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University of Texas at El PasoDigitalCommons@UTEP
Open Access Theses & Dissertations
2017-01-01
Optimization of Collagen Microneedle UsingTaguchi MethodAbhilash AdityaUniversity of Texas at El Paso, [email protected]
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Recommended CitationAditya, Abhilash, "Optimization of Collagen Microneedle Using Taguchi Method" (2017). Open Access Theses & Dissertations. 393.https://digitalcommons.utep.edu/open_etd/393
OPTIMIZATION OF COLLAGEN MICRONEEDLE USING TAGUCHI
METHOD
ABHILASH ADITYA,
Master's Program in Manufacturing Engineering
APPROVED:
Tzu – Liang (Bill) Tseng, Ph.D., Chair
Namsoo (Peter) Kim, Ph.D. Co-Chair
Amit. J. Lopes, Ph.D.
Charles Ambler, Ph.D.
Dean of the Graduate School
Copyright ©
by
Abhilash Aditya
2017
OPTIMIZATION OF COLLAGEN MICRONEEDLE USING TAGUCHI
METHOD
by
ABHILASH ADITYA, B.E
A THESIS
Presented to the Faculty of the Graduate School of
The University of Texas at El Paso
in Partial Fulfillment
of the Requirements
for the Degree of
MASTER OF SCIENCE
Department of Industrial, Manufacturing and Systems Engineering (IMSE)
THE UNIVERSITY OF TEXAS AT EL PASO
December 2017
iv
Acknowledgements
I would first like to thank my thesis advisors Dr. Tzu – Liang (Bill) Tseng and Dr.
Namsoo (Peter) Kim. Their doors were always open whenever I ran into a trouble spot or had a
question about my research or writing. They consistently allowed this paper to be my own work
but steered me in the right the direction whenever he thought I needed it. I would like to thank
Dr. Amit. J. Lopes for being a committee member and for their valuable guidance.
I would also like to acknowledge Monica Michel, Ph.D. for her support and I am
gratefully indebted to her very valuable comments on this thesis. I have received ample guidance
from my fellow researchers at PNE Lab that was instrumental in the completion of this thesis.
Finally, I must express my very profound gratitude to my parents for providing me with
unfailing support and continuous encouragement throughout my years of study and through the
process of researching and writing this thesis. This accomplishment would not have been
possible without them. Thank you.
v
Abstract
Research is conducted to create dissolving collagen microneedles and to optimize the parameters
for close to perfect dimension microneedles. A collagen microneedle patch has been developed
with a micro-manufacturing process (micro-molding process). The collagen microneedles,
varying in length from 300 to 600-μm may reach different targeted layers of the skin depending
on the application. As the microneedles penetrate the skin they dissolve, delivering collagen
through the epidermis. Such addition of collagen directly to the dermis layers allows it to be
absorbed, increasing skin's strength and resilience, since it is one of the natural components of
the younger looking skin. This process of collagen delivery system is painless, hygienic and easy
to use. The research included the preparation of various concentrations and modification in
various parameters in order to optimize the process of the microneedle patch preparation. Data is
collected from various experimental trials and statistical analysis is conducted using Taguchi L9
method. Parameters such as microneedle height, pressure drop, time in the vacuum oven, and
concentration are considered and analyzed to check which parameter has the greatest influence in
the production of in order to obtain the best microneedle patch. The dissolving collagen
microneedle process parameters are optimized for close to perfect dimensions.
vi
Table of Contents
Acknowledgements ........................................................................................................................ iv
Abstract ............................................................................................................................................v
Table of Contents ........................................................................................................................... vi
List of Tables ............................................................................................................................... viii
List of Figures ................................................................................................................................ ix
Optimization of collagen microneedles using Taguchi method ......................................................1
Chapter 1: Introduction ....................................................................................................................1
1.1Skin .............................................................................................................................................1
1.1.1 Epidermis .......................................................................................................................1
1.1.2 Dermis ............................................................................................................................2
1.1.3 Hypodermis ....................................................................................................................4
1.2 Collagen in skin .........................................................................................................................5
Chapter 2: Drug delivery through skin ............................................................................................7
2.1 Transdermal drug delivery ................................................................................................7
Chapter 3: Microneedles ................................................................................................................15
3.1 Microneedle Manufacturing .....................................................................................................19
3.1.1 Micro- electro discharge machining ............................................................................20
3.1.2 Reactive ion etching .....................................................................................................23
3.1.3 Micro-molding .............................................................................................................24
3.1.4 Drawing lithography ....................................................................................................26
3.1.5 Photo Lithography .......................................................................................................27
3.2 Materials ..................................................................................................................................29
3.2.1 Collagen .......................................................................................................................29
3.2.2 Polyvinylpyrrolidone ...................................................................................................31
vii
3.3 Procedure .................................................................................................................................32
Chapter 4: Statistical Analysis .......................................................................................................47
4.1 Taguchi Method ..............................................................................................................47
4.1.1 Control factors and levels ............................................................................................48
4.1.2 Procedure of microneedle preparation .........................................................................50
4.1.3 Data collection .............................................................................................................50
4.1.4 Validation .....................................................................................................................76
Chapter 5: Conclusion....................................................................................................................77
Chapter 6: Future Scope.................................................................................................................78
References ......................................................................................................................................79
Appendix ........................................................................................................................................83
Vita 84
viii
List of Tables
Table 2.1: Estimated global annual incidence, deaths, years of life lost, and cost resulting from
unsafe injection practices for hepatitis B, hepatitis C and human immunodeficiency virus (HIV)
infections using best case assumptions (i.e. minimizing disease burden and cost) ...................... 10 Table3.3(a): Microneedle mold dimensions ................................................................................. 34
Table 3.3(b): Parameters for the 15% and 30% collagen trials .................................................... 35 Table 3.3(c): Parameters for different ratios of collagen trials ..................................................... 37 Table 4.1.1(a): Factors and levels ................................................................................................. 48 Table4.1.1(b): Layout of L9 Orthogonal Array ............................................................................ 49 Table 4.1.1(c): Layout of L9 Orthogonal Array for microneedles ............................................... 49
Table 4.1.3(a): Ideal Volumes of microneedles ............................................................................ 52 Table 4.1.3(b): Actual volumes of the microneedles .................................................................... 69 Table 4.1.3(c): Trial volume ratios ............................................................................................... 70
Table 4.1.3(d): Means and standard deviation .............................................................................. 71
Table4.1.3(e): Signal to noise ratios ............................................................................................. 72 Table 4.1.3(f): Ranking of control factors .................................................................................... 73
ix
List of Figures
Figure 1.1.1:Division of epidermal layer of the skin3 ..................................................................... 2 Figure 1.1.2:Cross sectional view of dermis2 ................................................................................. 4 Figure 1.1.2 (a):Subcutaneous layer (Hypodermis)13 ..................................................................... 4 Figure 1.1.3 (b): Adipose tissue13 ................................................................................................... 5
Figure 1.2.1: Collagen in young and old skin14 .............................................................................. 6 Figure 2.1.1: Hypodermic needle in skin23 ..................................................................................... 8 Figure 2.1.2(a): Mortality estimates for HIV, Hepatitis B and Hepatitis C due to unsafe injections
(n = 1.301 million deaths). 28 ........................................................................................................ 10 Figure 2.1.2 (b): Cost of disease for Hepatitis and HIV (n= US$535 million).28 ......................... 11
Figure 2.1.3 (a): Needle stich injuries among male and female Health care workers. 29 ............. 12 Figure 2.1.3 (b): Needle stick injury among different groups of health care workers. 29 ............. 12 Figure 2.1.3 (c): Needle stick injury in different age groups among health care workers. 29 ....... 13
Figure 2.1.3 (d): Needle stick injury at different working stations. 29 .......................................... 13
Figure 3.1: Types of microneedles34 ............................................................................................. 16 Figure 3.2: Drug delivery methods from types of microneedles. (a) Solid (b) Coated (c)
Dissolving (d) Hollow32 ................................................................................................................ 17
Figure3.3: Thickness of the layers of the skin35 ........................................................................... 19 Figure 3.1.1(a): Electro discharge machining.43 ........................................................................... 21
Figure 3.1.1(b): Micro electro discharge machining process. 43 ................................................... 22 Figure 3.1.1(c): Microneedle after Micro EDM44 ......................................................................... 22 Figure3.1.2(a): Reactive ion etching47 .......................................................................................... 24
Figure 3.1.2(b): Silicon microneedles after reactive ion etching.48 .............................................. 24
Figure 3.1.3: (a) creation of master microneedle array; (b) Obtaining the master microneedle
array; (c) Preparing the microneedle mold; (d) Removing the microneedle mold; (e) Pouring the
microneedle material; (f) obtaining the final microneedle. 50 ....................................................... 25 Figure 3.1.4(a): Drawing lithography process39 ........................................................................... 26 Figure 3.1.4(b): The SEM image of thermosetting polymer after drawing lithography39 ............ 27 Figure 3.1.5(a): Micro lenses etched (a) Low magnification (b) High magnification (c) Array of
micro lenses51 ................................................................................................................................ 27
Figure 3.1.5(b): Photolithography process51 ................................................................................. 28 Figure 3.1.5(c): Effect of micro lens on the shape of the microneedle51 ...................................... 29 Figure 3.1.5(d): Microneedles fabricated by photolithography51 ................................................. 29 Figure 3.2.1(a): Molecular structure of collagen fiber54 ............................................................... 30 Figure3.2.2(a): (1) acetylene; (2) formaldehyde; (3) 2-butyne-1, 4-diol; (4) 1,4-butanediol; (5)
gamma- butyrolactone; (6) alpha-pyrrolidone; and (7) N-vinyl-2-pyrrolidone57 ......................... 31
Figure 3.2.2(b): Chemical structure of polyvinylpyrrolidone58 .................................................... 32 Figure 3.3(a): Preparation of PDMS microneedle mold ............................................................... 32 Figure 3.3(b): Micropoint Technologies ....................................................................................... 33 Figure3.3(c): Schematic representation and cross-sectional view of microneedle mold ............. 33
Figure3.3(d): PDMS microneedle mold from micropoint technologies ....................................... 34 Figure 3.3(e): Molten microneedle patch ...................................................................................... 36 Figure 3.3(f): Deteriorated microneedle patch.............................................................................. 38 Figure3.3 (g): 1:1 (collagen: PVP) 300 μm .................................................................................. 39
Figure 3.3(h): 1:1 (collagen: PVP) 400 μm .................................................................................. 39
x
Figure3.3(i): 1:1 (collagen: PVP) 500 μm .................................................................................... 40 Figure3.3(j): 1:1 (collagen: PVP) 600 μm .................................................................................... 40
Figure 3.3(k): 6:4 (collagen: PVP) 300 μm .................................................................................. 41 Figure 3.3(l): 6:4 (collagen: PVP) 400 μm ................................................................................... 42 Figure 3.3(m): 6:4 (collagen: PVP) 500 μm ................................................................................. 42 Figure 3.3(n): 7:3 (collagen: PVP) 300 μm .................................................................................. 43 Figure 3.3(o): 7:3 (collagen: PVP) 400 μm .................................................................................. 43 Figure3.3(p): 7:3 (collagen: PVP) 500 μm ................................................................................... 44
Figure3.3(q): 7:3 (collagen: PVP) 600 μm ................................................................................... 44 Figure 3.3(r): 8:2 (collagen: PVP) 400 μm ................................................................................... 45 Figure 3.3(s): 8:2 (collagen: PVP) 300 μm ................................................................................... 45 Figure4.1.3: Square pyramid......................................................................................................... 51
Figure 4.1.3(a): Experiment 1 Trial 1 ........................................................................................... 52 Figure 4.1.3(b): Experiment 1 Trial 2 ........................................................................................... 53 Figure 4.1.3(c): Experiment 1 Trial 3 ........................................................................................... 53 Figure 4.1.3(d): Experiment 2 Trial 1 ........................................................................................... 54 Figure 4.1.3(e): Experiment 2 Trial 2 ........................................................................................... 55
Figure 4.1.3(f): Experiment 2 Trial 3............................................................................................ 55 Figure 4.1.3(g): Experiment 3 Trial 1 ........................................................................................... 56
Figure 4.1.3(h): Experiment 3 Trial 2 ........................................................................................... 56 Figure 4.1.3(i): Experiment 3 Trial 3 ............................................................................................ 57 Figure 4.1.3(j): Experiment 4 Trial 1 ............................................................................................ 58
Figure 4.1.3(k): Experiment 4 Trial 2 ........................................................................................... 58 Figure 6 Figure 4.1.3(l): Experiment 4 Trial 3 ............................................................................. 59
Figure 4.1.3(m): Experiment 5 Trial 1 .......................................................................................... 60 Figure 7 Figure 4.1.3(n): Experiment 5 Trial 2 ............................................................................ 61
Figure 4.1.3(0): Experiment 5 Trial 3 ........................................................................................... 61 Figure 4.1.3(p): Experiment 6 Trial 1 ........................................................................................... 62 Figure 4.1.3(q): Experiment 6 Trial 2 ........................................................................................... 62
Figure 4.1.3(r): Experiment 6 Trial 3............................................................................................ 63 Figure 4.1.3(s): Experiment 7 Trial 1 ........................................................................................... 64
Figure 4.1.3(t): Experiment 7 Trial 2 ............................................................................................ 64 Figure 4.1.3(u): Experiment 7 Trial 3 ........................................................................................... 65
Figure 4.1.3(w): Experiment 8 Trial 2 .......................................................................................... 66 Figure 4.1.3(x): Experiment 8 Trial 3 ........................................................................................... 66 Figure 4.1.3(y): Experiment 9 Trial 1 ........................................................................................... 67 Figure 4.1.3(z): Experiment 9 Trial 2 ........................................................................................... 68
Figure 4.1.3(1): Experiment 9 Trial 3 ........................................................................................... 68 Figure 4.1.3(2): Mean of height .................................................................................................... 74 Figure 8 4.1.3(3): Mean of pressure drop ..................................................................................... 74
Figure 9 4.1.3(4): Mean of time .................................................................................................... 75 Figure 4.1.3(5): Mean of concentration ........................................................................................ 75
1
Optimization of collagen microneedles using Taguchi method
Chapter 1: Introduction
1.1Skin
Skin is the largest organ in the human body with an average surface area of 1.6–2 m2 and
accounts for about 15% of the total body weight of an adult. The skin provides protection from
the external environment preventing entry of pathogens. Apart from this, it prevents the water
loss, regulation of body temperature, enables sensations to be perceived, and plays a key role in
the synthesis of vitamin D.1 The skin is broadly divided into three parts: epidermis, dermis, and
hypodermis.2
1.1.1 EPIDERMIS
The epidermis is the topmost layer of the skin. The epidermis layer is divided, and the
layers are microscopically identified as Stratum Corneum, Stratum Lucidum, Stratum
Granulosum, Stratum Spinosum and Stratum Basale or Stratum Germinavatum (Figure 1.1.1).
The epidermis of thin skin consists of Stratum Basale, Stratum Spinosum, Stratum Granulosum
and Stratum Corneum. Whereas the epidermis of the thick skin consists of Stratum Basale,
Stratum Spinosum Stratum Granulosum, Stratum Lucidum and Stratum Corneum. The Stratum
Lucidum is a layer of rows of transparent, flattened, dead keratinocytes. The Stratum Corneum is
around 20 to 30 cell layers thick. It consists of cornified or horny cells and is up to 3/4 of the
epidermal thickness. It is prone to injuries from the external environment and mainly consists of
keratinocytes (flat squamous cells) containing a protein known as keratin. The Glycolipids in
2
between the cells help in preventing water loss. The Stratum Granulosum is 3 to 5 cells thick
consisting lamellar granules and Tonofibrils. This layer onwards, the cells start to die. Stratum
Spinosum is the thickest layer it is also known as the Spinous (prickly) layer. Stratum Basale or
Stratum Germinavatum is the deepest layer of all. This layer is constantly undergoing cell
division and helps in the formation of new keratinocytes that replace the cells in stratum
Corneum. This process takes about 27 days.3,4
Figure 1.1.1:Division of epidermal layer of the skin3
1.1.2 DERMIS
The dermis accounts for 90% of the weight of the skin and is also the foundation of the
skin organ system.5 It lies between the upper epidermis and the lower subcutaneous fat layer
(hypodermis) of the skin. This layer of skin serves as a structural and nutritional supporter of the
upper epidermis layer.6 The thickness of the dermis varies from one part of the body to the other.
The dermis on eyelids is about 0.6mm whereas the dermis in palm and sole, is about 3mm. The
thickness of the dermis changes even with respect to gender. The thickness of the dermis in
eyelids is thicker in men when compared to women.7
3
Usually, the dermis is thicker in women than men. The dermis gets thicker with age and
then at some point the thickness decreases. In women, the thickness grows until the age of 35
and then it decreases, whereas, in men, the thickness increases until the age of 55 and then
gradually decreases. In women, it is seen that the dermis thickness reaches its peak at the age of
32 or 33 and then gradually reduces till the age of 70 and then raises up to the age of 85.8
The matrix of collagen and elastic connective tissue builds the dermis. These two
components exist in varying quantities according to the depth of the dermis. This enables the
dermis to constantly undergo turnover and remodeling in normal skin, in pathologic processes,
and in response to external stimuli.9 Dermis can be divided into two layers such as papillary
layer and the reticular layer (Figure 1.1.2). The papillary layer consists of loose connective tissue
containing anchoring fibrils and numerous dermal cells. The reticular layer consists of compact
connective tissue containing large bundles of collagen and elastic fibers which provide strength
and resilience to the dermis.10 This layer also consists of sebaceous and sudoriferous glands,
nerves, and hair follicles.11
4
Figure 1.1.2:Cross sectional view of dermis2
1.1.3 HYPODERMIS
The hypodermis, also known as the subcutaneous layer (Figure 1.3 (a)), is the deepest
layer of the skin. This layer is made up of connective tissues and fat, with large blood vessels and
nerves present in this layer. It protects from external impact and maintains the body temperature.
The depth of the subcutaneous layer varies from one person to another. 11 The hypodermis
consists of fibroblasts, lymphocytes, mast cells and network of adipocytes, that is fat cells
arranged in the form of lobules. This layer stores readily available energy source, provides
mechanical cushion, and acts as a heat insulator.12
Adipose tissue (Figure 1.1.3 (b)) is composed of fat at a level of 60- 85%, with 90-99%
being triglycerides, 5-30% water and 2- 3% protein. The thickness of this tissue varies with
anatomical site, age, gender, race, and endocrine and nutritional status of the individual. This
layer consists of blood vessels that supply blood and lymphatic vessels for the dermis, nerves and
hair follicles.13
Figure 1.1.2 (a): Subcutaneous layer (Hypodermis)13
5
Figure 1.1.3 (b): Adipose tissue13
1.2 Collagen in skin
Collagen is one of the most important proteins of the skin, since the extra cellular matrix
of the dermis is functionally and structurally supported by collagen. The reduction of the
collagen content from the skin gives rise to the signs of aging (Figure 1.2.1).14 This reduction of
collagen in skin may be due to various reasons such as prolonged exposure to the sun causing
solar elastosis. This happens when the bonding between the dermis and epidermis decreases as
collagen levels are lower.15 Another reason of collagen depletion may be high serum glucose
levels in diabetic patients. Glycation is a big reason of decrease of collagen content in the skin.
The glycation products are dependent on oxidation reactions.14
The cutaneous aging process can be divided into two: extrinsic and intrinsic processes.
The solar elastosis can be categorized as an extrinsic process. The term ‘biological clock’ is used
to express a theory of aging. This would be the intrinsic process of aging. The degenerative
changes in the tissue may be due to the finite life span of the fibroblasts, causing cellular
senescence, and thereby altering the gene expression, in the so-called Hayflick phenomenon.
Another theory of free radicals is also known to have effects on the aging of the skin.14
6
Skin consists of collagen type I, III, V and VI, where VI being in the basement
membrane. Collagen type III is the most abundant in the papillary dermis, and type I is abundant
in the reticular dermis. The dermis also secretes proteoglycans such as decorin, biglycan,
fibromodulin, lumican, epiphycan, and keratocan. Any sort of mutation in these proteoglycans
can lead to defective fibrillogenesis and functions. The cross linking of collagen via the enzyme
Lysyl oxidase, and reduced activity of the enzyme can lead to collagen defects, as in Cutis laxa.5
Figure 1.2.1: Collagen in young and old skin14
7
Chapter 2: Drug delivery through skin
Drug delivery through the skin can be accomplished in one of two ways. They are by an
injection and through a transdermal patch system. Furthermore, the disadvantages of tablets,
such as difficulty in swallowing, poor wetting and dissolution properties which are involved in
orally administered drugs, may be overcome.16 Drug induced liver damage is one of the issues
caused by the orally administered drug delivery system. Poor absorption of the drugs and the
enzymatic degradation in the gastrointestinal tract or liver are not uncommon.17 Several surveys
have revealed the fact that a percentage of patients taking the drugs orally have suffered liver
injuries and a few may even have had liver transplants.18 Such facts make even more ideal the
possibility of transdermal drug delivery systems.
2.1 TRANSDERMAL DRUG DELIVERY
The transdermal drug delivery market worldwide is around US$2 Billion dollars
implying it is one of the most successful non-oral systemic drug delivery systems.19 Transdermal
drug delivery has certainly had an advantage over the oral administration of drugs especially
when considering the significant first-pass effect of the liver that can prematurely metabolize
drugs.20 One of the best-known drug delivery systems is the hypodermic needle. Charles Gabriel
Pravaz in France and by Alexander Wood in England in 1853 independently invented the
hypodermic needles.21
The hypodermic needles penetrate the skin and go through layers such as the epidermis,
dermis and the hypodermis (Figure 2.1.1). Such penetration serves as an effective way to deliver
the drugs Trans dermally. However, the needle comes into contact with the nerves and the blood
vessels. The hypodermic needle’s contact with the nerves induces pain, causing the receiver a
8
sense of discomfort. The large size of the hypodermic needle, which is manually inserted into the
skin, makes it difficult to deliver the drug to the exact target site within the skin.22
Figure 2.1.1: Hypodermic needle in skin23
The observation and the sensation of the hypodermic needle entering the body may cause
fear to few people. It can be said that such people are afraid of needle in other words they have
trypanophobia. It was seen in a study that hypodermic needles create stress-provoking stimulus
among children.24 There are 4 different types of fear of needles: vasovagal, associative, and
resistive and hyperalgesic. Vasovagal affects 50% of the people, which is just an inherent reflex
reaction. Associative type affects around 30% of the people who have witnessed the traumatic
painful medical procedure. Resistive type affects 20% of people who have experienced poor
handling or being forced physically or emotionally. Hyperalgesic type affects the other 10% of
the population due to hypersensitivity.25
The studies indicate that one quarter or 25% of the population suffers from fear of
needles. Most deaths from human history are caused by skin penetration caused by teeth, claws
and weapons. It can be said that the fear of needles can be due to the human evolution to survive
according to the etiology theory.26
9
Hypodermic needles are one of the most well known modes of administering drugs
through the skin, as well as a very effective method of delivering the drugs through the skin.
However, such penetration comes with serious health hazard such as an undesired transfer of
blood borne pathogens. In studies conducted, it has been seen that over 50 % of the injections
given in developing countries, are performed under the practice of unsafe administration
procedures.27 The morality estimates and the cost of the diseases are illustrated in Figure 2.1.1
(a) and (b).
Another use of hypodermic needles is while administering immunization vaccinations,
specifically for children. However, children are the most exposed victims to unsafe vaccination
methods, resulting in about 70-90% of children exposed to hepatitis B, which later become
chronic carriers. It’s been observed that 6-7% of the people get infected as adults and out of 20-
28% of them will die of causes related to hepatitis B.28 The annual incidence, deaths, years of life
lost, and cost resulting from unsafe injection practices for the diseases are shown in Table 2.1.
10
Table 2.1: Estimated global annual incidence, deaths, years of life lost, and cost resulting from
unsafe injection practices for hepatitis B, hepatitis C and human immunodeficiency
virus (HIV) infections using best case assumptions (i.e. minimizing disease burden
and cost)
Hepatitis B Hepatitis C HIV Total
Annual incidence
of infections
from unsafe
injections
(x 106)
8.2
2.3
0.1
10.6
Future deaths
(x 106)
1.0
0.2
0.1
1.3
No. of years of
life lost (x 106)
19.7
3.6
2.7
26.0
Direct medical
cost of
disease
(x 106 US$)
327
59
149
535
Figure 2.1.2(a): Mortality estimates for HIV, Hepatitis B and Hepatitis C due to unsafe injections
(n = 1.301 million deaths). 28
382
475
221
181
16 26China
India
Other Asia
Sub-Saharan Africa
Middle EasternCrescent
Former SocialistEconomies
11
Figure 2.1.2 (b): Cost of disease for Hepatitis and HIV (n= US$535 million).28
The blood borne pathogen transfer occurs not only due to unsafe vaccination procedures
but also due to sharp injuries. Healthcare workers fall victim to such injuries as they very closely
interact with patients with such chronic diseases. Needle stick injury is defined as injury caused
by objects such as hypodermic needles, blood collection needles, intravenous (IV) stylets and
needles used to connect parts of IV delivery systems.29
A study was conducted with 757 heath care workers including 350 nursing staff,
120 housekeeping staffs, 217 doctors and 70 medical laboratory technicians for needle stick
injuries and the following results as shown in Figure 2.1.3 (a), (b), (c) and (d) were found.
142
106140
87
1051
China
India
Other Asia
Sub-Saharan Africa
Middle Eastern Crescent
Former Socialist Economies
12
Figure 2.1.3 (a): Needle stich injuries among male and female Health care workers. 29
Figure 2.1.3 (b): Needle stick injury among different groups of health care workers. 29
26.83
73.17
Male Female
58.54
36.58
0 4.88
Nursing Staff
Housekeeping Staff
Doctors
Medical laboratoryTechnicians
13
Figure 2.1.3 (c): Needle stick injury in different age groups among health care workers. 29
Figure 2.1.3 (d): Needle stick injury at different working stations. 29
According to United States Department of Labor-Occupational Safety and Health
Administration [USDOL-OSHA] (in 2001), around 600,000 to 800,000 health care workers are
exposed to blood, with registered nurses sustaining most of such injuries. According to The
19.51
68.29
12.19
<20 years
20-30 years
>30 years
46.34
4.88
9.76
26.83
7.324.88
Wards
Laboratories
Out patient
Intensive care unit
Emergency unit
Others
14
Centers for Disease Control and Prevention [CDC], (1998a) such exposures include syphilis,
malaria, herpes and 20 more infections along with HIV and hepatitis. According to International
Health Care Worker Safety Center (1999), at least 1000 health care workers get infected
annually.30
National Institute for Occupational Safety and Health [NIOSH] has listed the specific designs of
the device, which are more dangerous to cause such needle stick injuries:30
• Devices with hollow-bore needles.
• Needle devices that need to be taken apart or manipulated by health care worker, like
blood-drawing devices that need to be detached after use.
• Syringes that retain an exposed needle after use.
• Needles that are attached to tubing-like butterflies that can be difficult to place in sharps
disposal containers.
Hypodermic needles are surely one of the best methods of drug delivery through the skin:
however, due the adverse drawbacks like leading to discomfort and put many in life-threatening
situations, there is a need to look for a better and safer way of drug delivery systems.
15
Chapter 3: Microneedles
The progress in the field of transdermal drug delivery has given rise to microneedles, the
new and effective way to deliver drugs through the skin. As the inability of the body to absorb
orally administered drugs is countered by the use of traditional hypodermic needles,17 and while
hypodermic needles are considered an the effective way to deliver the drugs to the body, most
people still relate the process of drug delivery through the hypodermic needles to feelings of pain
and discomfort.
Drug delivery through hypodermic needles is simply breaking the topmost and the
toughest layer of the skin. In other words, hypodermic needles increase the permeability of the
skin for the drugs to enter the body. Researchers have come up with a new process of
transdermal drug delivery method known as microneedles. Unlike hypodermic needles,
microneedles consist of a patch with numerous tiny needles of dimensions measured in microns.
These microneedles penetrate the skin allowing the drug to enter the body.31
The architecture of the microneedles is designed to overcome the disadvantages of the
hypodermic needles. The size of the microneedles is small enough to avoid the nerves (pain
receptors) causing no pain and discomfort, and strong enough to bypass the stratum corneum.32
Advancement in technology has enabled the production of microneedles of various sizes
and materials using Micro-Electro-Mechanical System, MEMS. Materials such as SU-8 (Epoxy
based negative photoresist), PMMA (Polymethylmethacrylate), PDMS (Polydimethylsiloxane,
silicon etc. are used as the materials of microneedles.33 Adding to this, the latest technologies
16
such as photolithography and ion etching are used for more accurate preparation of
microneedles. The type of microneedles varies depending on the type of usage and the form of
drug delivered. The types of microneedles that exists and are in use are: solid, coated, dissolving
and hollow microneedles (Figure 3.1).
Figure 3.1: Types of microneedles34
17
Figure 3.2: Drug delivery methods from types of microneedles. (a) Solid (b) Coated (c)
Dissolving (d) Hollow32
Each type of microneedles has their own unique way of delivering the drugs through the
skin. Starting with the solid microneedles, the patch has a microneedles array with no drugs in
the needles, either inside or coated. The solid microneedle patch is just designed to puncture the
skin, that is the stratum corneum to increase the permeability of the skin. The concentration of
the drug decides the number of micropores and size of the pores (Figure 3.2(a)). The advantage
of such type of microneedle is that there is no need of encapsulating or coating the drugs over the
microneedle. Due to the two-phase process, it can be complicated. The potency of the drug must
be high to make its way through the porous skin. Proper precise dosage of drugs cannot be
administered due to the application procedure.35
18
The coated microneedle is transdermal patch of microneedles whose needles are coated
with the drugs. When the coated microneedles penetrate the skin, the drugs already on the needle
get transferred to the skin (Figure 3.2(b)). The coated microneedles require efficient coating
procedure to coat the drugs on to the microneedles. Due to the minimal surface area, the dosage
of the drug is small, and because of the coating, the sharpness of the microneedles might be
reduced, furthermore, the drug may have to be reformulated before being coated on to the
microneedles. Still, it is important to note that the structural strength of the microneedles can be
retained after coating.35,32
In another type, the microneedle itself is made up of the drug material. The drug might
have to be reformulated to be a structural component of the microneedles. As the material takes
over the structural component, there might be fracture and deformation in the microneedle
geometry. It is often observed to have less sharpness as the drug material takes the shape of the
microneedle. The encapsulation or the absorption process of the drugs from the microneedle
might have an effect in the loss of a small amount of drug. Once the microneedle enters by
creating pores, the drug filled microneedles dissolves into the skin (Figure 3.2(c)). The waste can
be eliminated in this type of microneedles as the microneedles dissolve inside the skin.32,35
Hollow microneedles behave like micro-hypodermic needles (Figure 3.2(d)). In this case,
the microneedles are small and hollow. Due to the extreme design specifications, they are
comparatively complex to produce. Such complexity of design comes with many advantages
such as a delivery of high volume of drugs to start with, and it is possible to implement the use of
19
a controller of drug delivery. Addition of this microcontroller to the microneedle may provide
the ability to administer a large quantity of drug for a prolonged time. As there are no drugs that
need to be coated on to the microneedles nor the drugs are part of the structural component, there
is no necessity to reformulate the drugs. However, such microneedle type comes with a slight
complication, which is clogging of the hollow microneedle pathways. Also, leakage in the
microneedle array can be considered as a drawback.35 Moreover, such hollow microneedles can
be used more than just as a means of drug delivery, the hollow microneedle can be used to
remove bodily fluids as well with proper modifications.32
As mentioned above, the microneedles can be designed and constructed according to the
necessity. They can be produced in different heights to target different areas/ layers of the skin
(Figure 3.3). The description of the skin layer with the thickness is illustrated in the picture
below.
Figure3.3: Thickness of the layers of the skin35
3.1 Microneedle Manufacturing
20
The microneedles can be produced through various methods such as micro molding36,
electro discharge machining37, reactive ion etching process38, drawing lithography39,
photolithography40. Microneedles can be produced in different methods depending on the
material of the microneedles that needs to be produces and the desired design parameters.
The four types of microneedle such as solid, coated, dissolving and hollow microneedle
all have to be manufactured in different method suitable for its type. Some types of microneedle
such as hollow microneedle may need a combination of manufacturing methods to fabricate.
3.1.1 MICRO- ELECTRO DISCHARGE MACHINING
The micro electro discharge machine is method of precise machining process. Materials
such as conductive hardened material are usually machined through this process.41 Electro
discharge machining is a non-contact thermal machining process. Controlled and spatially
discreet high frequency electric discharge is used to remove the material.42
The electro discharge machining (EDM) control system produces calculated high
frequency electric discharge at a suitable depth for the required material removal from the work
piece. The electrode has three degrees of freedom, which helps in convenient movement of the
electrode over the work piece. A dielectric fluid is supplied continuously to wash away the
removed material from the work piece. When an insulating material is to me machined, an
assistive electrode is placed over the insulating material. The assistive electrode helps the
machining process to be carried out as usual (Figure 3.1.1(a)).
21
Figure 3.1.1(a): Electro discharge machining.43
The electrode of the EDM is brought close to the work piece with the assistive electrode.
The electrode on the EDM is the cathode and the work piece is the anode. The control system of
the EDM generates a high frequency electric discharge according to the desired etching rate and
the material. This discharge escalates the ionization process and the formation of electron
avalanche. This electron avalanche increases the temperature and melts the portion of the work
piece subjected to such condition. Thus, the molten material from the work piece is eroded and
washed away by the dielectric medium. This process is repeated multiple times until the work
piece acquires the desired shape (Figure 3.1.1(b) ). The microneedle manufactured by EDM
process is shown in Figure 3.1.1(c).
22
Figure 3.1.1(b): Micro electro discharge machining process. 43
Figure 3.1.1(c): Microneedle after Micro EDM44
The EDM is an effective and the precise process of material removal, but it has some
disadvantages, such as:42
• The threshold value of the resistivity of the material to be machined is 100 Ω.
23
• Material removal rate is much slower than the conventional material removal
processes.
• The energy efficiency is very low. About 18-50% of the input energy is used for
machining. It also has a very low flush rate of about 10-20% further disrupting the
material removal.
• It is an expensive process as the electrode gets consumed. Constant calibration is
necessary to avoid inaccuracies.
3.1.2 REACTIVE ION ETCHING
The reactive ion etching is a process of surface material removal in which a plasma is
created varying the ratio of the gas-flow, reaction pressure and radio frequency power (Figure
3.1.2(a)).45 Reactive ion etching provides high rate isotropic etching and it is possible to control
the etch rate by controlling the flow of gases such as oxygen, chlorine and fluorides.46 The work
piece will be placed on the electrode plate with an appropriate photo mask, and the required ratio
of gas mixtures are allowed to flow inside through the gas inlets. A radio frequency power
supplier powers the device while the gas mixture ionizes and forms plasma over the electrode
plate. The etching process that is occurring over the electrode plate may result in both isotropic
and anisotropic etching. The microneedle array etched by reactive ion etching is shown in the
Figure 3.1.2(b).
24
Figure3.1.2(a): Reactive ion etching47
Figure 3.1.2(b): Silicon microneedles after reactive ion etching.48
3.1.3 MICRO-MOLDING
Micro molding is one of the oldest fabrication processes. There are five types of mold
fabrication processes. They are Injection molding, reaction injection molding, hot embossing,
injection compression molding, and thermoforming.49 For the production of microneedle molds,
injection molding and hot embossing are the most predominantly used.
25
The production of microneedles through casting includes production of a master
microneedle array at the beginning. The micro EDM process or the reactive ion etching process
fabricates the master microneedle array. The master microneedle array is then hot embossed to
create a microneedle mold and then the required microneedle material in liquid state is poured
into the microneedle mold. It is then removed from the mold after solidifying (Fig 3.1.3 ).
Figure 3.1.3: (a) creation of master microneedle array; (b) Obtaining the master microneedle
array; (c) Preparing the microneedle mold; (d) Removing the microneedle mold; (e)
Pouring the microneedle material; (f) obtaining the final microneedle. 50
26
3.1.4 DRAWING LITHOGRAPHY
Drawing lithography is used to produce ultra-high aspect ratio microneedles from
thermosetting polymer on a 2D plate to 3D microneedle up to a height of 2 millimeters. A plate
is spin coated and heated before the thermosetting polymer gets into contact. Pillars of
thermosetting polymers of required dimensions and arrangement are put in contact with the plate.
The first drawing is performed to elongate the thermosetting polymer. The heat from the plate
makes the thermosetting polymer stick and when elongated, the diameter of the polymer
decreases. At the required length, the drawing stops and the elongated polymer is thermally
cured. The elongated polymer then becomes strong and hard. The second drawing is performed
to break the thermosetting polymer to form needles (Figure 3.1.4(a)).39 The microneedles
prepared by the drawing lithography is shown in the Figure 3.1.4(b).
Figure 3.1.4(a): Drawing lithography process39
27
Figure 3.1.4(b): The SEM image of thermosetting polymer after drawing lithography39
3.1.5 PHOTO LITHOGRAPHY
The photolithography fabrication process involves the use of light to cure/prepare the
microneedles. The light rays are subjected to bending after passing through a glass substrate. The
glass substrate is etched to form a concave shape to make the light converge (Figure 3.1.5(c)).
Figure 3.1.5(a): Micro lenses etched (a) Low magnification (b) High magnification (c) Array of
micro lenses51
The glass substrate is coated with chromium and then a photo mask is placed on it, and
then undergoes etching of craters in the form of concave lenses. An array of micro lenses result
at the end of the procedure (Figure 3.1.5(a)).51
28
Figure 3.1.5(b): Photolithography process51
The etched glass substrate is then placed over the microneedle polymer and exposed to
the ultraviolet rays. The micro lenses on the glass substrate make the UV rays bend and converge
in the shape of the microneedles (Figure 3.1.5(b)). The chromium residue on the glass substrate
further enhances the focal length of the glass substrate making the microneedle shape much
better.51 Figure 3.1.5(d) shows the microneedles produced by photolithography.
29
Figure 3.1.5(c): Effect of micro lens on the shape of the microneedle51
Figure 3.1.5(d): Microneedles fabricated by photolithography51
3.2 Materials
3.2.1 COLLAGEN
The present thesis concentrates on the production of collagen microneedles using
hydrolyzed collagen. Collagen is classified according to its structure and supramolecular
organization and include fibril-forming, fibril- associated collagens with interruptions in triple
30
helix (FACITs), network-forming collagens, anchoring fibrils or transmembrane collagens
(Figure 3.2.1(a)).52 The complexities and diversities in the structure lead to further
characterization of collagen types. There is one common characteristic of all the collagen family,
that is the right-handed triple helix α-chain (Fig). Three identical chains (homotrimers) can form
a triple helix as in types II, III, VII, VIII, X, XIII, XV, XVII, XXIII, XXV collagen and by two
or three different chains (heterotrimers) as in types I, IV, V, VI, IX, and XI collagen.53
Figure 3.2.1(a): Molecular structure of collagen fiber54
Collagen consists of about 33% of amino acid residues, 12-14% of proline, a little less than 14%
of 4-hydroksyproline, and about 1.5% of 4-hydroksylysine. Tryptophan and cysteine were not
noticed. Collagen share a repeating pattern Gly-X-Y in which the X and Y positions are
frequently occupied by proline (Pro) and 4-hydroksyproline (Hyp) residues.54
Bovine hide, bone, pigskin or fish bones and fish skin are the main source for the
collagen peptides. Marine sources are alternatives for bovine or porcine and they are not
associated with the prions related to risk of Bovine Spongiform Encefalopathy.55 The hydrolyzed
collagen is obtained by a process of controlled hydrolysis to obtain soluble peptides. At first the
raw material is washed, homogenized and demineralized with diluted mineral acid or alkaline.
31
The raw material is extracted in several stages with warm water. Further enzymatic degradation
of gelatin results in a final product, which is hydrolyzed collagen. Hydrolyzed collagen varies
from one another due to their molecular weight which ranges from 2 to 6 kDa and is less than the
average molecular weight of the peptones.54
3.2.2 POLYVINYLPYRROLIDONE
Polyvinylpyrrolidone (PVP) (Figure 3.2.3(b)) is a water-soluble polymer. About 27% of
the global produce of polyvinylpyrrolidone is used for pharmaceutical purposes, 26% in
cosmetics, 5% in food, 8% in coating, detergents, adhesives, 2% in consumer products, 1% or
less in textiles, polish, petroleum, agriculture, petroleum, chemical manufacturing and enzymes.
PVP is available both in solid and liquid state.56
PVP is polymerized starting with formaldehyde and acetylene, and proceeds through 2-
butyne-1, 4-diol and y-butyro- lactone to a-pyrrolidone and N-vinyl-2-pyrrolidone (the
monomer) (Figure 3.2.2(a)).57
Figure3.2.2 (a): (1) acetylene; (2) formaldehyde; (3) 2-butyne-1, 4-diol; (4) 1,4-butanediol; (5)
gamma- butyrolactone; (6) alpha-pyrrolidone; and (7) N-vinyl-2-pyrrolidone57
32
Figure 3.2.2(b): Chemical structure of polyvinylpyrrolidone58
3.3 Procedure
The microneedles, on which this thesis was focused on, were prepared by using micro
molding procedure. The material of the molds used for the microneedle fabrication is
Polydimethylsiloxane (PDMS). PDMS is a non-reactive polymer and will not interact with the
collagen solution. The PDMS is also flexible and can withstand up to 2000C, making it a
favorable material for the microneedle molds.
The PDMS molds were prepared by a micro molding process. The mold with the master
microneedle array is prepared, on to which the molten PDMS is poured. The mold is allowed to
cure and solidify. Once the solidification is completed, the PDMS microneedle mold is carefully
removed (Figure3.3(a)).
Figure 3.3(a): Preparation of PDMS microneedle mold
33
The PDMS molds are readily available in the market and were used to prepare the
microneedles for this thesis, obtained from a company called Micropoint Technologies (Figure
3.3(b)), which manufactures the PDMS microneedle molds (Figure 3.3(d)). The microneedles in
the molds have a height varying from 300 to 500 microns (Table3.3(a)) and the dimensional
representation of the microneedle molds is shown in Figure 3.3(c).
Figure 3.3(b): Micropoint Technologies
Figure3.3(c): Schematic representation and cross-sectional view of microneedle mold
34
Figure3.3 (d): PDMS microneedle mold from micropoint technologies
Table3.3 (a): Microneedle mold dimensions
Height (μm)
Base (μm)
Pitch (μm)
Patch size (mm)
Array size
300 100 500 8 x 8 10 x 10
400 150 500 8 x 8 10 x 10
500 200 500 8 x 8 10 x 10
600 200 500 8 x 8 10 x 10
The collagen solution was prepared by dissolving hydrolyzed collagen with distilled
water. Parameters such as time, pressure drop, and temperature were varied, and the microneedle
patches were observed. Experiments we conducted initially with 15% and 30% (Wt./Vol.) ratio
of collagen with distilled water (Table 3.3(b)).
35
Table 3.3(b): Parameters for the 15% and 30% collagen trials
Pressure drop
-0.06Mpa
-005Mpa
-0.04Mpa
-0.03Mpa
-0.02Mpa
0Mpa
Temperature
250C
300C
350C
-180C
Time 1hour to 6 hours
The experiments were conducted using the different heights of microneedles and by
varying the pressure drop, temperature and the time. The experiments were conducted at room
conditions as well. The pressure drop was varied from 0Mpa to -0.06Mpa in the intervals of
0.01Mpa. The experiments were run for a duration ranging from an hour to 6 hours. The
temperature was varying from 250C to 350C in the intervals of 50C. Many combinations of
pressure drop, temperature and time were tried, and the outcomes were observed.
The initial experimental microneedle samples showed microneedles on the patch scarcely
seen. Bubble formation on the microneedle patch was one of the major drawbacks of the
experiments. The bubbles deformed the patch and destroyed the microneedles. The Bubble
formation was observed to be maximum at -0.06Mpa pressure drop and minimum at -0.02Mpa.
36
The microneedle samples were very brittle, and they crystallized when kept for longer durations,
and the samples were flexible and sticky when kept for shorter durations.
An experiment was conducted using both 15% and 30% collagen, and kept in freezer at -
180C for 6 hours. The microneedle sample from this experiment was close to perfect. But, the
microneedle patch melted within seconds due to the increase to room temperature (Figure
3.3(e)).
Figure 3.3(e): Molten microneedle patch
The concentration of collagen and distilled water was modified along with other
parameters such as pressure drop, temperature and time. The samples are observed for the
different combinations of the parameters (Table 3.3(c)).
37
Table 3.3(c): Parameters for different ratios of collagen trials
Pressure drop
-0.06MPa
-0.04MPa
-0.02MPa
0MPa
Temperature
25°C
30°C
35°C
-18°C
Time 4 Hour to 12 Hours
Different ratios of collagen with water such as 1:1; 1.5:1; 1.8:1; 2:1; 3:1 were used for the
experiments and modified along with the other parameters. Unlike the previous trials the
pressure drop was changed in increments of 0.02MPa. The other values of the parameters
remained the same. The experiments were conducted with different combinations of
concentration of collagen, pressure drop, temperature and time.
After conducting new experiments, it was observed that the bubble formation was
comparatively reduced. Furthermore, microneedle creation was successful due to the reduction in
38
bubble formation. The reason behind improved microneedle formation and reduction in bubbles
can be due to the increased concentration of collagen.
Despite the improvements, the microneedle samples were still highly inconsistent when
trying to recreate the microneedles with similar parameters. The microneedles formation is seen
on the sample patches, but it deteriorates in time (Figure 3.3(f)).
Figure 3.3(f): Deteriorated microneedle patch
The microneedle mixture was modified by adding polyvinylpyrrolidone (PVP) to
strengthen the weak structure of the sample patches. A 1:1 ratio of collagen to PVP was prepared
by mixing 0.5 g of hydrolyzed collagen and 0.5 g of PVP and dissolved in 10ml of distilled water
to prepare 10% collagen and PVP solution. This solution is then poured into the microneedle
mold and allowed to solidify (Figure 3.3 (g), (h), (i) and (j)).
39
Figure3.3 (g): 1:1 (collagen: PVP) 300 μm
Figure 3.3(h): 1:1 (collagen: PVP) 400 μm
40
Figure3.3(i): 1:1 (collagen: PVP) 500 μm
Figure3.3 (j): 1:1 (collagen: PVP) 600 μm
The 10% solution of 1:1 collagen and PVP mixture gave one of the best visual results.
The microneedle samples were measured, and images were obtained. Unlike the previous
experiments, the structural strength of the microneedle samples was stronger as it could retain its
41
structure. The deterioration of the microneedle structure in time was eliminated. The addition of
PVP provides enhanced structural support.
The ratio of collagen and PVP were varied and experiments were run to check the
behavior of the microneedle patch and to obtain the optimal structure of the microneedles on the
sample patches. Experiments with collagen to PVP ratios of 6:4; 7:3 and 8:2 were conducted and
the microneedle structure were observed (Figure 3.3(k), (l), (m), (n), (o), (p), (q), (r) and (s)).
All the ratios of collagen and PVP mixture are 1g and are dissolved in 10ml of distilled
water to make it 10% (Wt./Vol.) solution. Such solutions were then poured into the molds and
solidified and carefully removed. The microneedle samples are then obtained and measured.
6:4 RATIO OF COLLAGEN TO PVP
Figure 3.3(k): 6:4 (collagen: PVP) 300 μm
42
Figure 3.3(l): 6:4 (collagen: PVP) 400 μm
Figure 3.3(m): 6:4 (collagen: PVP) 500 μm
7:3 RATIO OF COLLAGEN TO PVP
43
Figure 3.3(n): 7:3 (collagen: PVP) 300 μm
Figure 3.3(o): 7:3 (collagen: PVP) 400 μm
44
Figure3.3 (p): 7:3 (collagen: PVP) 500 μm
Figure3.3 (q): 7:3 (collagen: PVP) 600 μm
8:2 RATIO OF COLLAGEN TO PVP
45
Figure 3.3(r): 8:2 (collagen: PVP) 400 μm
Figure 3.3(s): 8:2 (collagen: PVP) 300 μm
46
After the measurement of all different ratios of collagen and PVP, 7:3 ratio off collagen
and PVP gave the best structurally sound microneedle samples. Thus the 7:3 ratio of collagen
and PVP will be used for the statistical analysis.
47
Chapter 4: Statistical Analysis
4.1 TAGUCHI METHOD
Dr. Genechi Taguchi introduced a statistical approach to improve the quality of goods
manufactured. It is a design to study variation and used in various fields of engineering for
statistical analysis.58 Taguchi defines quality as "The quality of a product is the (minimum) loss
imparted by the product to the society from the time product is shipped".59 Taguchi method is
used to optimize the process parameters making it a powerful and a unique quality improvement
tool when compared to other traditional practices.60 Taguchi method is used to obtain robust
design whose definition of robustness according to Taguchi is “a product whose performance is
minimally sensitive to factors causing variability (at the lowest possible cost)”.61
Taguchi method employs Design of experiment’s orthogonal array theory to analyze
large number of variables with small number of data.59 The Taguchi method uses the loss
function to analyze the pattern of deviation performance parameters from the target value. Such
loss function is translated to signal to noise ratio (S/N ratio). The signal to noise ratio can be of
three types nominal-the-best, larger-the-better, and smaller-the-better.58
The steps involved in Taguchi method are as follows.60
• Identify the main function and its side effects.
• Identify the noise factors, testing condition and quality characteristics.
• Identify the objective function to be optimized.
48
• Identify the control factors and their levels.
• Select a suitable Orthogonal Array and construct the Matrix Conduct the Matrix
experiment.
• Examine the data; predict the optimum control factor levels and its performance.
• Conduct the verification experiment.
4.1.1 CONTROL FACTORS AND LEVELS
The statistical analysis is conducted considering the microneedle height, pressure drop,
amount of time spent by the microneedle molds inside the vacuum oven and the concentration of
the collagen and PVP mixture with distilled water. The Table 4.1.1(a) shows the factors and the
levels designated for each factor.
Table 4.1.1(a): Factors and levels
Factors
Level
A
Microneedle
Height (μm)
B
Pressure Drop
(MPa)
C
Time (Hours)
D
Concentration
of Collagen &
PVP in
Distilled Water
(%)
1 300 -0.02 12 8
2 400 -0.03 18 10
3 500 -0.04 24 12
The alphabets A, B, C and D are the four parameters and the numbers 1,2 and 3 are the
levels. According to Taguchi method, an experiment with four factors and three levels can
49
implement Taguchi L9 method. An experiment with four factors and three levels usually has to
conduct 81 (34= 81) (Factors- s; level –k) (sk) experiments to check all the combinations of the
factors.
Using Taguchi L9 method, the statistical analysis can be performed using only 9
experiments. Taguchi L9 method has a unique orthogonal array with 9 rows with specific
arrangement of the factors. The Taguchi method tries to find the pattern using such arrangement
of control factors to replicate the result of 81 experiments. The Table 4.1.1(b) shows the
arrangements of the control factors in the Taguchi L9 orthogonal array.
Table4.1.1 (b): Layout of L9 Orthogonal Array
Experiment No
Control factor 1
Control factor 2
Control factor 3
Control factor 4
1 1 1 1 1
2 1 2 2 2
3 1 3 3 3
4 2 1 2 3
5 2 2 3 1
6 2 3 1 2
7 3 1 3 2
8 3 2 1 3
9 3 3 2 1
The corresponding values of the control factors are plugged in to represent the Taguchi
orthogonal array for the collagen microneedles, which is shown in the Table 4.1.1(c).
Table 4.1.1(c): Layout of L9 Orthogonal Array for microneedles
No. Of Experiments
A
Microneedle
Height (μm)
B
Pressure Drop
(MPa)
C
Time (Hours)
D Concentration of Collagen &
PVP in Distilled
Water (%)
50
4.1.2 PROCEDURE OF MICRONEEDLE PREPARATION
The experiments are conducted according to the control factors shown in the table. 0.7g
of collagen is mixed with 0.3 g of PVP to prepare 7:3 ratios of collagen and PVP mixture. This
mixture is dissolved in 12.5 ml, 10 ml and 8.333ml of distilled water to prepare 8%, 10% and
12% (Wt./Vol.) concentration of the collagen and PVP in distilled water.
The solution is poured into corresponding molds according to the Taguchi experimental
design and maintained in the appropriate conditions. All the molds began with 100μl of the
solution and then after 3 hours, another 60μl of solution is poured into the mold. The solution is
then allowed to continue according to the Taguchi experimental design. Once the procedure is
completed, the microneedle patches are carefully removed from the molds and are let to rest for
6 hours before the measurements are taken.
4.1.3 DATA COLLECTION
1 300 -0.02 12 8
2 300 -0.03 18 10
3 300 -0.04 24 12
4 400 -0.02 18 12
5 400 -0.03 24 8
6 400 -0.04 12 10
7 500 -0.02 24 10
8 500 -0.03 12 12
9 500 -0.04 18 8
51
The performance parameters are dependent on the control factors. Performance parameter
for each experiment is calculated and noted. The performance parameter for this experiment is
the volume ratios of the microneedle. The shape of the microneedle is a square pyramid. The
volume ratio is the ratio of the actual volume to the ideal volume of the microneedles.
Figure4.1.3: Square pyramid
The ‘h’ represents the height and ‘b’ represents the base of the square pyramid shaped
microneedle (Figure 4.1.3). The volume of the square pyramid can be calculated using the
formula involving the height and the base of the square pyramid as shown in the equation below.
The volumes of the different sizes of the microneedle is calculated and considered as the
ideal volume. The Table 4.1.3(a) shows the volumes of the different sizes of the microneedles.
52
Table 4.1.3(a): Ideal Volumes of microneedles
Microneedle Height (μm)
Microneedle Base (μm)
Microneedle Volume (μm3)
300 100 1
400 150 2
500 200 6.67
The microneedles are observed under the microscope and measured for the height and the
base. Three such measurements are taken from one microneedle patch from an experiment. The
following figures are obtained under the microscope.
Experiment1
Figure 4.1.3(a): Experiment 1 Trial 1
53
Figure 4.1.3(b): Experiment 1 Trial 2
Figure 4.1.3(c): Experiment 1 Trial 3
54
Experiment 2
Figure 4.1.3(d): Experiment 2 Trial 1
55
Figure 4.1.3(e): Experiment 2 Trial 2
Figure 4.1.3(f): Experiment 2 Trial 3
56
Experiment 3
Figure 4.1.3(g): Experiment 3 Trial 1
Figure 4.1.3(h): Experiment 3 Trial 2
57
Figure 4.1.3 (i): Experiment 3 Trial 3
Experiment 4
58
Figure 4.1.3(j): Experiment 4 Trial 1
Figure 4.1.3(k): Experiment 4 Trial 2
59
Figure 6 Figure 4.1.3(l): Experiment 4 Trial
60
Experiment 5
Figure 4.1.3(m): Experiment 5 Trial 1
61
Figure 7 Figure 4.1.3(n): Experiment 5 Trial 2
Figure 4.1.3(0): Experiment 5 Trial 3
Experiment 6
62
Figure 4.1.3(p): Experiment 6 Trial 1
Figure 4.1.3(q): Experiment 6 Trial 2
63
Figure 4.1.3(r): Experiment 6 Trial 3
Experiment 7
64
Figure 4.1.3(s): Experiment 7 Trial 1
Figure 4.1.3(t): Experiment 7 Trial 2
65
Figure 4.1.3(u): Experiment 7 Trial 3
Experiment 8
Figure 4.1.3(v): Experiment 8 Trial 1
66
Figure 4.1.3(w): Experiment 8 Trial 2
Figure 4.1.3(x): Experiment 8 Trial 3
67
Experiment 9
Figure 4.1.3(y): Experiment 9 Trial 1
68
Figure 4.1.3(z): Experiment 9 Trial 2
Figure 4.1.3(1): Experiment 9 Trial 3
69
The actual volumes are calculated for the corresponding microneedle patches. The
volume ratios are represented as follows.
The measured height and the base of the microneedles are used to calculate the actual
volumes and are shown in the Table 4.1.3(b).
Table 4.1.3(b): Actual volumes of the microneedles
Experiment No. Microneedle Height
(μm)
Microneedle Base
(μm)
Microneedle
Volume (μm3)
1 299.32 99.78 0.993
290.49 98.25 0.935
298.47 99.73 0.99
2 293.51 106.43 1.11
260.30 97.60 0.827
299.47 99.01 0.979
3 285.34 107.04 1.09
295.04 109.70 1.18
270.73 99.95 0.902
4 383.01 147.60 2.78
300.85 148.78 2.22
311.23 149.70 2.32
5 371.48 140.07 2.43
377.19 147.58 2.74
369.77 138.98 2.38
6 394.72 145.72 2.79
70
380.34 148.47 2.81
374.45 149.90 2.8
7 440.42 190.68 5.34
464.32 187.85 5.46
412.89 179.5 4.43
8 485.60 178.09 5.13
498.19 186.06 5.75
486.52 186.39 5.63
9 489.08 195.16 6.21
454.40 173.17 4.54
499.70 199.88 6.65
The average volumes ratios of each experiment are calculated are tabulated as the three
trial volume ratios for the experiments. The Table 4.1.3(c) below shows the calculated trial
volumes of the experiments.
Table 4.1.3(c): Trial volume ratios
No. Of
Experiment
s
A
Microneedl
e Height
(μm)
B
Pressur
e Drop
(MPa)
C
Time
(Hours
)
D
Concentratio
n of Collagen
& PVP in
Distilled
Water (%)
Trial 1
(Volum
e ratio)
Trial 2
(Volum
e ratio)
Trial 3
(Volum
e ratio)
1 300 -0.02 12 8 0.993 0.935 0.99
2 300 -0.03 18 10 1.11 0.827 0.979
3 300 -0.04 24 12 1.09 1.18 0.902
4 400 -0.02 18 12 0.926 0.74 0.773
5 400 -0.03 24 8 0.81 0.913 0.793
71
6 400 -0.04 12 10 0.93 0.936 0.933
7 500 -0.02 24 10 0.8 0.818 0.664
8 500 -0.03 12 12 0.769 0.862 0.844
9 500 -0.04 18 8 0.931 0.680 0.997
There exist some factors that cannot be controlled such as change in the microneedle
mold dimension and human error. Such errors are considered as noise. Therefore, the signal to
noise ratio is calculated. The below formula represents the signal to noise ratio (SN).
Where X is the mean and SD is the standard deviation of the trial volumes. The Table
4.1.3(d) shows the mean and the standard deviation of the trail volumes of the experiments.
Table 4.1.3(d): Means and standard deviation
Trial 1
(Volume ratio)
Trial 2
(Volume ratio)
Trial 3
(Volume ratio)
Mean (X) Standard
deviation (SD)
0.993 0.935 0.99 0.972 0.032
1.11 0.827 0.979 0.972 0.141
1.09 1.18 0.902 1.057 0.141
72
0.926 0.74 0.773 0.813 0.099
0.81 0.913 0.793 0.838 0.064
0.93 0.936 0.933 0.933 0.003
0.8 0.818 0.664 0.76 0.084
0.769 0.862 0.844 0.825 0.049
0.931 0.680 0.997 0.869 0.167
The means and the signal to noise ratio of all the control troll factors must be calculated
to check which factor gives the best results. The Table 4.1.3(e) explains the method of
calculation of the means and the signal to noise ratios of the experiments.
Table4.1.3 (e): Signal to noise ratios
No. Of
Experiment
s
A
Microneedl
e Height
(μm)
B
Pressure
Drop
(MPa)
C
Time
(Hours)
D
Concentration
of Collagen &
PVP in
Distilled
Water (%)
Mean (X)
Signal to
noise ratios
(SN)
1 300 -0.02 12 8 X1= 0.972 SN1=2.965
2 300 -0.03 18 10 X2=0.972 SN2=1.676
3 300 -0.04 24 12 X3= 1.057 SN3=1.749
4 400 -0.02 18 12 X4=0.813 SN4=1.828
5 400 -0.03 24 8 X5=0.838 SN5=2.234
6 400 -0.04 12 10 X6=0.933 SN6=4.985
7 500 -0.02 24 10 X7=0.76 SN7=1.913
8 500 -0.03 12 12 X8=0.825 SN8=2.452
9 500 -0.04 18 8 X9=0.869 SN9=1.432
The mean of the first control factor (A1), which is the height of 300 microns, are
calculated by the considering the corresponding values of the means.
73
= 1.0007
Similarly, for the control factor B1, which is pressure drop of -0.02MPa, the
corresponding means are used to calculate the mean.
𝑀𝑒𝑎𝑛 𝑜𝑓 𝐵1 (−0.02𝑀𝑝𝑎) =0.972+0.813+0.76
3 = 0.848
After calculating the means of all the control factors, they are tabulated, and the lowest
values are subtracted from the higher values (Table 4.1.3(f)). According to these values, the
ranks are given for the control factor, which gives the best results.
Table 4.1.3(f): Ranking of control factors
Level Height Pressure drop Time Concentration
1 1.0007 0.848 0.910 0.893
2 0.861 0.878 0.888 0.888
3 0.818 0.9532 0.885 0.898
Delta 0.1827 0.1052 0.025 0.005
Rank 1 2 3 4
74
Figure 4.1.3(2): Mean of height
Figure 8 4.1.3(3): Mean of pressure drop
0.7
0.75
0.8
0.85
0.9
0.95
1
300 400 500
Height
Mean
0.7
0.75
0.8
0.85
0.9
0.95
1
0.02 0.03 0.04
Pressure drop
Mean
75
Figure 9 4.1.3(4): Mean of time
Figure 4.1.3(5): Mean of concentration
0.7
0.75
0.8
0.85
0.9
0.95
1
12 18 24
Time
Mean
0.7
0.75
0.8
0.85
0.9
0.95
1
8% 10% 12%
Concentration
Mean
76
4.1.4 VALIDATION
The Figure 4.1.3 (2), (3), (4) and (5) show the variation of the means with respect to the
control factors. It is seen that the height and the pressure drop plays the very important role in the
collagen microneedle production. The variation of the means of the time and concentration is
low and the effect on the formation of the microneedle is negligible.
According to the graphs the microneedles with 300 microns height and the pressure drop
of -0.04Mpa gives the optimistic results. From the 9 experiments conducted, it is evident that the
300 microns microneedles have close to perfect dimensions. The experiments conducted with
pressure drop of -0.04Mpa gives the best results compared to other pressure drops.
77
Chapter 5: Conclusion
The dissolving collagen microneedles was created using micro molding process. Various
parameters such as pressure, temperature, and duration of solidification was experimented.
Dissolving collagen microneedles with PVP additives was created and Taguchi L9 experimental
design was created. The microneedle height, pressure drop, time of solidification and the
concentration of the collagen and PVP in distilled water were considered as the control factors.
The control charts of the means of the control factors were studied. The signal to noise
ratio values suggested the experimental results were correct. The results indicated the control
factors, microneedle height and the pressure drop plays an important role in microneedle
formation. It was also observed that the microneedle height of 300 microns consistently gives
close to perfect dimension microneedles. Further, the pressure drop of -0.04Mpa below the
atmospheric pressure produces the microneedles close to perfect dimensions.
78
Chapter 6: Future Scope
The microneedle is an advanced method of drug delivery system, which can eliminate the
traditional practice of hypodermic drug delivery system. Studies can be conducted to produce
100% collagen dissolving microneedles. Materials such as nicotine and BOTOX can be
administered through the microneedles with more research on microneedles. The potential of
microneedles can be extended to deliver numerous pharmaceutical drugs for patients who use
hypodermic needles. Use of 3D printers instead of traditional methods of microneedle production
must be focused on.
79
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Appendix
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84
Vita
Abhilash Aditya was born in Bengaluru, Karnataka, India. Graduated from JSS Academy
of Technical Education in 2012 with Bachelors in Mechanical Engineering. After graduating he
got an internship opportunity at Precision Press Products and worked on sheet metal works. The
internship involved learning about inventory and product management. Then he later decided to
join The University of Texas at El Paso to pursue Masters of Science in Manufacturing
Engineering. During the course, he worked as a graduate research assistant under few professors.
His work involved 3D printers, statistical analysis, predictive modeling and biomaterials. He also
holds green belt in Lean-Six Sigma. Apart from academics, he was also involved in student
organizations and served as the President of Indian Student Association (ISA). He completed a
Master’s Degree in Manufacturing Engineering in the fall of 2017.
Permanent address: 1532 Upson Drive
El Paso, Texas, 79902
This thesis was typed by Abhilash Aditya.
