DU3 high sensitivity PMT dtectors, 4 diode lasers
(405/488/561/640 nm), and a motorized microscope stage with 3 axis navigation (X, Y and
Z). A 488 nm laser setting was used to capture images.
All images were analyzed with ImageJ software.
Perfusion Testing
A liquid perfusion setup was assembled in house to confirm that the pores were
continuous and would allow for steady and predicable outflow. This was done using a
column-based water pressure system in conjunction with several test sample fixtures. In the
first sample fixture setup (Figure 16), a cylindrical sample formed in the small end of a
pipette was secured via super glue back into a pipette after dissolution of the nanofibers. The
pipette was filled with a dye and the tip of the sample was dipped into a collection vial. The
second perfusion setup achieved a higher back end pressure on the device. A test sample was
secured this time in the large end of a pipette with superglue while other side of the pipette
was superglued into a 64 inch long tube filled with water and dye. Perfusion was confirmed
with visual confirmation of dye on the exposed end of the test sample.
30
Figure 17 – A perfusion test setup
including a pipette filled with red dye,
a nanoporous filter secured with super
glue in the tip of the pipette which is
partially submerged in a collection bath
where dye is collecting, indicating
successful perfusion.
Figure 18 – Water column
perfusion assembly.
31
The third perfusion setup (shown in Figure 18, 19) achieved a greater number of
pores exposed to the pressure of the water column. First, a flat sample was prepared by
sandwiching a rectangular mesh between a 3:2 MMA/PEGDMA mixture. The nanofibers
were dissolved out from the bulk polymer and the porous sample was imbedded in a strong
fast-cast urethane (Master Fast-Cast Urethane, Dynacast) inside a cylindrical mold. Once set,
a 1mm thick cross section of the epoxy cylinder containing the porous sample was cut on a
lathe. This thin cylinder was placed between two flanged tube fittings and sealed with
silicone gasket sealer on either side. This apparatus was inserted on the bottom of the water
column and perfused with a 1:1 mixture of dimethylformamide (DMF) and water, which was
collected in a vial. Visual confirmation of fluid in the collecting vial was used to qualify
perfusion.
Figure 19 – (Left) Schematic of the third type of perfusion setup. Perfusion fluid
enters from a water column into a flanged aluminum fixture. A casted urethane disc
containing a nanoporous MMA/PEGDMA sample is placed in the flow path and
sealed with a silicone disc between each aluminum fixture. Fluid drains through the
nanopores out of the other end and collects in a vial for further testing. (Right)
Picture of the test disc used for perfusion qualification. Two rectangular pieces are
seen imbedded in the disc, each of which contain nanopores. Using two pieces of
porous sample yields a higher perfusion rate which will be easily detectable by
visual confirmation.
32
This test was performed for 90 hours with two different discs. One contained large
pores fabricated from a sacrificial template made with 20% PVA electrospinning solution
and one contained small pores with a 10% solution. Evaporation was accounted for by using
a 10% glucose solution as perfusion fluid and back-calculating flow rate through dry weight
of the fluid which perfused over the 90 hours.
The length of tubing necessary was determined by the amount of pressure needed to
simulate physiological eye pressure for a given sample length. This value is reported to be
between 15-30 mmHg for glaucomic patients. In order to simulate a 1mm long clinical
device using a 6mm long test sample, a back pressure of 90-180 mmHg was needed. For
example, a 6mm long test sample requires six time the eye pressure in order to simulate a
1mm clinical device. In this study, pressure of 20 mmHg was considered a disease-state IOP,
requiring 64 inches of water pressure for a 6mm long test device. All perfusion setups were
also tested with a control sample of identical geometry and no nanochannels to confirm
efficacy of the setup.
Figure 20 - Picture of the test fixture in line
with a water column. Fluid will drain from the
water column through the porous section of
the test disc, and ultimately collect in a vial.
33
CHAPTER III
RESULTS
Electrospinning
Using the house-made electrospinning apparatus, PVA nanofibers were fabricated in
a variety of sizes topological morphologies. In general, as the concentration of PVA inside
the syringe was increased, the fiber diameters increased and fiber formation was more stable.
b
.
Figure 21 – SEM images of PVA nanofiber mats collected on a flat plate. All parameters
were identical besides a change in PVA solution from 8%, 10%, 15% and 20%
corresponding to a-c respectively.
34
At a low concentration, fibers formed in ribbon like morphologies (Figure 21.a). At a
concentration of 10%, the fibers stabilize (Figure 21.b).At higher concentrations of 15% and
20%, fiber diameters increase (Figure 21.c, Figure 21.d). Fiber measurements for Mowiol 8-
88 and Mowiol 10-98 are given in Table 1 and Table 2, respectively.
There were stark differences in the fiber formation between the two types of PVA
used in this study Mowiol 8-88 and Mowiol 10-98. While Mowiol 10-98 yielded higher fiber
diameters at a given concentration, its high level of hydrolysis hampered its ability to
dissolve in water, therefore limiting the maximum fiber diameter achievable. The high
hydrolysis of Mowiol 10-98 also made the fibers produced at lower concentrations more
stable (Figure 22).
35
Figure 22 – Graph showing results of average fiber diameters of various
concentrations of two samples of PVA with different degrees of hydrolysis. The
PVA with a high degree of hydrolysis produces stable fibers at lower concentrations,
and produces higher fiber diameters compared to the low hydrolysis sample of the
same concentration. The low hydrolysis PVA is able to be dissolved in water at
higher concentrations and is able to produce an overall higher fiber diameter.
36
Table 1 – Fiber diameter measurements of Mowiol 8-
88 for concentrations of 8%, 10%, 15%, 20% and 25%
(w/w). All measurements in nanometers.
Measurement 8% 10% 15% 20% 25%
1 152 217 205 407 263
2 210 106 142 468 436
3 77 132 173 520 712
4 105 106 226 414 431
5 143 94 116 229 323
6 175 126 94 213 421
7 118 67 15 288 190
8 139 177 157 576 612
9 196 137 106 379 487
10 66 54 195 325 237
11 180 70 95 223 229
12 261 152 94 402 351
13 193 164 91 523 276
14 141 142 229 269 446
15 114 76 124 380 569
Mean 151.3 121 147 374 399
SD 52.3 46 49 116 152
Min 66 54 91 213 190
Max 261 217 229 576 712
37
Table 2 – Fiber diameter measurements of Mowiol 10-98 of
concentrations 8%, 10% and 13% (w/w). All measurements in
nanometers.
Measurement 8% 10% 13%
1 124 122 349
2 71 171 371
3 90 155 239
4 82 112 236
5 81 310 290
6 51 140 272
7 86 207 445
8 71 93 338
9 82 152 196
10 90 94 201
11 71 75 298
12 81 222 202
13 78 124 259
14 66 176 331
15 81 99 223
Mean 80 150 283
SD 16 61 72
Min 51 75 196
Max 124 310 445
38
Alternative fiber collectors yielded fiber morphologies which may be useful in the
final design of a transcorneal drainage device. Using the pin gap electrode collector, arrays of
aligned fibers were achieved (Figure 23). Collecting fibers on a wire was a useful way to
form and easily manipulate a fiber array in a cylindrical shape. As shown in Figure 24, fibers
are able to be collected on a wire with a stable and randomly oriented morphology.
Figure 23 – (left) SEM image of aligned PVA nanofibers collected on a pin gap electrode.
(Right) Laser scanning topology microscopy of aligned PVA nanofibers collected on a pin
gap electrode. Color indicates depth with red being closest to the camera.
39
The investigation into electrospinning proved that tailorable fiber arrays of various
morphologies and sizes are able to be made easily and reliably using several different
collectors.
Figure 24 – (Left) SEM image of PVA nanofibers collected on a 25
micron wire. The diameter of fibers collected around the fiber is
approximately 200 microns. (Right) Magnified SEM image of the fibers
pictured on the left. Fiber morphology indicates stable, branching fibers.
40
Nanofiber/Polymer Composite
Nanofibers produced via electrospinning PVA on a 25 micron wire were successfully
incorporated into a cylindrically shaped memory polymer composite. These samples were
made in a variety of shapes and with several shape memory polymers depending on the final
use. Pictured in figure 25 is a tert-butyl acrylate (tBA) with di(ethylene glycol)
dimethacrylate (DEGDMA) crosslinker shape memory polymer and a nanofiber-bearing 25
micron wire composite which was formed in a 100 micron ID capillary tube. This sample
was prepared to show viability of creating a sample mimicking the size of a filter which
would be incorporated into a glaucoma drainage device.
Porous LCE
Cylindrical samples of pure LCE were fabricated in pipettes to show the change in
diameter with exposure to heat. The sample shown in Figure 26 starts at 0 oC (Left) and
contracts over 2 minutes of exposure to heat (Right). The diameter of the cylinder changes
from 0.14 in to 0.20 in, a change of 43% the original diameter. The change in shape
Figure 25 – A cylindrical
composite (D = 100 micron) of
nanofibers on a wire with tBA and
DEGDMA crosslinker.
41
represents a molecular-level reorganization of the mesogens from a monodomain (Figure 26
Left) to polydomain (Figure 26 Right).
Porous cylindrical samples created with LCE were used to confirm the existence of
channels within the sample. Cross sections of cylindrical nanoporous LCE samples, taken in
the center of the sample, were analyzed under SEM imaging (Figure 27). The surface shown
was within 100 microns of the wire, where hollow channels were expected to have taken the
place of PVA nanofibers. The holes seen in the surface have both circular and elliptical
topology, resulting from the random orientation of the fibers at this cross section.
Figure 26 – A cylindrical sample of LCE transitioning phases due to heating. (Left)
Cylindrical sample starts at 0 oC and a diameter of 0.14 in. The sample is in the
monodomain.(Right) After heating of the sample for 2 minutes, the sample transitions to
polydomain and has a diameter of 0.2 in.
42
Figure 27 – SEM image of the surface of a cross sectioned cylindrical nanoporous LCE.
The image was taken from a sample which used the fiber-on-a-wire approach to
composite formation. The topology and size of the holes seen in this picture indicate that
they were indeed formed from dissolved nanofibers. Some holes are elliptical in shape,
indicating that a cylindrical fiber was passing through that cross section at an angle.
43
Fluorescent Imaging
Flattened composites for fluorescent imaging were also successfully created. These
were made using several methods. The first was using a 1mm thick spacer between two glass
slides. This produced an approximately 1mm thick rectangular sample. The second way in
which flattened samples were created was without any spacer at all. These samples were
approximately 80 microns in thickness. Using these two styles of sample thicknesses, images
shown in Figure 28 were captured using fluorescent confocal microscopy. Both samples were
created with nanofibers formed from electrospinning a 20% w/w solution of PVA. Fiber
measurements from the 80 micron thick sample are shown in Table 3.
Figure 28 – Fluorescent microscopy image showing nanochannels infiltrated with a 100
μM solution of fluorescein. Images from two samples are shown. On the left is a 1mm
thick sample and the right shows an 80 micron thick sample. Both samples were made
with nanofibers formed from electrospinning a 20% w/w concentration of PVA.
44
The average fiber diameter of nanochannels are compared to the sacrificial nanofiber
template in Figure 29. The average diameters were taken from a small area of the samples,
and merely approximate the total average diameter with 15 measurements from each image.
Using a two sample t-test, the means are significantly similar (P < .01).
Table 3 – Fiber diameter measurements
taken from the 80 micron thick sample
containing nanochannels formed from
20% w/w concentration PVA solution
electrospun on a flat plate collector.
Measurements are in nanometers.
Measurement Diameter
1 684
2 568
3 546
4 312
5 621
6 403
7 494
8 287
9 462
10 403
11 479
12 408
13 305
14 429
15 456
Mean 457
SD 114
Min 305
Max 684
45
Perfusion Testing
Perfusion was shown using three test fixtures. The first was a simple pipet apparatus.
Figure 30 shows two pipette perfusion tests after 48 hours. The red dye was perfused through
a 6mm porous cylinder, and is clearly seen to be collecting in the water bath. The green dye
was used in a control setup where the 6mm cylinder did not contain any pores. The lack of
green dye in the collecting bath shows that no dye is escaping the pipette around the edges of
the cylinder.
Figure 29 – Bar graph comparing the average diameter of electrospun fibers and
nanochannels (taken from fluorescent images). Both were formed from a 20%
w/w PVA electrospinning solution.
0
100
200
300
400
500
600
Average Diameter of Sacrificial Fibers and Nanochannels
Electrospun Mat
Nanochannels
46
The second perfusion setup used a water column to increase the pressure behind the
filter in order to reach physiological conditions. The thicker cylinder diameter of this setup
made handling and securing test samples in place with ease. Results were obtained using a 5x
magnification microscope and confirmation of a blue dye reaching the other side of the filter.
The results in Figure 31 show blue dye along the edges of a copper wire, with some blue dye
surrounding the wire. It is apparent that the dye traveled through the filter and wetted the
exposed face. Because flow was extremely slow, the water in the dye was able to evaporate,
depositing spots of solid dye on the face of the sample.
Figure 30 – A pipette based dye perfusion test. The red
dye was perfused through a porous cylindrical sample,
while the green dye was used in a control setup with a
cylinder containing no pores. Perfusion is confirmed in the
red test while the green dye did not penetrate the fixture.
47
The third perfusion test was performed using a disc apparatus which minimized the
thickness of the sample the liquid perfused. Confirmation of perfusion was attained by visual
confirmation of fluid reaching the back side of the fixture. A collection vial was placed
beneath the test fixture, but after 48 hours fluid was still captured inside the aluminum test
fixture, held in place through capillary action. Figure 32 shows the downstream end of the
aluminum test fixture after 48 hours. Fluid is visible inside the test fixture, indicating it
passed through the porous disc.
The dry weight of the evaporated perfusion fluid was used to provide a more
comprehensive qualitative analysis. The table of measurements and conversion into flow rate
Figure 31 - Enhanced contrast image of the end
of the large cylinder/water column test fixture
after 48 hours. The blue seen next to the copper
wire shows that dye has perfused through the
cylinder. Blue dye seen around the wire is the
result of surface wetting followed by
evaporation of the dye.
48
is listed in table 4. The results indicate that, as expected, less fluid passes through a sample of
smaller pores compared to a sample containing larger pores.
Figure 32 – Fluid collected in the downstream
side of the disc perfusion apparatus. Image was
taken after 48 hours of continuous perfusion
Table 4 – Data obtained from 90 hour perfusion studies of two discs with different sized
pores. Large and small pores were formed with a sacrificial template made from 20%
and 10% PVA electrospinning solution, respectively.
Pore Size (Concentration) Dry Weight Volume Fluid Perfused Flow Rate
Large Pores (20%) 0.822 g 8,220 ul 1.52 ul/min
Small Pores (10%) 0.371 g 3,710 ul 0.68 ul/min
49
CHAPTER IV
DISCUSSION
Trans-corneal drainage is on the forefront of glaucoma device industry interest. It
offers a predictable outflow of AH while avoiding bleb formation and complications
associated with invasive surgery such as scarring and inflammation. Implanting a trans-
corneal drainage device requires minimal surgical skill and is therefore well suited for
treatment of glaucoma in developing countries. There are several additional considerations
when designing a trans-corneal drainage device compared to traditional ab interno devices,
which are not exposed directly to the environment. First of all, the trans-corneal device must
provide a microbial barrier. Secondly, the device must rely solely on its intrinsic drainage
properties to manage IOP – there is no downstream pressure barrier. Lastly, the trans-corneal
device must be secured in place without the use of barbs or sutures as many ab interno
devices are. Fabrication of a trans-corneal drainage device as proposed in this investigation
meets all three of these requirements, as well as offering additional advantages for users and
surgeons.
The first aim of this study was creating tailorable nanofibers via electrospinning. It is
widely reported in literature that electrospinning solution is the most sensitive parameter in
changing the diameters of nanofibers produced(43-46). It was found that by raising the
concentration of PVA (Mowiol 8-88) in the electrospinning solution from 10% to 25%, fibers
from 54 nm to 712 nm could be fabricated, respectively.
Fundamental information about the nature of electrospinning was also gained in this
process. By using two types of PVA, Mowiol 8-88 and Mowiol 10-98, relationships between
50
average molecular weight and fiber morphology were observed, as well as relationships
between degree of hydrolysis and solubility of PVA in water. While using 8% w/w
concentrations of both Mowiol 8-88 and Mowiol 10-98, drastically different fiber
morphologies were observed. While solutions of Mowiol 10-98 produced fibers with a
unique, branching and cylindrical morphology, solutions of Mowiol 8-88 produced fibers
with a flattened, ribbon like morphology. As indicated by Tao, J, this was directly related to
the entanglement concentration of the type of PVA used. Mowiol 10-98 would yield an
entangled solution at lower concentrations due to the increased hydrophilic interactions.
These would create stable nanofibers upon electrospinning while the same concentration of
Mowiol 8-88 would produce flattened fibers. This is an important observation because only
unique, cylindrical fibers will be suitable to achieve a drainage device with tailorable,
controllable and predictable outflow.
The relationship between degree of hydrolysis of the PVA and its ability to dissolve
in water was counter intuitive, but allowed fibers of higher diameters to be fabricated when
taken advantage of. The manufacturing of PVA is achieved by hydrolyzing
poly(vinylacetate), a polymer which is not water soluble (Figure 33). Contrary to common
sense, increasing the degree of hydrolysis does not always increase the water solubility. At
Figure 33 – Hydrolysis of poly(vinylacetate) into
poly(vinylalcohol) (1)
51
very high (99%) levels of hydrolysis, solid PVA forms very stable crystal structures which
are require heating above 100 oC in water to dissolve. At a lower level of hydrolysis (85%),
the remaining acetate groups act as steric hindrance to tight crystal structure formation and
allow for much easier dissolution of PVA in water at temperature below 100 oC. The
majority of this study was performed with lower hydrolysis Mowiol 8-88 due to its ability to
dissolve at high concentration and thus create a wide range of fiber diameters.
The difference in fiber diameters between the two types of PVA at an electrospinning
solution concentration of 10% can be explained by the difference in surface tension between
the solutions (47). The highly hydrolyzed PVA is more hydrophilic and thus has an increased
surface tension compared to a less hydrophilic polymer solution. This increase in surface
tension alters the Rayleigh instability relationships during splaying of the polymer jet in the
electric field. This alteration causes earlier gelation of the fiber jet as it travels to the
collector, thus increasing the size of fibers collected.
The use of multiple fiber collector geometries allowed manipulation of the fiber array
patterns from randomly oriented to fully aligned. This is advantageous because when used in
device fabrication, these two array patterns will yield very different drainage properties. As
discussed earlier, the Hagan-Poiseuille equation dictates fluid flow through a single pipe. It
states that drainage rate is proportional to the length of this tube. Considering with just one
nanochannel in a 1mm long drainage device, path length fluid will travel if the nanochannel
was formed with unaligned fibers will be much greater than 1mm. However, if an aligned
fiber array is used to fabricate the drainage device, the path length of fluid flow through the
device will be very close to, if not exactly, 1mm. In this study, unaligned fiber arrays were
studied due to their ease of handling compared to unaligned fiber which generally are formed
52
between two electrodes (hanging in air) rather than directly on a collector. Future studies
may investigate a manufacturing technique that allows for aligned fibers to be incorporated
into the device.
The use of varying concentrations of PVA solution yielded tailorable and predictable
nanofiber morphologies and topologies. This is the basis of obtaining a viable trans-corneal
drainage device as the size and morphology of the fibers impacts not only the drainage rate,
but the physical barrier to microbes on the surface of the eye. As the average size of corneal
bound microbes is about 1 micron, any concentration of low hydrolysis PVA would be able
to create a physical barrier to microbes if used to fabricate a sacrificial template for a GDD.
Synthesis of LCE was a simple one pot click reaction which required minimal
chemical synthesis skill. Following Yakacki et al, the TAMAP reaction produced and
elastomer which would set within 15-20 minutes of adding catalyst. This was helpful because
the reaction mixture was able to be poured into a mold around PVA nanofibers when it still
had a relatively low viscosity. Therefore the reaction mixture was able to fully penetrate the
nanofiber web before setting.
The change of diameter of the cylinder shown in Figure 27 gives an example of the
shape memory properties of this material. The chemistry used to synthesize these cylinders
can be easily tailored to achieve a thinner initial diameter by decreasing the crosslink density
during the first stage of the reaction. This, however, is at the expense of expansive strength of
the polymer. Outside the scope of this study, but still of significant importance to the final
product, would an investigation of the fixity and relaxation strength of the shape memory
behavior of a cylindrical LCE.
53
In this investigation, only the first stage of the reaction was used. This was effective
to observe whether the fiber mesh was being fully infiltrated by LCE monomers. In a second
stage reaction, a cylindrical sample would be stretched lengthwise and cured with a UV light.
Upon heating the sample up to body temperature, the device would expand both lengthwise
and axially (see Figure 11). The strength at which the device locks in place is crucial for
effective placement of the device as IOP swings of around 15 mmHG upon inversion of the
head (48).
Visual confirmation of pores via several modes of imaging showed that once samples
were treated to 48 hours of 60 oC water, or alternatively 30 minutes of sonication and 24
hours of 60 oC water, fibers were able to be dissolved out of the bulk LCE. In addition to
LCE, MMA/DEGDMA was a useful bulk polymer to fabricate test samples. This polymer is
glassy at room temperature, where LCE is quite rubbery. The hardness of MMA/DEGDMA
made it possible to create rigid cylinders which could be reliably superglued into testing
fixtures. Its amorphous nature differs from the LCE polydomain substructure and makes the
MMA/DEGDMA optically clear while the LCE is opaque. This allowed the fluorescent
images to be taken with MMA/DEGDMA (Figure 25) while the cross section SEM images
clearly showed nanopores in the LCE sample (Figure 24). The mechanical and optical
differences not only proved convenient, but the use of two different materials shows the
robustness of the manufacturing process.
The fluorescent image seen on the right side of Figure 25 is a powerful visual which
clearly shows fluid infiltrating the nanochannels. This indicates that fluid would pass through
a drainage device manufactured in the same manner. This image differs from the left side of
Figure 25 because the microscope used to take these images creates 15 micron deep Z-stacks
54
starting from the bottom of the sample. When imaging a 1mm thick sample, the camera only
captures the bottom most fibers in the sample. The 80 micron thick sample shows many more
fibers within the 15 micron deep window that was available to image. These images show
that fluid may be easily infiltrated into the nanochannels created after dissolution of
nanofibers embedded in a shape memory polymer.
To prove without a doubt that fluid may pass through a nanoporous drainage device
fabricated with an electrospun sacrificial template, water column perfusion tests were
performed with several indicators and a variety of test fixtures.
The first perfusion test involved supergluing a porous MMA/DEGDMA cylindrical
sample into the tip of a pipette. This was a delicate process that required glue around the
entire edge of the pipette tip while not allowing any glue to touch the flat top or bottom
surfaces of the sample as the leave the channels clear. Once the sample was secured and
sealed into the tip of the pipette and the pipette was filled with a colored dye, the tip of the
filter was dipped into a collection bath to wait for colored dye to perfuse the sample.
Encouraging results were obtained from these tests, with an indicator dye appearing to
perfuse through the nanochannels into a collection bath. While using a control sample
containing no pores, dye was not able to perfuse into the collection vial, proving a robust seal
around the cylinder.
The two other perfusion set ups also showed qualitative perfusion, with visual
confirmation being the indication of a positive result. In the future, a more elaborate
perfusion setup would be needed to quantify the flow rate at a given pressure. A
programmable syringe pump would be needed to obtain these results, such as Pump 11 Plus
by Harvard Apparatus.
55
CHAPTER V
CONCLUSION
The purpose of this investigation was to create a manufacturing technique for a
tailorable trans-corneal glaucoma drainage device using a sacrificial nanofiber template
within a shape switchable liquid crystal elastomer. After fabrication of an electrospinning
power unit, several concentrations of poly(vinylalcohol) were used to create sacrificial
nanofiber templates on flat plate and wire collectors. SEM images of these arrays showed
various morphologies and topologies of the fibers, including randomly oriented and highly
aligned. The diameters of the fibers produced varied from around 50 nm to 750 nm, which
once dissolved out of a bulk polymer yielded nanochannels of the same diameter. It was
shown in imaging and perfusion tests that not only could these nanochannels be infiltrated
with a liquid, a fluid may pass through them in a controlled fashion.
There is one instance in the literature published by Bellan et al which uses sacrificial
electrospun nanofibers as a template for nanochannels in a bulk polymer (38). The current
study expands on this in several ways. While Bellan et al focus on creating these channels in
a poly(dimethylsiloxane), or PDMS, substrate, the current study uses a functional shape
memory elastomer. This implies greater potential for end usage of such a device including,
but not limited to, a trans-corneal glaucoma drainage device. Additionally, the study
performed by Bellan et al did not deeply explore the relationships between electrospun fiber
morphology and the various electrospinning parameters such as solution concentration.
These relationships play a key role in the tunable nature of such a device, especially
considering the Hagen-Pouseuille equation which dictates flow as a function of channel
diameter to the fourth power.
56
There were limitations throughout this study that, if alleviated, would allow the
proper qualification of this transcorneal drainage device. Of particular importance is the
perfusion testing. These tests were designed to indicate a binary result – did the fluid pass
through or not. There was little means of measuring exact flow through test fixtures using the
low-tech solutions that were obtained for little to no cost. As a result, there was no way to
quantify the drainage rate of a trans-corneal filter containing convoluted and tortuous
nanochannels paths. As indicated previously, the Hagen-Pouseuille equation gives the
relationship between flow, pressure, and channel diameter. Another equation would better
suit this scenario if such quantifiable drainage information was available. The Darcy equation
relates flow and pressure inside a porous medium. The tortuous nature of the nanochannels
create a scenario that mimics a porous medium closer than a group of pipes. The Darcy
equation contains a K factor, which is derived from perfusion data. If an advanced perfusion
set-up (Pump 11 Plus, Harvard Apparatus) was available, a quantifiable relationship between
PVA solution concentration used in electrospinning and drainage rate of a trans-corneal
device could be derived.
Another limitation of the study was time. Aligned fiber arrays may prove necessary in
future studies if the drainage rates of filter devices using randomly oriented fiber arrays
proved too slow. Because of the different nature of aligned fibers (which collect between to
grounded units opposed to directly on a collector), separate manufacturing technique as well
as manufacturing fixtures must still be designed.
Lowering IOP in patients with glaucoma remains the cornerstone of limiting risk of
vision loss, and while there are many approaches to this, no current strategy is without
complications. This study proposed a unique solution to replace the gold standard both in
surgical and drainage device treatments of Glaucoma. A trans-corneal device which is
57
replaceable, tunable and easily deployed into patients has the potential to greatly reduce the
number of those who lose their vision from the disease. It will especially make an impact in
areas of the world where ophthalmic surgeons and surgical arenas are not readily available.
58
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