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SKIN PENETRATION ENHANCEMENT OF HYDROPHILIC MACROMOLECULES USING
MICRONEEDLES COMBINATION WITH ELECTROPORATION AND SONOPHORESIS
By Miss Maleenart Petchsangsai
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Program in Pharmaceutical Technology
Graduate School, Silpakorn University Academic Year 2014
Copyright of Graduate School, Silpakorn University
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SKIN PENETRATION ENHANCEMENT OF HYDROPHILIC MACROMOLECULES USING MICRONEEDLES COMBINATION WITH ELECTROPORATION AND SONOPHORESIS
By Miss Maleenart Petchsangsai
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Program in Pharmaceutical Technology
Graduate School, Silpakorn University Academic Year 2014
Copyright of Graduate School, Silpakorn University
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การเพิม่การซึมผ่านผวิหนงัของสารโมเลกลุใหญ่ที่ชอบนํา้โดยใช้ไมโครนีเดิลส์ร่วมกบั อเิลก็โทรพอเรชันและซอนอฟอรีซิส
โดย นางสาวมาลนีาฎ เพชรแสงใส
วทิยานิพนธ์นีเ้ป็นส่วนหนึง่ของการศึกษาตามหลกัสูตรปริญญาเภสัชศาสตรดุษฎีบัณฑติ
สาขาวชิาเทคโนโลยเีภสัชกรรม บัณฑติวทิยาลยั มหาวทิยาลยัศิลปากร
ปีการศึกษา 2557 ลขิสิทธิ์ของบัณฑติวทิยาลยั มหาวทิยาลยัศิลปากร
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The Graduate School, Silpakorn University has approved and accredited the Thesis title of “Skin penetration enhancement of hydrophilic macromolecules using microneedles combination with electroporation and sonophoresis” submitted by Miss Maleenart Petchsangsai as a partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmaceutical Technology.
............................................................................
(Assistant Professor Panjai Tantatsanawong, Ph.D.)
Dean of Graduate School
........../..................../.......... The Thesis Advisors
1. Associate Professor Tanasait Ngawhirunpat, Ph.D. 2. Associate Professor Praneet Opanasopit, Ph.D.
The Thesis Examination Committee ………………………….….…….. Chairman (Associate Professor Suwannee Panomsuk, Ph.D.) ............../................./............. ………………………….….…….. Member (Professor Uday B. Kompella, Ph.D.) ............../................./............. ………………………….….…….. Member (Associate Professor Tanasait Ngawhirunpat, Ph.D.) ............../................./............. ………………………….….…….. Member (Associate Professor Praneet Opanasopit, Ph.D.) ............../................./.............
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53354806 : MAJOR : PHARMACEUTICAL TECHNOLOGY KEY WORD :MICRONEEDLES / ELECTROPORATION / SONOPHORESIS /
MACROMOLECULES MALEENART PETCHSANGSAI : SKIN PENETRATION ENHANCEMENT OF HYDROPHILIC MACROMOLECULES USING MICRONEEDLES COMBINATION WITH ELECTROPORATION AND SONOPHORESIS. THESIS ADVISORS : ASSOC. PROF. TANASAIT NGAWHIRUNPAT, Ph.D., AND ASSOC. PROF. PRANEET OPANASOPIT, Ph.D.117 pp.
The objective of the present work was to investigate the effects of three combinatorial techniques (microneedles (MN), electroporation (EP), and sonophoresis (SN)) on the in vitro skin permeation of the hydrophilic macromolecular compound, fluorescein isothiocyanate-dextran (FD-4; molecular weight (MW) 4.4 kDa). Assessment of the in vitro skin permeation of FD-4 was performed in porcine skin. MN, EP, and SN were used as physical enhancement methods, given the potential of their various mechanisms. The total cumulative amount of FD-4 that permeated through treated skin using two or three combined methods, i.e. MN + EP, MN + SN, EP + SN, and MN + EP + SN, was investigated. Microconduits created by MN alone and in combination with the other techniques were observed under confocal laser scanning microscopy (CLSM). The histology of the treated skin was examined. In vitro skin permeation experiments revealed that all of single methods significantly improved skin penetration of FD-4 compared with passive delivery. The amount of FD-4 permeated across MN-treated skin depended on insertion force applied. An increased insertion force resulted in an increase in the amount of FD-4 permeated. Electroporation significantly enhanced FD-4 permeated when the voltage was higher than 200 V. Moreover, an increase in pulse voltage led to an increase in FD-4 permeated. The application of 20 kHz and intensities between 1.7 - 6.1 W/cm2 in continuous mode for 2 min, significantly enhanced the permeation through SN-treated skin. Nonetheless, the range of intensities applied in this study did not affect the skin permeation of FD-4. The two combined methods significantly enhanced FD-4 permeation across the treated-skin compared with individual methods, except for EP + SN. The increased amount of FD-4 permeated using MN + EP went along with an increased pulse voltage. The MN pretreatment followed by SN vividly facilitated the transport of FD-4. However, the amount of FD-4 permeated following EP + SN method was less than MN alone. The total cumulative amount of FD-4 permeated using three combined techniques (MN + EP + SN) was greater than that observed using a single method or two combinations (MN + EP, MN + SN, EP + SN).The images obtained from CLSM studies demonstrated that microchannels created by the two or three combined methods were much larger than those by MN alone. The histological images indicated no noticeable damage in the skin treated with all of the enhancement methods was observed. These results suggest that MN + EP + SN may serve as a potentially effective combination strategy to transdermal delivery of various hydrophilic macromolecules without causing structural alterations or skin damage. In addition, the results provide a useful knowledge for developing suitable skin enhancement methods using MN, EP, and SN. Department of Pharmaceutical Technology Graduate School, Silpakorn University Student's signature ........................................ Academic Year 2014 Thesis Advisors' signature 1. ........................... 2. ...........................
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53354806 :สาขาวิชาเทคโนโลยเีภสัชกรรม
คาํสาํคญั : ไมโครนีเดิลส์ / อิเลก็โทรพอเรชนั / ซอนอฟอรีซิส / สารโมเลกุลใหญ่ มาลีนาฎ เพชรแสงใส : การเพิ ่มการซึมผ่านผิวหนงัของสารโมเลกุลใหญ่ท่ีชอบนํ้ าโดยใชไ้มโครนีเดิลส์ร่วมกบัอิเลก็โทรพอเรชนัและซอนอฟอรีซิส. อาจารยท่ี์ปรึกษาวิทยานิพนธ ์ : ภก.รศ.ดร.ธนะเศรษฐ์ งา้วหิรัญพฒัน์ และ ภญ.รศ.ดร.ปราณีต โอปณะโสภิต. 117 หนา้ การวิจยัน้ีมีวตัถุประสงคเ์พื่อศึกษาผลของเทคนิคการเพิ่มการซึมผ่านผิวหนงั 3 เทคนิค ไดแ้ก ่ ไมโครนีเดิลส์ อิเลก็โทรพอเรชนัและซอนอฟอรีซิส ต่อการซึมผ่านผิวหนงัของสารตน้แบบ ฟลูออเรสซินไอโซไธโอไซยาเนตเดกซ์แทรน นํ้ าหนกัโมเลกุล 4,400 ดลัตนั (FD-4) การประเมินการซึมผ่านผิวหนงัหมูภายนอกร่างกายดาํเนินการโดยใชเ้ทคนิคเด่ียว และใช ้2 หรือ 3 เทคนิคร่วมกนั ไดแ้ก่ ไมโครนีเดิลส์ร่วมกบัอิเลก็โทรพอเรชนัหรือซอนอฟอรีซิส อิเลก็โทรพอเรชนัร่วมกบัซอนอฟอรีซิส และไมโครนีเดิลส์ร่วมกบัอิเลก็โทรพอเรชนัและซอนอฟอรีซิส ทาํการศึกษาลกัษณะของรูท่ีเกิดข้ึนบนผิวหนงัภายใตก้ลอ้งจุลทรรศน์แบบคอนโฟคอลชนิดท่ีใช้เลเซอร์ในการสแกน และศึกษาจุลกายวิภาคศาสตร์ของผิวหนงัท่ีไดรั้บการทดสอบดว้ยเทคนิคดงักล่าว ผลการศึกษาพบวา่ เทคนิคการเพิ่มการซึมผ่านทั้ง 3 เทคนิค สามารถเพิ ่มการซึมผ่านของสารตน้แบบไดอ้ยา่งมีนยัสาํคญั ปริมาณการซึมผ่านโดยใชไ้มโครนีเดิลส์ข้ึนกบัแรงกดท่ีใช ้ การเพิ ่มแรงกดส่งผลใหก้ารซึมผ่านเพิ ่มข้ึน การใชอ้ิเลก็โทรพอเรชนั เพิ ่มการซึมผ่านอยา่งมีนยัสาํคญัเม่ือใชค้วามต่างศกัยไ์ฟฟ้ามากกว่า 200 โวลต ์ และปริมาณการซึมผ่านเพิ ่มข้ึนเม่ือเพิ ่มความต่างศกัยไ์ฟฟ้า ซอนอฟอรีซิสคลื่นความถี่ต ํ่า (20 กิโลเฮิรตซ์) เพิ ่มการซึมผ่านอยา่งมีนยัสาํคญัเม่ือใชอ้ยา่งต่อเน่ืองเป็นเวลา 2 นาที โดยผลการเพิ่มไม่ข้ึนกบัความเขม้ของพลงังานท่ีใช ้ (1.7 - 6.1 วตัตต่์อตารางเซนติเมตร) การใช ้ 2 เทคนิคร่วมกนัมีประสิทธิภาพในการเพิ ่มการซึมผ่านไดม้ากกว่าการใช้เทคนิคเด่ียว ยกเวน้การใชอ้ิเลก็โทรพอเรชนัร่วมกบัซอนอฟอรีซิส ปริมาณการซึมผ่านของสารตน้แบบเม่ือใชไ้มโครนีเดิลส์ร่วมกบัอิเลก็โทรพอเรชนัข้ึนกบัความต่างศกัยท่ี์ใช ้ โดยการซึมผ่านมากขึ้นเม่ือความต่างศกัยไ์ฟฟ้าสูงข้ึน เทคนิคไมโครนีเดิลส์ร่วมกบัซอนอฟอรีซิสเพิ ่มการซึมผ่านโดยไม่ข้ึนกบัความเขม้ของพลงังานท่ีใช ้การใช ้3 เทคนิคร่วมกนัสามารถเพิ ่มการซึมผ่านไดม้ากท่ีสุด เม่ือเปรียบเทียบกบัการใชเ้ทคนิคเด่ียวและ 2 เทคนิคร่วมกนั จากภาพถ่ายดว้ยกลอ้งจุลทรรศน์แบบคอนโฟคอลชนิดท่ีใชเ้ลเซอร์ในการสแกนพบวา่ขนาดของรูบนผิวหนงัท่ีเกิดข้ึนจากการใช ้ 2 หรือ 3 เทคนิค มีขนาดใหญ่กว่าการใชไ้มโครนีเดิลส์เพียงอยา่งเดียว จากการศึกษาจุลกายวิภาคศาสตร์ไม่พบความผิดปกติเกิดข้ึนบนผิวหนงัท่ีทดสอบดว้ยเทคนิคดงักล่าว จากการศึกษาคร้ังน้ีแสดงใหเ้ห็นถึงศกัยภาพของเทคนิคเพิ ่มการซึมผ่านท่ีใช ้ 3 เทคนิคร่วมกนัในการนาํส่งสารโมเลกุลใหญ่ท่ีมีคุณสมบติัชอบนํ้ า โดยไม่ทาํใหเ้กิดอนัตรายแก่ผิวหนงั และใหข้อ้มูลท่ีเป็นประโยชน์ต่อการพฒันาระบบนาํส่งทางผิวหนงัเม่ือใชไ้มโครนีเดิลส์ อิเลก็โทรพอเรชนั และซอนอฟอรีซิส ภาควิชาเทคโนโลยเีภสัชกรรม บณัฑิตวิทยาลยั มหาวิทยาลยัศิลปากร ลายมือช่ือนกัศึกษา....................................... ปีการศึกษา 2557 ลายมือช่ืออาจารยท่ี์ปรึกษาวิทยานิพนธ์ 1. ........................... 2. .............................
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Acknowledgments
First and foremost, I would like to express my very great appreciation to my
thesis advisor, Assoc. Prof. Dr. Tanasait Ngawhirunpat, for his patient supervision,
enthusiastic encouragement, and useful notice of this work. I would also like to thank
my thesis co-advisor, Assoc. Prof. Dr. Praneet Opanasopit, for her informative advice
and kind help in keeping my research progress on schedule. For my oral defense
committee, I wish to thank Assoc. Prof. Dr. Suwannee Panomsuk, for her valuable
time and insightful questions.
My sincere gratitude also goes to Prof. Uday B. Kompella for giving me a
very great opportunity to work at his laboratory, University of Colorado Anschutz
Medical Campus, Colorado, USA. A professional advice and knowledge given by him
have been enlightening me in working not only now but also in the future. I would
like to express my thanks to Assoc. Prof. Dr. David Bourne for his valuable time and
insightful commend. I wish to acknowledge the generous help provided by Assoc.
Prof. Dr. Robert I. Scheinman during my visit. My special thanks are extended to the
members of Kompella's lab for hugely contributing in my work and personal life.
I would also like to acknowledge the following institutes; the Thailand
Research Funds through the Golden Jubilee Ph.D. Program (Grant No.
PHD/0019/2553), Faculty of Pharmacy, and the Graduate School, Silpakorn
University for financial support throughout my study.
I am particularly grateful for the kind assistance given by every teachers
and staffs in faculty of Pharmacy, Silpakorn University. I very much appreciate all my
friends and members of the Pharmaceutical Development of Green Innovation Group
(PDGIG). They have been a source of friendships and considerate collaboration. I
wish to thank my family for their spiritual supports and unconditional love. I could
never have achieved my thesis without the encouragement from them.
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TABLE OF CONTENTS
Page
English Abstract................................................................................................... iv
Thai Abstract........................................................................................................ v
Acknowledgements.............................................................................................. vi
List of Tables........................................................................................................ viii
List of Figures....................................................................................................... ix
List of Abbreviations............................................................................................ xi
Chapter
1 Introduction.............................................................................................. 1
2 Literature reviews..................................................................................... 8
3 Materials and methods.............................................................................. 46
4 Results and discussion.............................................................................. 57
5 Conclusions............................................................................................... 80
Bibliography......................................................................................................... 82
Appendix............................................................................................................... 94
Biography.............................................................................................................. 113
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LIST OF TABLES
Table Page
1 The ideal properties of a molecule showing good penetrating through
stratum corneum......................................................................... 18
2 Operating features of MN, EP, and SN in transdermal drug delivery... 44
3 The rate of FD-4 permeation across skins treated with MN using
various insertion forces.............................................................. 62
4 The rate of FD-4 permeation across skins treated with MN followed
by EP using various pulse voltages............................................ 66
5 The permeation rate of FD-4 across skins treated with different
enhancement techniques............................................................ 73
6 The cumulative amount of FD-4 permeated using MN with various
insertion forces........................................................................... 100
7 The cumulative amount of FD-4 permeated using EP with various
pulse voltages............................................................................. 101
8 The cumulative amount of FD-4 permeated using MN + EP with
various pulse voltages................................................................ 102
9 The cumulative amount of FD-4 permeated using SN with various
ultrasound dintensities................................................................ 103
10 The cumulative amount of FD-4 permeated using MN + SN with
various ultrasound intensities..................................................... 104
11 The cumulative amount of FD-4 permeated using EP, SN, and EP +
SN............................................................................................... 105
12 The cumulative amount of FD-4 permeated using all enhancement
methods...................................................................................... 106
13 The cumulative amount of FD permeated.............................................. 107
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LIST OF FIGURES
Figure Page
1 Schematic diagram representing a cross-section of skin showing the
different cell layers and appendage............................................... 10
2 Schematic diagram of the stratum corneum: corneocytes as bricks, and
intercellular lipids as mortar......................................................... 12
3 Histological cross-section of the skin....................................................... 13
4 Schematic diagram representing the three possible penetration
pathways....................................................................................... 14
5 Some strategies for enhancing transdermal drug delivery........................ 16
6 Microneedles delivery to the skin............................................................. 22
7 The possible sites that cavitation occurs such as coupling medium, skin
surface and inside of corneocytes....................................... 33
8 Three possible mechanisms that inertial cavitation may enhance
stratum corneum permeability................................................... 35
9 A schematic diagram of the MN array (a), a homemade MN array
fabricated by nine acupuncture needles (b)................................ 50
10 A schematic diagram of the MN + EP array (a) and a homemade MN +
EP array which each of needle was systematically connected by
copper wire (b).............................................................................. 51
11 Illustration of the in vitro skin permeation study with MN + EP (a) and
MN + SN (b)................................................................................. 54
12 Scanning electron microscope (SEM) images of pores created by MN
array (a) and an individual hole created in porcine skin by MN
(b).................................................................................................. 60
13 In vitro permeation profiles of FD-4 in porcine skin pierced with MN
array using various insertion forces.............................................. 63
14 In vitro permeation profiles of FD-4 across porcine skin after treatment
with MN + EP using various pulse voltages................................. 64
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Figure Page
15 In vitro permeation profiles of FD-4 across porcine skin after treatment
with SN using various intensities.................................................. 68
16 In vitro permeation profiles of FD-4 across MN-treated skin after the
application of SN with various intensities.................................... 70
17 In vitro permeation profiles of FD-4 across porcine skin following
treatment with various enhancement methods.............................. 72
18 In vitro permeation profiles of FD with various MW across porcine
skin................................................................................................ 75
19 CLSM image of untreated skin (passive delivery) at different depth
representing fluorescent signal observed on the skin surface ...... 77
20 CLSM images of a microchannel created by MN (a), MN + EP (b), MN
+ SN (c), and MN + EP + SN (d) at various depths..................... 78
21 Histological images of treated skin after H&E staining........................... 79
22 Calibration curve of FD-4 for the in vitro skin permeation study............ 96
23 Calibration curve of FD-20 for the in vitro skin permeation study.......... 97
24 Calibration curve of FD-40 for the in vitro skin permeation study.......... 98
25 CLSM image of MN-treated skin at different depth representing
fluorescent signal observed on the skin surface............................ 109
26 CLSM image of MN + EP-treated skin at different depth representing
fluorescent signal observed on the skin surface............................ 110
27 CLSM image of MN + SN-treated skin at different depth representing
fluorescent signal observed on the skin surface............................ 111
28 CLSM image of MN + EP + SN-treated skin at different depth
representing fluorescent signal observed on the skin surface....... 112
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LIST OF ABBREVIATIONS
%w/w percent weight by weight
C degree Celsius
g microgram(s)
L microliter(s)
m micrometer(s)
μM micromolar(s)
AVG average
cm centimeter(s)
cm2 square centimeter(s)
Conc. concentration
Da Dalton
e.g. exemplī grātiā (Latin); for example
EP electroporation
et al. and others
etc. et cetera (Latin); and other things/ and so forth
FD-4 fluorescein isothiocyanate (FITC)-dextrans MW 4,400
FD-20 fluorescein isothiocyanate (FITC)-dextrans MW 20,000
FD-40 fluorescein isothiocyanate (FITC)-dextrans MW 40,000
G (needle) gauge
g gram(s)
h hour(s)
i.e. id est (Latin); that is
IP iontophoresis
kDa kilodalton
kg kilogram(s)
L liter(s)
LTR localized transport region
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LIST OF ABBREVIATIONS
m meter(s)
m2 square meter(s)
mA milliampere(s)
mg milligram(s)
min minute(s)
mL milliliter(s)
mm millimeter(s)
MN microneedles
ms millisecond(s)
MW molecular weight
nm nanometer(s)
PBS phosphate buffer solution
pH potentia hydrogenii (Latin); power of hydrogen
R2 coefficient of determination
s second(s)
S.E. standard error
SN sonophoresis
V volt(s)
W watt(s)
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CHAPTER 1
INTRODUCTION
1.1 Statement and significance of the research problem
Transdermal drug delivery system (TDDS) is an appealing alternative to
minimize and avoid the limitations allied with oral and parenteral administration of
drugs. TDDS allows the permeation of drugs across the skin and into the systemic
circulation thus avoiding the hepatic first-pass effect observed during oral
administration (Alexander et al., 2012). This system is able to achieve sustained
release of drug. Hence it reduces inconvenience of frequent oral administration and
improves patient compliance (Arora, Prausnitz, and Mitragotri, 2008). Moreover,
TDDS has been promising to overcome the problem of poor macromolecular
bioavailability obtaining from oral delivery system by reducing gastrointestinal
enzymatic degradation. As compared to parenteral delivery, TDDS is capable of
avoiding pain problem, bacterial infection, accidental needle-sticks, and possible side
effects due to transiently high plasma drug concentration that might meet the
problems during injection. Nevertheless, limitations of TDDS are significantly
associated with the skin's barrier function which restricts the amount of drug absorbed
across the skin. Of course, it is limited to potent drug molecules requiring a daily dose
of 10 mg or less (Arora, Prausnitz, and Mitragotri, 2008; Herwadkar and Banga,
2012).
Skin is the largest organ in our bodies which covers an area of
approximately 2 m2 and provides the contact between the bodies and the external
environment. The main function of skin is to act as a barrier for preventing the loss of
tissue water. Though skin's outermost layer, stratum corneum, is only 15–20 μm in
thickness, it provides this significant barrier and has an extreme effect on TDDS
(Hadgraft, 2001). There are few sorts of drugs with appropriate physicochemical
properties which can effortlessly penetrate across the skin. This means that
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its molecular weight should be less than 500 Da. Furthermore, the drug should have
adequate solubility in both lipophilic and hydrophilic environments. Gaining access to
systemic circulation, the drug has to cross the lipoidal area in the stratum corneum,
and then penetrate through more aqueous environments, the deeper skin layer, i.e.
viable epidermis. As a result, its partition coefficient should be in the range between 1
and 3 (Barry, 2001; Hadgraft and Lane, 2005; Donnelly, Raj Singh, and Woolfson,
2010). Consequently, the barrier properties do not permit hydrophilic macromolecules
such as peptides and proteins to permeate passively. Therefore, chemical or physical
enhancement techniques are required to assist transport of these macromolecules
across skin (Herwadkar and Banga, 2012).
Among of chemical techniques, chemical penetration enhancer is widely
used. The principal mechanisms are modification of the stratum corneum barrier by
lipid extraction, protein modification, and partitioning promotion. Some molecules,
e.g. azone, fatty acids, alcohol, terpenes, and DMSO, can disrupt lipid organization in
the stratum corneum resulting in an increase of drug's diffusion coefficient. As ionic
surfactants can well interact with keratin in corneocytes, they loosen the protein
structure. Consequently, skin permeability of drug facilitated using such surfactant
increases. Many solvents, e.g. propylene glycol, ethanol, change its soluble properties
by altering the chemical environments in stratum corneum resulting in an increase of
the partition of second molecules in stratum corneum. Nanotechnology-based carriers,
i.e. liposome, nanoparticles, can be alternative techniques for facilitating the skin
permeation of hydrophilic macromolecules by encapsulating such molecules in these
carriers. Furthermore, the lipophilic prodrugs show the improvement of the partition
of the drug into the skin (Barry, 2001; Herwadkar and Banga, 2012).
Physical enhancement methods, such as microneedles, ablation,
iontophoresis, electroporation, sonophoresis etc., have also been investigated.
Although a mutual achievement is to improve skin permeability, the implementations
and mechanisms of these methods are different (Barry, 2001; Herwadkar and Banga,
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2012). In this research, microneedles (MN), electroporation (EP), and sonophoresis
(SN) are intentionally highlighted.
MN are micron-sized needles which can breach stratum corneum to create
holes big enough for various molecules to pass through. They can be made of several
materials such as metals, silicon, plastic, glass, biodegradable polymer, sugar etc. The
average needles length ranges from 100 to 1,500 μm which is long enough to pierce
the stratum corneum and upper layers of viable epidermis. Nevertheless, they do not
penetrate into the deeper dermis layer where plenty of nerve ending are located. In
other words, they are a minimally invasive manner without causing pain.
Interestingly, as compared to macromolecular size, the holes are much larger in
dimensions so that macromolecules can penetrate through the skin effortlessly.
Additionally, holes created by MN are believed to be aqueous which facilitate the
transport of hydrophilic macromolecules (Prausnitz, 2004; Herwadkar and Banga,
2012). MN can be commonly classified into four groups; (i) solid MN (ii) drug-coated
MN (iii) dissolving MN, and (iv) hollow MN. Practically, each of these MN is
capable of facilitating skin permeation of the macromolecular drugs by different
ways. Solid MN can be used as skin pretreatment, after inserting and removing of
solid MN, a drug formulation will be applied and allowed passive diffusion. Drug-
coated MN can be fabricated by coating solid MN with a water-soluble formulation
containing drugs. After insertion, the drug coating is dissolved off and diffused into
the skin, after which the MN is removed. Dissolving MN are made from water-
soluble materials, such as hydrophilic polymers and sugars that will completely
dissolve in the skin after insertion. This MN is used as skin pretreatment, drug
encapsulated within MN can be released after MN insertion. Hollow MN containing a
hollow bore is similar to hypodermic needle. Nevertheless, the size of hollow MN is
much smaller than that of hypodermic needle. It facilitates the drug transport by
pressure-driven flow using either syringe or actuator (Prausnitz, 2004; Kim, Park, and
Prausnitz, 2012). Henry et al. (1998) explored the first study using solid MN in order
to deliver small hydrophilic molecules, i.e. calcein, in cadaver skin. The results
showed that the skin permeability of calcein increased by three orders of magnitude
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(Henry et al., 1998). Later, McAllister et al. (2003) demonstrated the skin
permeability of different compounds, i.e. insulin, bovine serum albumin (BSA), and
latex nanoparticles (25 and 50 nm in radius) using solid MN. The permeation
increased by orders of magnitude compared to the absence of MN. Additionally,
single glass hollow MN was used to deliver insulin into the skin of in vivo diabetic
hairless rats. The results showed that blood glucose level dropped up to 70% after 30
min of infusion (McAllister et al., 2003). To enhance the skin permeability of
compounds, the combination of MN and other enhancement methods were
investigated. Wu et al. (2007) has reported an increased skin permeation of
fluorescein isothiocyanate (FITC)-dextrans, FD, with various molecular weights (3.8,
10.1, 39.0, 71.2, and 200.0 kDa) using a combination of MN pretreatment and
iontophoresis. This method significantly enhanced FD flux compared with MN
pretreatment alone or iontophoresis alone (Wu, Todo, and Sugibayashi, 2007).
Additionally, the combination of MN and EP, so called in-skin EP, shown a
synergistic effect of the skin permeation of FD-4 compared to MN alone or
conventional EP (Yan, Todo, and Sugibayashi, 2010). Furthermore, the skin
permeation of calcein and BSA can be enhanced using an array of hollow MN which
pierced the stratum corneum and the fluid media was then given the energy by SN
(Chen, Wei, and Iliescu, 2010). Furthermore, drug-loaded dissolving MN made from
a copolymer of methylvinylether and maleic anhydride (PMVE/MAH) showed the
potential for delivering macromolecules (BSA and insulin) across the neonatal
porcine skin when they was used in the combination with iontophoresis. However,
this combination did not enhance the skin permeation of small molecule loaded MN,
i.e. theophylline, methylene blue, and fluorescein sodium (Garland et al., 2012).
Apparently, MN promises the potential to overcome the problem associated with the
stratum corneum barrier. Moreover, the combinational techniques demonstrated the
synergistic effect of MN and other physical enhancement methods.
EP or electropermeabilization is the transitory structural perturbation of
lipid bilayer membranes due to the application of high voltage pulses within the very
short time (μs-ms). Electrical exposure causes a rearrangement of lipid bilayer in the
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cell membrane or in more complex structure such as stratum corneum. Electric field
occurred during current application induces aqueous pore formation and provides a
local driving force for facilitating the transport of molecules. The principal
mechanisms of drug transport through permeabilized skin are electrophoresis,
diffusion, and electroosmosis. The main driving force occurred during high voltage
pulses application provides electrophoretic movement of charged molecules.
Enhanced passive diffusion through highly permeabilized skin facilitates the transport
of either neutral molecules or drug added after current application. In contrast to
iontophoresis, the drug transport via electroosmosis is limited due to the short time of
current application. So, the influence of electroosmosis on drug transport is quite low.
Up to now, there are various molecules with a wide range of molecular weight that
can be successfully delivered using EP such as fentanyl (Vanbever et al., 1998),
timolol (Denet and Préat, 2003), salmon calcitonin (Chang et al., 2000), parathyroid
hormone (Chang et al., 2000; Medi and Singh, 2003), fluorescein isothiocyanate
(FITC)-dextrans (FD) (Tokudome and Sugibayashi, 2003), DNA (Guo et al., 2011).
SN, also known as phonophoresis, is the use of ultrasound to deliver
therapeutic compounds through the skin. The used frequencies can be classified into
two groups; (i) low-frequency SN (LFS); 20-100 kHz and (ii) high-frequency SN
(HFS); 0.7-16 MHz. The main mechanism of SN for facilitating skin permeation is
still unclear. However, the accepted mechanism is cavitation. The cavitation occurs
within cavities near the corneocytes of the stratum corneum leading to disorganization
of the stratum corneum lipid bilayers. As a result, the skin permeability is increased.
However, there are some different points in the mechanism of HFS and LFS. Namely,
HFS mainly contributes to cavitational formation within the stratum corneum and skin
appendages (hair follicles shafts and sweat glands). On the contrary, LFS leads to a
microjet outside the skin (Polat et al., 2011). The first use of SN to increase
percutaneous absorption of cortisol was reported by Fellinger and Schmid in 1954.
From that time, at least 150 research works related to SN were explored. There are a
number of molecules which can be successfully delivered such as triamcinolone
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acetonide (Yang et al., 2005), clobetasol 17-propionate (Fang et al., 1999), insulin
(Boucaud et al., 2002), gold nanoparticles and quantum dots (Seto et al., 2010).
Although, each enhancement method and their combinations promise a
great effectiveness for improving skin permeation, there have not been the research
works related to the use of three combined methods. The objective of this study was
to investigate the skin permeation of hydrophilic macromolecules by using three skin
enhancement methods, i.e. microneedles (MN), electroporation (EP), and
sonophoresis (SN). Fluorescein isothiocyanate (FITC)-dextrans (FITC-dextran) was
used as model macromolecule. First of all, the effect of insertion force on skin
permeation using MN alone was investigated. Simultaneously, the effects of electrical
protocol using EP alone, e.g. pulse voltage, pulse duration, and number of pulse, as
well as the effects of ultrasonic parameter were evaluated. Thereafter, the amount of
hydrophilic macromolecules permeated using combination of these three techniques,
MN + EP, MN + SN, EP + SN, and MN + EP + SN, were also evaluated with
appropriated conditions.
1.2 Objective of this research
1. To evaluate the potential of physical enhancement methods including
microneedles, electroporation, and sonophoresis for facilitating the transport of
hydrophilic macromolecules through the skin barrier
2. To examine the effect of several parameters associated with each of
physical enhancement method, e.g. insertion force, electrical parameters, and
ultrasonic parameters, on the in vitro skin permeation of hydrophilic macromolecules
3. To determine the effect of combinational enhancement techniques for
improving the skin permeation of hydrophilic macromolecules
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1.3 The research hypothesis
1. Physical enhancement methods, i.e. microneedles, electroporation, and
sonophoresis, can enhance the transport of hydrophilic macromolecular compounds
through the skin barrier.
2. Several parameters, e.g. insertion force, electrical parameters, and
ultrasonic parameters, have an influence on the skin permeation of hydrophilic
macromolecules.
3. The combinational enhancement techniques can facilitate the transport
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CHAPTER 2
LITERATURE REVIEWS
2.1 The structure and function of skin
2.1.1 Function of skin
2.1.2 Physiology of skin
2.1.2.1 Epidermis
2.1.2.2 Dermis and appendageal structures
2.2 Penetration pathways
2.3 Basic mathematical principles in skin permeation
2.4 Advantages and disadvantages of transdermal drug delivery
2.4.1 Advantages
2.4.2 Disadvantages
2.5 Skin permeation enhancement methods
2.5.1 Drug or vehicle interaction
2.5.1.1 Drug or prodrug
2.5.1.2 Chemical potential
2.5.1.3 Ion pairs or coacervates
2.5.1.4 Eutectic system
2.5.2 Vesicles and particles
2.5.2.1 Liposome and analogs
2.5.2.2 High velocity particles
2.5.3 Stratum corneum modified
2.5.3.1 Hydration
2.5.3.2 Chemical penetration enhancers
2.5.4 Stratum corneum bypassed or removed
2.5.4.1 Microneedles
2.5.4.1.1 Fabrication of MN
2.5.4.1.2 Solid MN
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2.5.4.1.3 Coated MN
2.5.4.1.4 Dissolving MN
2.5.4.1.5 Hollow MN
2.5.4.1.6 Applications of MN
2.5.4.1.7 Sterility of MN
2.5.4.1.8 Skin resealing after MN insertion
2.5.4.1.9 Advantages and disadvantages of MN
2.5.4.2 Ablation
2.5.4.2.1 Thermal ablation
2.5.4.2.2 Radiofrequency ablation
2.5.4.3 Follicular delivery
2.5.5 Electrically-assisted methods
2.5.5.1 Ultrasound
2.5.5.1.1 Factors affecting ultrasonic cavitation
2.5.5.1.2 Mechanisms of skin enhancement using ultrasound
2.5.5.1.3 Applications of SN for transdermal drug delivery
2.5.5.2 Iontophoresis
2.5.5.3 Electroporation
2.5.5.3.1 Pathways of drug transport through electroporated
skin
2.5.5.3.2 Mechanisms of molecular transport
2.5.5.3.3 Parameters affecting skin delivery using EP
2.5.5.3.4 Applications of EP
2.5.5.3.5 Safety of skin EP
2.5.5.4 Magnetophoresis
2.5.5.5 Photomechanical wave
2.6 Combination methods
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2.1 The structure and function of skin
2.1.1 Function of skin
The skin covers an area of approximately 2 m2 and provides the contact
between our bodies and the external environment. It prevents the loss of water and the
admission of foreign materials.
2.1.2 Physiology of skin
Macroscopically, skin comprises two main layers, i.e. epidermis and
dermis which includes appendageal structures (Figure 1).
Figure 1 Schematic diagram representing a cross-section of skin showing the
different cell layers and appendage
2.1.2.1 Epidermis
The epidermis is a stratified, squamous, keratinizing
epithelium. Keratinocytes are the major cellular component (> 90%) and are
responsible for the barrier function of the skin. The epidermis can be classified into 5
layers indicating steps of keratinocyte differentiation, i.e. (i) stratum basale, (ii)
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stratum spinosum, (iii) stratum granulosum, (iv) stratumlucidum, and (v) stratum
corneum.
Considering from inside to outside of the body, first layer,
stratumbasale or basal layer is responsible for the continual renewal of the epidermis.
Proliferation of the stem cells in the stratum basale creates new keratinocytes which
then propel existing cells towards the surface. This cycle normally occurs every 20-30
days.
The upper layer is stratum spinosum. Keratinocytes in this layer
maintain a complete set of organelles and membrane-coating granules (or lamellar
bodies). The next layer is stratum granulosum or granular layer which more flattened
keratinocytes are still viable. More lamellar bodies are also found in the upper part of
granular cells.
Inthe stratum lucidum, the transitional layer before becoming
the stratum corneum, the cellular organelles are collapsed leaving keratin filaments in
the granular layer. The lamellar bodies fuse with cell membrane and also release their
lipid contents into the intercellular space. These contents can arrange into
multilamellar domain.
The outermost layer is stratum corneum. Although the stratum
corneum is only 15–20 μm thick, it provides a very effective barrier to penetration
(Hadgraft 2001). As the stratum corneum is a rate-limiting step to skin penetration, its
structure is elaborated in detail.
The stratum corneum is composed of two main compartments,
i.e. the corneocytes embedded in intercellular lipid bilayer. This makes its comparison
to a “brick and mortar system” as shown in Figure 2.
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Figure 2 Schematic diagram of the stratum corneum: corneocytes as bricks and
intercellular lipids as mortar
The corneocytes stack up to 18 - 20 layers depending on the
anatomic location in the body. Each of which is about 0.5 μm in thickness.
Consequently, stratum corneum has the thickness of 10 μm approximately. The
histological cross-section of skin representing the stacks of corneocytes is shown in
Figure 3. The lipophilic matrix filling the tortuous pathway between the stacked
corneocytesprovides the permeability barrier. These lipids are mixture of about 13
species of ceramides, cholesterol, and free fatty acids in an equal ratio. Ceramides are
responsible for formation of the compact intercellular lamellae leading to barrier
function of stratum corneum. Moreover, they also exhibit distinctive signaling
function for cell proliferation and programmed cell death. Changes in intercellular
lipid composition and/ or organization can lead to a higher skin permeability (Menon,
Cleary, and Lane, 2012).
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2.1.2.2 Dermis and appendageal structures
The dermis, the inner and larger (90%) skin layer, comprises
primarily connective tissue, i.e. collagen and elastic fibers, and provides support to the
epidermis. The thickness of dermis is about 1-4 mm. The dermis incorporates blood
and lymphatic vessels and nerve endings. The extensive microvasculature network
found in the dermis represents the site of resorption for drugs absorbed across the
epidermis. The dermis also supports skin's appendageal structures, specifically the
hair follicles and sweat glands.
Figure 3 Histological cross-section of the skin. The outermost layer of the epidermis
is stratum corneum which is composed of dead corneocytes embedded in a lipid
matrix. Below the stratum corneum, the viable epidermis is located, which is
comprised of keratinocytes.
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2.2 Penetration pathways
Penetrat ion pathways can be classified into 3 ways, namely, (i)
intercellular, (ii) transcellular, and (iii) appendageal pathway including sweat ducts
and hair follicles as displayed in Figure 4. The appendageal pathway does not seem to
be important to skin permeation because it only covers about 0.1% of the skin surface.
However, this pathway may facilitate the penetration of ions and large hydrophilic
molecules (Barry, 2001; Hadgraft, 2001).
The intercellular route is now considered to be the major pathway for
permeation of most drugs across the stratum corneum. This might be assumed that a
molecule moving via the transcellular route must partition into and diffuse through the
keratinocyte. Before moving to the next keratinocyte, the molecule must partition and
diffuse through the 4-20 lipid lamellae bilayers between each keratinocyte. This order
of partitioning into and diffusing across multiple hydrophilic and hydrophobic
regions,is hostile for most drugs. For this reason, the intercellular route have been the
targeted route for improving transdermal drug delivery by manipulating the solubility
in the lipid bilayers or changing the highly ordered structure of this region (Benson,
2005).
Figure 4 Schematic diagram representing the three possible penetration pathways;
appendageal, intercellular, and transcellular route
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2.3 Basic mathematical principles in skin permeation
Since all compounds are thought to transfer the skin by a passive diffusion
mechanism (to date, no active transport mechanisms have been reported), it is then
possible to apply the Fick’s first laws of diffusion to the data obtained from the skin
permeation experiments. This law is used to predict the rate at which substances
penetrate the skin. The law is to assume that the rate of transport of drug passing
through a unit area is proportional to the concentration gradient;
= −
Where J is the rate of transfer per unit area, C is the concentration of
diffusing substance, x is the space coordinate, and D is the diffusion coefficient
(Yamashita and Hashida, 2003).
2.4 Advantages and disadvantages of transdermal drug delivery
2.4.1 Advantages
The benefits of skin delivery include (i)reducing a significant first-pass
effect of the liver that can prematurely metabolize drugs, (ii) maintaining drug levels
in the systemic circulation within the therapeutic window, due to the extended release
of drug provided by the system, (iii) reducing a frequent administration, thus
improving patient compliance and acceptability, and (iv) simply terminating the
treatment by removing the patch.
2.4.2 Disadvantages
The limitations are associated with the skin's barrier function, which
extremelydefines the amount of drug delivered through skin. Hence, molecules with
specific physicochemical properties can penetrate the skin.
(1)
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Figure 5 Some strategies for enhancing transdermal drug delivery (Barry 2001)
METHODS FOR ENHANCING TRANSDERMAL DRUG DELIVERY
Drug and vehicle interactions
Drug or prodrug
Chemical potential
Ion pairs or coacervates
Eutectic systems
Vesicles and particles
Liposomes and analogs
High velocity particles
Stratum corneum modified
Hydration
Chemical enhancer
Stratum corneum bypassed or
removed
Microneedle array
Ablation
Follicular delivery
Electrically assisted methods
Ultrasound
Iontophoresis
Electroporation
Magnetophoresis
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2.5 Skin permeation enhancement methods
Several enhancement methods used to overcome skin's barrier function can
be categorized based on two strategies, i.e. increasing skin permeability and/ or
providing driving force acting on the drug (Figure 5) (Alexander et al., 2012).
According to the objectives of this study, only three enhancement methods, i.e.
microneedles (MN), electroporation (EP), and sonophoresis (SN) will be elaborated in
the next sessions. The other strategies will be briefly described.
2.5.1 Drug or vehicle interaction
2.5.1.1 Drug or prodrug
Due to the barrier function of the stratum corneum, only the
drug with proper physicochemical properties can translocate across the skin barrier at
an acceptable rate. Those physicochemical properties of drug can be summarized in
Table 1 (Naik, Kalia, and Guy, 2000).
The partition coefficient is one of the important factors to
permeation of molecule in the first stratum corneum layer. The use of prodrugs,
through the addition of a cleavable chemical group that typically increases drug
lipophilicity, can facilitate the transfer of a drug across the skin. Prodrugs can be
gained by adding, for example, alkyl side chains with enzymatically cleavable linkers,
such as esters or carbonates (Naik, Kalia, and Guy, 2000; Barry, 2001; Prausnitz and
Langer, 2008).
2.5.1.2 Chemical potential
For maximum penetration rate, the drug should be at its highest
thermodynamic activity by increasing the concentration of the dissolved drug. When
the amount of drug dissolved in vehicle exceeds its equilibrium solubility, it reaches a
state of supersaturation. The maximum flux may then increase (Naik, Kalia, and Guy,
2000; Barry, 2001; Alexander et al., 2012).
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Table 1 The ideal properties of a molecule showing good penetrating through stratum
corneum
Aqueous solubility > 1 mg/mL
Lipophilicity 10 <Ko/w*< 1,000
Molecular weight < 500 Da
Melting point < 200 C
pH of saturated aqueous solution pH 5-9
Dose deliverable < 10 mg/day
*Ko/w ; oil-water partition coefficient
2.5.1.3 Ion pairs or coacervates
Charged molecules do not easily penetrate stratum corneum.
One technique improving their permeation is that adding an oppositely charged
species, charged molecules become lipophilic ion pairs. These complexes then pass
into the lipid in stratum corneum more easily since charge temporarily neutralizes.
Coacervation is a form of ion pairing, resulting in the lipophilic
complex. The complexes may then partition into the stratum corneum, where they
diffuse, dissociate, and pass into viable tissues.
However, the enhancement effect of both two strategies is
moderate (Barry, 2001; Alexander et al., 2012).
2.5.1.4 Eutectic system
Change of solid drugs into a highly concentrated oily state at
ambient temperature displays enhanced skin permeation due to their high
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thermodynamic activity in the vehicle. Melting point of a drug is inversely
proportional to its solubility in lipids. As a result, lowering the melting point exhibits
increased skin permeation (Alexander et al., 2012).
2.5.2 Vesicles and particles
2.5.2.1 Liposome and analogs
Liposome and its analogs, i.e. transfersomes, ethosomes, and
niosomes, have been used as chemical enhancers. The advantages of these molecules
over conventionally chemical enhancer are not only increasedskin permeability, but
also increaseddrug solubilization in the formulation and drug partitioning into the skin
(Barry, 2001; Prausnitz and Langer, 2008).
2.5.2.2 High velocity particles
The use of a supersonic shock wave of helium gas canfiresolid
particles (20 - 100 μm) through stratum corneum into lower skin layers, so-called The
PowderJect system.The claimed advantages of the system are pain-free delivery
(particles are too small to triggerpain receptors in skin), improved efficacy and
bioavailability, targeting to a specific tissue such as a vaccine delivered to epidermal
cells, sustained release or fast release, accurate dosing, safety (the device avoids skin
damage or infection from needles) (Barry, 2001).
2.5.3 Stratum corneum modified
2.5.3.1 Hydration
Hydration of stratum corneum may increase the skin
permeation of most substances. Water in formulation, i.e. W/O or O/W emulsion, acts
by opening up the compact structure of stratum corneum. Moisturising factors,
occlusive films, hydrophobic ointments, and patch prevent water loss resulting in
raised hydration. Drug bioavailability improves afterward (Barry, 2001).
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2.5.3.2 Chemical penetration enhancers
Substances that temporarily emasculate the skin's barrier
function can enhance skin permeation rate of various compounds. Chemical
penetration enhancers can be categorized into three groups based on the influence on
the stratum corneumbarrier,so-called the lipid-protein-partitioning concept. First
group, the enhancers acting as lipid action, the enhancer disrupts the organization of
intracellular lipid bilayer which highly ordered, making it permeable. When chemical
enhancer moves into the lipid bilayer, it will rotate, vibrate and translocate in the
bilayer, resulting in forming of microcavities and an increase of free volume available
for drug to diffuse. The example of enhancers is Azone®, terpenes, fatty acids,
DMSO, and alcohols. Moreover, some solvents such as DMSO, ethanol, and micellar
solutions may extract lipids in the stratum corneum. making it more permeable
through forming aqueous microcavities (Barry, 2001; Prausnitz and Langer, 2008).
The second, protein modification, substances such as ionic
surfactants, DMSO, and decylmethylsulphoxide, can interact with keratin in
corneocytes and thus open up the compact protein structure. Consequently, the
stratum corneum is more permeable. The last group, the enhancers promote the
partition of drug into the skin, partitioning promotion. Solvents, e.g. ethanol,
propylene glycol, entering the stratum corneum, change its solution properties by
altering the chemical environment, and thus increase partition of a second molecule
into the stratum corneum.
Although chemical enhancer obviously increases skin
permeation, it typically correlates with increased skin irritation. A small amount of
these enhancers that increase skin permeability without irritation has been used to
deliver a number of small molecules, however it has limited effect on delivery of
hydrophilic compounds or macromolecules (Barry, 2001; Prausnitz and Langer,
2008).
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2.5.4 Stratum corneum bypassed or removed
2.5.4.1 Microneedles
Microneedles (MN) are micron-sized needles that can breach
the stratum corneum to create holes large enough for various molecules to pass
through. The average needle lengths range from 100 to 1,500 μm, which is long
enough to pierce the stratum corneum and upper layers of viable epidermis.
Nevertheless, the needles do not puncture the deeper dermis layer, where numerous
nerve endings are located. In other words, the needles are minimally invasive and do
not create a sense of pain. Interestingly, the needle holes are considerably larger than
macromolecular sizes, therefore, the macromolecules can penetrate the skin
effortlessly without size restriction. Microparticles with a size of 2 μm and higher
have also been shown to be delivered via microchannels created by MN. Additionally,
MN holes are thought to be aqueous, thereby facilitating hydrophilic macromolecules
transport (Prausnitz, 2004; Herwadkar and Banga, 2012).
2.5.4.1.1 Fabrication of MN
MN can be categorized into four groups, i.e. solid MN, drug-
coated MN, dissolving MN, and hollow MN based on the application. As shown in
Figure 6, each of these MN designs has different mechanisms to deliver the drugs into
skin.
2.5.4.1.2 Solid MN
Solid MN can be used as a pretreatment in order to first create
pore in the skin. After that, it will be removed and then the drug will be applied over
the pores. The formulations can be either a conventional formulation, such as cream,
ointment, gel, lotion, or transdermal patch (Alexander et al., 2012).
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Figure 6 Microneedles delivery to the skin. MN is first applied to the skin (a) and
then used for drug delivery (b).
Solid MN can be fabricated out of various materials such as
silicon (Henry et al., 1998; McAllister et al., 2003; Pearton et al., 2008),
biodegradable polymer, e.g. polylactic acid (PLA) (Park, Allen, and Prausnitz, 2005),
polyglycolic acid (PGA) (McAllister et al., 2003; Park, Allen, and Prausnitz, 2005),
poly-lactic-co-glycolic acid (PLGA) (Park, Allen, and Prausnitz, 2005), and
polycarbonate (Oh et al., 2008), metal, e.g. stainless steel (Martanto et al., 2004; Gill
et al., 2008; Verbaan et al., 2008; Wermeling et al., 2008). Stainless steel displays
many advantages, such as providing sufficient mechanical strength, easily cutting
using a laser, and FDA-approved safety (Gill and Prausnitz, 2007).
Various techniques have been used for fabricating solid MN.
For instance, silicon MN with the length of 150 μm was made using a reactive ion
etching techniques. The benefit of this technique was to ready to modify the length of
needles as required (Henry et al., 1998). For MN made from biodegradable polymer,
micro-electromechanical masking and etching were first adapted to make a
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micromold. After that polymer MN was filled using a micromold and a vacuum-based
method. The 600 μm-long MN arrays with 120 needles per array were successfully
fabricated out of this technique (Park, Allen, and Prausnitz, 2005). Stainless steel
metal sheet was cut using an infrared laser, providing 1,000 μm long-MN with 105
needles per array (Martanto et al., 2004).
Solid MN should have sufficient mechanical strength by
choosing a proper material In addition, the design of a suitable geometry contributed
to the reduction of force needed to insert MN into skin as well as an increase in skin
permeability (Davis et al., 2004). Teo et al. (2005) reported that the transport of
fluorescence molecules following the application of MN with 150 μm diameter was
10-folds higher than that of MN with 50 μm diameter. They concluded that the larger
diameter provided the higher skin permeation (Teo et al., 2005).
2.5.4.1.3 Coated MN
Solid MN can also be coated with a drug typically using a
water-soluble formulation. After insertion of MN into the skin, the drug coating is
dissolved off and diffused into the skin, after which the MN is removed. Coated MN
can reduce the step of administration as compared to solid MN, however; the amount
of drug coated is limited which is less than 1 mg per coating (Kalluri and Banga,
2011b; Alexander et al., 2012).
MN has been coated by a variety of processes, most of which
involve dipping or spraying the MN using an aqueous solution. For instance, single
MN and solid MN arrays, made from stainless steel were uniformly coated using
micron-scale dip coating process with the drug solution containing calcein, vitamin B,
BSA, DNA, vaccine, and nanoparticles. To improve coating outcome and gain more
amount of formulation on the surface of MN, reduced surface tension of drug solution
by adding surfactant, i.e. Lutrol F-68 NF, and increased viscosity by adding
carboxymethylcellulose sodium salt (CMC), were utilized (Gill and Prausnitz, 2007).
Further study, pDNA-coated MN was also coated by dip coating process. The stability
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of formulation coated was improved by adding a stabilizing agent, i.e. sugar, in order
to protect the drug from damage during drying process and storage (Pearton et al.,
2012). Another technique was spray-coating. Marie G. et al. (2011) demonstrated the
successful spray-coating method using atomizer. Two polymers,
hydroxypropylmethylcellulose (HPMC) and CMC, can be coated on silicon MN;
however, the latter required a surfactant (Tween 80) to help wetting of the MN
surface (McGrath et al., 2011).
2.5.4.1.4 Dissolving MN
Dissolving MN are fabricated out of hydrophilic polymers cast
in an aqueous solution at room temperature and at atmospheric pressure or under
vacuum. After insertion, the polymer is dissolved and thus encapsulated-drug in MN
shaft is released. The advantage of dissolving MN over solid or coated MN is no
sharp waste after the application. Nonetheless, it needs much time to fully dissolve, at
least 5 min, and the mechanical strength is insufficient due to the breadth of MN (Chu
and Prausnitz, 2011; Kim, Park, and Prausnitz, 2012).
The preparation method of dissolving MN is based on casting
an aqueous solution to fill a micromold. During the process, vacuum or centrifugation
may be used. Lee et al. (2008) prepared dissolving MN from biocompatible polymer,
e.g. CMC and amylopectin, using casting method with centrifugation. The model
drugs encapsulated were BSA, sulforhodamine B, and lysozyme (Lee, Park, and
Prausnitz, 2008). In addition, the novel design of dissolving MN, for example, the
separable arrowhead MN has been reported. This comprised of two portions, i.e.
micron-sized sharp tip made from water-soluble polymer, e.g. polyvinylalcohol
(PVA), polyvinylpyrrolidone (PVP) (arrowhead), and blunted-metal shaft. The drug
(influenza virus and sulforhodamine B) was first loaded into micromold and then the
solution of polymer was filled by casting method. The blunted-metal MN was
fabricated using an infrared laser. The metal shaft was covered on the micromold
cavities containing polymer arrowhead and gentle force was then applied to link the
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two portions together. After insertion into the skin, the sharp tip was rapidly
disassociated from needle shaft within seconds and let the drug to be released in the
skin (Chu and Prausnitz, 2011).
2.5.4.1.5 Hollow MN
Similar to hypodermic injection, hollow MN permits pressure-
driven flow of a liquid formulation through microconduits created by the needles. To
generate the flow of liquid, the device, e.g. a syringe, a micro-gear pump, needs to be
used. Although, the rate and pressure for delivering liquid formulation can be
adjustable, i.e. making a rapid bolus inject ion or a slow diffusion, only the
formulation in the form of liquid can be applied, solid formulation cannot be used
without reconstitution. As a result, the drugs which are unstable in a liquid phase,
miss the opportunity to use with hollow MN (Kim, Park, and Prausnitz, 2012).
Hollow MN could be fabricated by assembling commercially
hypodermic needles (30 G) (Verbaan et al., 2007) or using single needles (33 G)
(Wonglertnirant et al., 2010). It could also be prepared from borosilicate glass pipette
using fire-pulling instrument (Martanto, Jason S. Moore, et al., 2006). Hollow metal
MN was fabricated using an ultraviolet laser to make a mold from polyethylene
terephthalate or Mylar. After that nickel was electroplated onto the mold (Davis et
al., 2005).
2.5.4.1.6 Applications of MN
Due to the barrier layer, stratum corneum, is just micron thick,
MN has been developed to overcome this barrier without going to the deeper layer.
By bypassing the barrier, MN has been shown to dramatically increase the number of
compounds that can move across the skin including low and high MW drug, vaccine
and protein.
Traditionally, low MW compounds have larger diffusion
coefficient compared to macromolecular compounds. As a result, they diffuse into the
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skin more readily. For example, calcein (623 Da) was shown to increase the skin
permeation by more than 1,000-fold when MN was inserted into the skin and left in
place. Meanwhile, the skin permeation was increased 10,000-fold when MN was
inserted and immediately removed. That was not plugging the pores, hence, the skin
permeation was higher than remaining MN in the skin (Henry et al., 1998). Next
study demonstrated the potential of super-short solid MN (70-80 μm) for delivering
galanthamine (287 Da). Their results showed that the amount of drug peameated
depends on insertion force and time (Wei-Ze et al., 2010). Additionally, MN can
facilitate the transport of docetaxel incorporated in elastic liposomes (Qiu et al.,
2008).
Biotherapeutic drugs, e.g. peptides, proteins, DNA, and siRNA,
are large molecules that cannot penetrate transdermally by passive diffusion. MN has
been shown to improve the delivery of those molecules. For example, insulin, one
study has shown that application of insulin solution after MN pretreatment can reduce
blood glucose level in in vivo diabetic rats (Martanto et al., 2004). PTH administered
with coated MN has progressed through Phase I and Phase II clinical trials for
osteoporosis treatment (Daddona et al., 2011). The delivery of plasmid DNA and
siRNA have been accomplished using MN. siRNA can be loaded in dissolving MN
and silenced reporter gene expression in a transgenic reporter mouse model
(Gonzalez-Gonzalez et al., 2010). Furthermore, sustained release pDNA hydrogel
formulation was delivered after MN pretreatment and the gene was successfully
expressed in ex vivo human skin (Pearton et al., 2008).
Interestingly, MN can promote vaccine delivery and hence,
improve immunogenic response. For example, diphthelia toxoid adjuvanted with
cholera toxin can be delivered after skin pretreatment with MN. This strategy resulted
in the similar level of immune response as compared to subcutaneous injection (Ding
et al., 2009). An array of hollow MN, the Micronjet MN, was used to deliver
influenza vaccine in healthy adult. their results demonstrated that intradermal
vaccination using 3 μg or 6 μg of influenza vaccine induced similar immune
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responses compared to intramuscular injection of 15 μg of vaccine (Van Damme et
al., 2009).
2.5.4.1.7 Sterility of MN
If the MN product needs an approval from Food and Drug
Administration (FDA), sterility of the MN tends to be required for regulatory
approval. This can be done by various methods such as ethylene oxide gas
sterilization, -irradiation, and aseptic technique using ethanol (Kalluri and Banga,
2011b). For example, MN made out of stainless steel was firstly assembled into a
patch under a laminar flow hood followed by ethylene oxide gas sterilization
(Wermeling et al., 2008). The -irradiation method may cause oxidation of
parathyroid hormone (PTH 1-34) coated on MN, leading to instability of product
which could be reduced by optimizing the irradiation dose and temperature during
sterilization process (Ameri, Wang, and Maa, 2010).
2.5.4.1.8 Skin resealing after MN insertion
The duration for microconduits created in skin to close is one
of the important factors since it has effects on skin irritation, infection, and efficacy of
delivery. Kalluri and Bangka (2011) studied pore closure in a hairless rat model after
puncture with 600 μm-long MN. Their result revealed that skin's barrier function
recovered within 3-4 h and complete pore closure was observed within 15 h in vivo.
Nonetheless, the duration for skin resealing depended on occlusive condition that may
be extended (Kalluri and Banga, 2011a).
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2.5.4.1.9 Advantages and disadvantages of MN
Advantages of MN
1. As MN can be fabricated to be long enough to penetrate the stratum
corneum but short enough not to puncture nerve endings, consequently, MN can
reduce the chances of pain, infection, and injury, leading to greater satisfaction of the
patients.
2. Drug administered using MN strategies can be a controlled-release
dosage form, such as patch, and administered at constant rate for a longer period, so
frequent dosage is not required.
3. Administration of drug via transdermal route using MN can avoid first-
pass effect.
4. MN allow rapid penetration of drugs into the systemic circulation.
5. MN can be used for delivering a wide range of drug molecules, ranging
from low MW to high MW, and also proteins and DNA.
6. Single-use needles are easily disposable and potentially biodegradable.
Disadvantages of MN
1. The needles are very small, so the MN tips can be broken off and left
under the skin.
2. MN may cause skin irritation.
2.5.4.2 Ablation
The principle of ablation is to simply remove the stratum
corneum barrier using several strategies such as thermal ablation, laser ablation,
radiofrequency ablation.
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2.5.4.2.1 Thermal ablation
Thermal ablation enhances drug delivery by using thermal
energy for very short time, i.e. fractions of a second, to create microchannels on the
surface of skin. An electric pulse is converted to thermal energy, thereby causing
localized high temperature on the stratum corneum surface which vaporizes the
region. This process creates microchannels which have 50-200 μm in width and 30-50
μm in depth which facilitate transport of molecules. There has been a patented
technology, the PassPort patch, developed by Altea therapeutics (Atlanta, GA)
(Kalluri and Banga, 2011b).
2.5.4.2.2 Radiofrequency ablation
Radiofrequency ablation associates with the creation of
aqueous transport pathways in the skin. Upon application, radiofrequency waves
(100–500 kHz) cause the microelectrodes to vibrate on the skin surface, resulting in
localized heating and ablation of the stratum corneum in those regions. It has been
reported that this process creates about 102 micropathways/cm2 of skin which are ~50
μm deep and ~30–50 μm wide (Kalluri and Banga, 2011b).
2.5.4.3 Follicular delivery
The pilosebaceous unit (hair follicle, hair shaft, and sebaceous
gland) provides a route that bypasses intact the stratum corneum. However, cross-
sectional area of this route is very small (Barry, 2001).
2.5.5 Electrically-assisted methods
2.5.5.1 Ultrasound
The use of ultrasound to deliver therapeutic compounds
through the skin is generally referred to as sonophoresis (SN) and also known as
phonophoresis. An ultrasound wave is a longitudinal compression wave with
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frequency above that of the audible range of human hearing (above 20 kHz).
Ultrasound waves are made by generating an electric signal which is then amplified
before being sent to the ultrasound horn. After reaching the horn, it is converted into a
mechanical wave by piezoelectric crystals (which change their static dimensions in
response to an electric field). Hence the mechanical wave moves through the tip of the
transducer, which is then transmitted to the desired or coupling medium (Polat et al.,
2011).
2.5.5.1.1 Factors affecting ultrasonic cavitation
Amplitude
The amplitude of ultrasound is proportional to the displacement
of the ultrasound horn during each half cycle (Polat et al., 2011). An increase in
amplitude results in an increase in drug flux. Aldwaikat and Alarjah (2015) reported
the effect of various amplitudes, i.e. 10%, 15%, and 20%, on skin permeation of
diclofenac sodium through Epiderm skin culture following LFS application. The flux
of diclofenac sodium increased when the amplitudes increased; however there was no
significant difference on the flux obtained from 10% and 15% amplitudes (Aldwaikat
and Alarjah, 2015).
Frequency
The frequency of ultrasound corresponds to the number of
times that the transducer is displaced per second of application time. Commonly used
frequencies for SN are generally separated into two groups, i.e. (i) low-frequency
sonophoresis (LFS), which includes frequencies in the range of 20–100 kHz, and (ii)
high-frequency sonophoresis (HFS), which includes frequencies in the range of 0.7–
16 MHz, but most commonly 1–3 MHz, so-called therapeutic frequency. These
distinctive differences of frequency are made because the enhancement mechanisms
of LFS and HFS are dissimilar. The range of frequencies between ~100 kHz and 700
kHz (intermediate ultrasound), is not included in either (i) or (ii) above because this
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range has not been investigated in the field of transdermal drug delivery. The most
likely reason is the lack of commercially available ultrasound equipment in the range
of 100–700 kHz.
Transdermal drug delivery with LFS should be more effective
than HFS in enhancing skin permeability. These could be explained that, at HFS, the
production of bubbles becomes more difficult than at LFS. As a result, the effect of
cavitation on the skin, which is created during HFS, is less than that of LFS (Polat et
al., 2011). Aldwaikat and Alarjah (2015) also compared the influence of frequency of
skin permeation of diclofenac sodium using 20 kHz as LFS and 1 MHz as HFS. The
reduced frequency from 1 MHz to 20 kHz contributed to the increased skin
permeability (Aldwaikat and Alarjah, 2015).
The ultrasound duty cycle
The duty cycle is the ratio of the time that ultrasound is on. It is
typically displayed as the percentages, such as 10% (e.g. 0.1 s ON and 0.9 s OFF),
50% (e.g. 5 s ON and 5 s OFF). Furthermore, ultrasound can be continuously applied
without stopping in the middle of treatment. The pulsing mode is more common than
the continuous mode because it can reduce thermal effects associated with ultrasonic
energy by allowing time for heat to dissipate from the coupling medium during
treatment (Polat et al., 2011). However, the discontinuous mode may be less effective
than the continuous mode. The application of 50% duty cycle reduced skin
permeation of diclofenac sodium as compared to 100% (total application time was 5
min) (Aldwaikat and Alarjah, 2015).
The distance between ultrasound horn and skin
Many researchers have been conducted the experiments using
the distance between ultrasound horn and skin ranging from 0 to 4 cm. However, the
distances between 0.3 cm and 1 cm are commonly used (Polat et al., 2011). An
extended distance between the ultrasound horn and the skin caused a decreased skin
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permeation. The skin permeation of ketoprofen was reduced when the distance was
changed from 0.3 to 0.6 cm. This could be due to the reduction of cavitation effect
(section 2.5.5.1.2) (Herwadkar et al., 2012).
Treatment time
Total treatment times can be extremely varied, from a few
seconds to days (Polat et al., 2011). There have been several research works
demonstrated that an increase in treatment time resulted in an improved skin
permeability. The efficacy of ultrasound-mediated insulin delivery when used
discontinuous mode increased with an increased total application time from 6 to 18
min. The blood glucose level was significantly reduced at the highest application time
(60% from initial glucose level) (Boucaud et al., 2002). Nonetheless, at specific
condition, for example, the skin permeation of diclofenac sodium following
continuous application of 1 MHz for 10 and 20 min were not significantly different.
In the same study, the frequency was changed to 20 kHz and application time was
shorten to 3 and 5 min. The results showed that an increase in application time from 3
to 5 min contributed to an improved skin permeation (Aldwaikat and Alarjah, 2015).
The compositions of coupling medium
Formulation of the coupling medium has an effect on SN-
mediated skin delivery. The coupling medium containing a chemical enhancer, e.g.
sodium lauryl sulfate can enhance the transdermal delivery. The transport of
ketoprofen across SN-treated skin using 0.1% sodium lauryl sulfate as coupling
medium was higher than that of using water (Herwadkar et al., 2012).
2.5.5.1.2 Mechanisms of skin enhancement using ultrasound
Although all the mechanisms are not fully understood, it is
accepted that the main mechanism is cavitation. Nevertheless, there have been some
literatures that stated about thermal effect associated with ultrasonic energy (Machet
et al., 1998; Polat et al., 2011).
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Cavitation
Cavitation refers to the creation of cavities as well as extension,
contraction, and distortion of bubbles in a liquid medium. Cavitation occurs due to the
nucleation of small gaseous cavities during acoustic pressure cycles which is
unpredictable in terms of size, type, and shape. Cavitation can be further classified
into two categories, i.e. stable and inertial cavitation. (Park et al., 2014).
Figure 7 The possible sites that cavitation occurs such as coupling medium, skin
surface, and inside of corneocytes
Stable cavitation corresponds to bubbles formed at moderately
low ultrasonic intensities (1-3 W/cm2) which oscillate about some equilibrium size for
many acoustic cycles. Oscillation of bubbles around asymmetric boundary conditions
leads to a phenomenon called microstreaming, which can generate high velocity
gradients and hydrodynamic shear stresses. Microstreaming can affect a cell
membrane by causing shear stresses over a cell membrane, resulting in tension and
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stretching on cell membrane. This contributes to the creation of transient
microchannels, allowing delivery of compounds across the skin. Figure 7 shows the
possible sites in which the cavitation may occur. It can be formed in intracellular
and/or intercellular structure of stratum corneum including in the coupling medium at
the skin surface (Park et al., 2014).
Inertial cavitation corresponds to the violent growth and
collapse of bubbles that can occur within a period of a single cycle or a several cycles.
Cavitation increases with decreasing frequency. Lower frequency provides more time
for bubbles to grow in the expansion cycle and then generates either shock waves in a
liquid medium or micro-jet near the skin surface. If the bubble collapses spherically, it
will generate high-pressure cores that emit shock waves. The shock waves can cause
structural change in the keratinocytes-lipid interface region. As a result, the shock
waves can create the diffusional pathways for delivering molecules through the skin
(Figure 8 (a)). When a bubble collapses asymmetrically near a boundary, it typically
creates a micro-jet, which can only be on the skin surface or penetrate into the skin.
The effect of micro-jet can disrupt lipid bilayers of stratum corneum as well (Figure 8
(b-c)) (Tang et al., 2002; Park et al., 2014).
Thermal effect
An attenuation of ultrasound wave leads to an increased
temperature of the medium that the wave moves across. This is because ultrasound
energy is partially absorbed. Thermal effect can enhance skin permeability by four
possible mechanisms, i.e. (i) increasing the kinetic energy and diffusivity of permeant,
(ii) dilating hair follicles and sweat glands, (iii) facilitating drug absorption, and (iv)
improving blood circulation under the treated area (in in vivo experiments).
Merino et al. (2003) reported the effect of increased
temperature of skin after LFS (20 kHz) was applied. When skin's temperature was
increased by 20C, the delivery of mannitol was enhanced 35-folds. Compared to the
skin heated to the same level, without using ultrasound, the delivery of mannitol was
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only 25%. These results suggested that thermal energy alone may not play a
significant role in transdermal drug delivery, even though the increased temperature
may influence skin permeability. However, recent studies, especially those conducted
with LFS at higher amplitudes, have paid more attention to controlling and
minimizing thermal effects. This is because significant increases in temperature, and
sustained exposure to high temperatures, can lead to many harmful side effects, such
as epidermal detachment, burns, and necrosis of the viable epidermis or underlying
tissues (Merino et al., 2003; Polat et al., 2011; Park et al., 2014).
Figure 8 Three possible mechanisms that inertial cavitation may enhance stratum
corneum permeability. (a) Spherical collapse near the stratum corneum surface
generating shock waves, (b) Asymmetrical collapse causing a micro-jet on the skin
surface, and (c) a micro-jet penetrating into the stratum corneum.
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2.5.5.1.3 Applications of SN for transdermal drug delivery
The number o f st ud ies have been demo nst r a t ed t he
effectiveness of ultrasound for delivering a wide range of molecules, such as lidocaine
(Tachibana and Tachibana, 1993), mannitol (Machet et al., 1998; Tang, Blankschtein,
and Langer, 2002; Merino et al., 2003), clobetasol 17-propionate (Fang et al., 1999),
fentanyl (Boucaud et al., 2001), caffeine (Boucaud et al., 2001), dexamethasone
(Saliba et al., 2007), diclofenac (Aldwaikat and Alarjah, 2015), ketoprofen
(Herwadkar et al., 2012), FD-4 and FD-40 (Morimoto et al., 2005).
2.5.5.2 Iontophoresis
Iontophoresis is an application of pulsatile or continuous low-
voltage current (typically, < 0.5 mA/cm2of skin) to the formulation in order to
electrically drive drug molecules across the skin. The charged molecules are placed
under an electrode with the same polarity, i.e. positively charged drug is placed under
anode. Hence, when current is applied, the charged drug is forced to run from the
electrode into the layers of skin. The two main mechanisms of drug transport are
electrophoresis and electroosmosis. The charged drugs are moved via electrophoresis,
while weakly charged, uncharged compounds, and large polar molecule can be moved
by electroosmotic flow of water generated by the movement of mobile cations (e.g.
Na+). Iontophoresis is suitable for small molecules that carry a charge and some
macromolecules up to a few thousand Da, but is limited to the molecules larger than
15 kDa. Nevertheless, the maximum delivery rate depends on the maximum current
which is limited by skin irritation and pain (Barry, 2001; Kalluri and Banga, 2011b).
2.5.5.3 Electroporation
Electroporation (EP) is the application of short, high-voltage
electrical pulses (50 to 1,500 V) to promote transdermal drug delivery of molecules.
For example, electrical parameters used are typically tens to hundreds of volts
(normally higher than 50 V) for microseconds to milliseconds with intervals between
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pulses of a few seconds to a minute. EP of mammalian skin results in reversible phase
transition and fluidization of intercellular lipid bilayer in the stratum corneum, leading
to the creation of transient pores. This is due to jule heating and localized temperature
that rises over 60C. The temporary aqueous pathways are created for facilitating the
transport of hydrophilic compounds and macromolecules (Denet, Vanbever, and
Préat, 2004; Kalluri and Banga, 2011b; Wong, 2014).
2.5.5.3.1 Pathways of drug transport through
electroporated skin
After high voltage application, the transport will occur through
localized transport regions (LTR) which covers between 0.02% and 25% of the skin
surface. Size and number of LTR depend on pulsing protocol. The size increases with
an increase in pulse duration and number of pulses. LTR's diameter ranges between
0.1 mm for short pulse and 0.2-2.5 mm for long pulse. In contrast, voltage will affect
the number of LTR. Short duration and high voltage pulse will increase the number of
LTR more than long duration with medium voltage (Denet, Vanbever, and Préat,
2004; Zorec et al., 2013).
2.5.5.3.2 Mechanisms of molecular transport
The transport of molecules through transiently permeabilized
skin by EP results from three different mechanisms at different times. First, enhanced
diffusion which is existed during and after pulses.Due to the transport of neutral
molecules can be impeded by electrophoresis during pulsing, enhanced diffusion
through permeabilized skin could play a significant role in the transport of such
molecules. This evidence could be supported by Vanbever et al. (1998). The transport
of mannitol (neutral molecule) after pulsing was similar to that both during and after
pulsing. This could be due to prolonged permeabilization of the skin caused by EP
after pulsing promoted the molecular transport (Vanbever, Leroy, and Préat, 1998).
Second, electrophoretic movement only occurs during pulsing, caused by the
electrically driving force. This mechanism is important for charged molecules.
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Prausnitz et al. (1993) showed the evidence that the transport of calcein (-4) was
dropped with the reversed electrod polarity which was opposed to the electrophoretic
direction (Prausnitz et al., 1993). Finally, electroosmosis; however it is slightly
appeared during pulsing (Denet, Vanbever, and Préat, 2004). Vanbever et al. (1998)
hypothesized that if electroosmosis played an important role in the molecular
transport, the higher transport would be obtained when the anode was in the donor
compartment. However, their results showed that whether anode or cathode was in the
donor compartment, the cumulative amount of mannitol was not different. This
confirmed that electroosmosis might have little effect on the molecular transport
(Vanbever, Leroy, and Préat, 1998).
2.5.5.3.3 Parameters affecting skin delivery using EP
Electrical parameters
Pulse wave forms
Wave forms can be divided into two types, i.e. exponential
decay wave and square wave. The characteristics of exponential decay wave are a
short high voltage at the beginning, followed by a long low voltage tail. On the
contrary, the square wave is characterized by high or low voltage configuration
(Wong, 2014). Each type is used in different ways. Exponential decay wave is used
for transdermal delivery, while square wave is used for electrochemotherapy and gene
therapy.
The predominant benefits of exponential decay wave are to
prolong the high permeability state of electroporated skin and promote the effect of
electrophoretic movement. However, the duration of exponential decay pulses varies
based on skin resistance and EP set-up, e.g. electrode, conducting medium. These
result in poor clinical reproducibility and affect the consequence of transdermal drug
delivery. In other words, square wave pulses provide constant voltage and duration
which is suitable for the therapies that require a precise control and reproducibility of
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drug transport such as chemotherapy and DNA transfer (Denet, Vanbever, and Préat,
2004).
Voltage, duration, and number of pulse
After EP application, the transdermal voltage will be a fraction,
i.e. 10-50%, of the voltage applied through the electrodes, depending on the resistance
of the skin and drug reservoir. Thus, it affects the efficiency of EP (Denet, Vanbever,
and Préat, 2004). For instance, transdermal voltages were decreased from 400 V to 86
V and 250 V to 54 V or 22% of applied voltages for human dermatomed skin (Denet
and Préat, 2003). As any of these parameters are increased, i.e. voltage, duration,
number of pulse, flux is increased. For example, Prausnitz et al. (1993) demonstrated
the transport of calcein (-4 charge) was increased with an increased voltage from 55 V
to 165 V (Prausnitz et al., 1993). Moreover, the transport of metoprolol increased with
an increased pulse voltage (from 24 V to 250 V) and duration (from 80 ms to 700 ms)
(Vanbever and Préat, 1995). The flux of timolol across human dermatomed skin was
increased when high voltage pulse (400 V) * 10 pulses with 10 ms was used as
compared to low voltage pulse (250 V) * pulses with 100 and 200 ms. This indicated
that an increase in voltage resulted in an increased skin permeability of timolol (Denet
and Préat, 2003).
Design of electrode
Electrode designs have an important effect on the efficacy of
drug transport because they determine the distribution and intensity of electric field in
the skin. The transport of benzoate following EP application depended on electrode
designs. The simplest shape, plate-plate electrode, gave the highest electric field
intensity as compared to two other electrode designs, needle-needle and ring-needle,
which corresponded to the cumulative amount of benzoate permeated (Mori et al.,
2003). However, this type could stimulate nerves and muscles in the skin as well as
burn the superficial layer of skin. On the contrary, an interweaving electrode permits
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the electric field to be localized within the superficial layers of the skin, thereby
avoiding undesirable effects in underlying tissues (Denet, Vanbever, and Préat, 2004).
Physicochemical properties of molecules
Charge of molecules
Charged molecules can highly pass through electroporated
skinsince electrophoretic movement is the principal mechanism for skin EP.
Therefore,an increase in charge of drugs in the formulation results in an increase in
the transport. Regardless of MW of permeant, at the same condition, the flux of polar
molecules could be ranked as follow; calcein (-4 charge) > Lucifer (-2 charge)
>erythrosin derivative (-1 charge). Given, pKa of drug molecules as well as pH of
formulation are important factors to molecular transport across electroporated skin
(Prausnitz et al., 1993).
Lipophilicity
EP can enhance the transport of both lipophilic and hydrophilic
molecules. Nevertheless, an increase in lipophilicity of drug such as making prodrug,
tends to decrease the enhancement ratio of drug. Sung et al. (2003) demonstrated the
transdermal delivery of nalbuphine and its prodrug. The enhancement ratio decreased
with an increased lipophilicity of prodrug as determined by log P, nalbuphine (log P =
0.17 > nalbuphine propionate (log P = 1.05) > nalbuphine enanthate log P = (1.94))
(Sung et al., 2003).
Molecular weight
Molecular weight has an effect on the drug transport through
electroporated skin. The higher molecular weight, the lower skin permeation is
obtained. For example,the cumulative amount of FITC and FD across hairless rat skin
using 100 V and 150 ms duration, was decreased with a rise of their MW as follow;
FITC (MW = 389.4 Da) > FD-4.4 (MW = 4.4 kDa) > FD-12 (MW = 12 kDa) > FD-
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38 (MW = 38 kDa). It could also be noted that skin penetration of FD which have
their MW up to 38kDa could be achieved using EP (Lombry, Dujardin, and Préat,
2000).
Formulation of drug reservoir
Buffer ions
The ions present in the drug reservoir, such as buffer ions, may
battle with the drug needed to be delivered for the electrophoretic movement. The
enhancement ratio of timolol after EP (400 V and 10 ms) decreased from 21.8 to 15.3
where ionic strength in phosphate buffer increased from 125 mM to 525 mM (Denet
and Préat, 2003).
2.5.5.3.4 Applications of EP
EP can enhance the transdermal delivery of a wide range of
molecules, including small and large molecules, hydrophilic and lipophilic molecules,
and charged or even neutral molecules. For example, timolol can be applied using
square wave pulse. The results showed that the transport of timolol enhanced by 1-2
orders of magnitudes as compared to passive diffusion. The higher enhancement
factor was obtained when using phosphate buffer with lower ionic strength. 10 pulses
of 400 V - 10 ms were more sufficient than 10 pulses of low voltage - long duration,
i.e. 250 V - 100-200 ms. Though the current lasted for only 10 s, the transport
remained high after pulsing for at least 6 h. This result supported the mechanism
associated with the enhanced passive diffusion after post-pulsing (Denet and Préat,
2003). Transdermal delivery of FD-4, doxorubicin and fentanyl were shown to be
increased with an increase in pulse voltage up to 570 V. Moreover, the study
demonstrated that the delivery was restricted to the area contacting with the
electrodes. This could reduce possible side effects (Blagus et al., 2013).
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2.5.5.3.5 Safety of skin EP
Although the stratum corneum still has a much higher electrical
resistance than underlying skin and deeper tissues, an electrical field applied to the
skin will concentrate in the stratum corneum and will be much lower in the viable
tissues, which could be protected from adverse effects. Safety of skin EP is needed to
be considered before going to clinical stage. There have been various methods used
for evaluating safety of skin, such as skin viability using MTT assay, histological
examination using microscopy, and skin barrier properties using TEWL measurement.
Medi and Singh (2006) investigated the safety of EP on in vivo
rabbits. The application of 100 V, 200 V, and 300 V (5 pulses and 10 ms) to skin did
not cause any changes in skin viability as compared to control group. TEWL values
increased immediately after EP with all voltages applied; however, the values
gradually decreased to the similar level as control within a week. This could be noted
that electroporated skin could recover after high voltage pulse application.
Corresponding to their histological examination, the images of skin samples revealed
the detachment of stratum corneum layer after EP (100 V). However, the recovery of
stratum corneum to the normal state could be seen after 1 week of EP (Medi and
Singh, 2006).
2.5.5.4 Magnetophoresis
Magnetophoresis uses a magnetic field (5 to 300 mT) to
enhance skin delivery. The drug with diamagnetic property such as lidocaine can be
repelled away from the magnetic field and migrated into the skin (Wong, 2014). The
skin permeation of lidocaine across porcine epidermis following the application of
magnetic field was higher than that of passive diffusion. In addition, the skin
permeation increased with an increase in the magnetic field strength (Murthy,
Sammeta, and Bowers, 2010).
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2.5.5.5 Photomechanical wave
Erbium-doped yttrium aluminium garnet, Qswitched ruby and
carbon dioxide lasers have been used to irradiate the drugs and thus stress the stratum
corneum. The permeation occurs via ablation mechanism or lipid bilayer disruption in
a process ranging from nanoseconds to a few microseconds with pressure amplitude
in the hundreds of atmospheres (300 to 1000 bar). Skin permeability can be promoted
through instant evaporation of water by laser thereby forming microchannels in skin
for transdermal drug delivery. The channel depth is limited to less than 200 μm to
avoid pain and bleeding (Barry, 2001; Wong, 2014).
2.6 Operating features of MN, EP, and SN in transdermal drug
delivery
According to the objective of this study, only three physical enhancement
methods have been evaluated. As a result, some of information related to operating
features of those techniques are summarized in Table 2.
2.6 Combination methods
Many groups of researchers are still working on the single or combined
strategies to improve the rate and extent of drug delivered through skin. Those
strategies should be used in practical way without damaging the skin. Several
combined strategies such as (i) chemical enhancer combined with either MN, IP, EP
or SN, (ii) MN combined with IP, EP or SN, (iii) IP combined with either EP or SN,
and (iv) EP combined with SN, have been shown to work synergistically. A few
examples associated with MN combined with other physical methods are explained
below.
MN create additional aqueous pores in the skin through which the applied
current is conducted. They can be combined with either IP, EP or SN, so as to utilize
electrical driving forces (in case of IP and EP) and ultrasonic energy (in case of SN)
to improve drug transport.
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Table 2 Operating features of MN, EP, and SN in transdermal drug delivery
MN EP SN
Operating
condition
- Manual
- Actuator with constant
pressure
- Voltage : 50 - 1,500 V
- Pulse duration : μs - ms
- Time between pulse : a few
seconds to a minutes
- Frequency : LFS (20 - 100 kHz), HFS
(0.7 - 16 MHz), but most commonly used
is therapeutic frequency (1 - 3 MHz)
- Treatment time : minutes to hours
Drug type Low MW - high MW Low MW - high MW Low MW - high MW
Mechanism
of action
Bypass the stratum corneum
and create an aqueous
transport pathway in the skin
- Electrophoresis,
electroosmosis and diffusion
- Ultrasound cavitation induces
disorganization of lipid bilayer in stratum
corneum.
Adverse
effects
Skin irritation in some cases EP is regarded as mild and
reversible on skin tissue. The
most common side effect is
muscle contraction.
- No permanent damage to the skin or
underlying tissues.
- The most frequent adverse effects during
or after SN are skin erythema, pain, and
tinnitus.
- In comparison to HFS, LFS lacks the
safety records due to the short term of
studies.
MN pretreatment combined with IP has been studied by Wu et al. (2007).
This method significantly enhances the flux of FD compared with MN pretreatment or
IP alone (Wu, Todo, and Sugibayashi, 2007). Later, Kumar and Banga (2012)
reported increased calcein and human growth hormone flux using the same
combination (Kumar and Banga, 2012).Two advantages of these combined strategies
are the ability to control the flux of drugs through the modulation of the applied
current, as well as a reduction in the lag time needed for drugs to penetrate the skin
(Schoellhammer, Blankschtein, and Langer, 2014).
Furthermore, drug-loaded dissolving MN created from a methylvinylether
and maleic anhydride (PMVE/MAH) copolymer demonstrated the potential for
delivering BSA and insulin across neonatal porcine skin when used in conjunction
with IP. However, this combination did not enhance the ability of small molecule-
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loaded MN, e.g. theophylline, methylene blue, and fluorescein sodium, to permeate
skin (Garland et al., 2012). This is because little disruption of the stratum corneum is
necessary to enhance the delivery of small molecules (Wong, 2014).
Additionally, the combination of MN and EP as a pretreatment, which is
also referred to as in-skin EP, displayed a synergistic effect on FD-4 skin permeation
compared with MN alone or conventional EP. This study showed a 140-fold
enhancement in delivery over control samples, and an almost 7-fold enhancement
over EP alone (Yan, Todo, and Sugibayashi, 2010).
In addition to the combined method described above, skin permeation of
calcein and BSA was enhanced using an array of hollow MN in conjunction with
LFS. The increased delivery rate and amount of drug delivered are attributed to
stratum corneum fracturing using hollow MN. Thereafter, the increased flow rates of
media containing solutions of each model drug result from ultrasound energy which is
simultaneous treatment (Chen, Wei, and Iliescu, 2010).
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CHAPTER 3
MATERIALS AND METHODS
3.1 Materials
3.2 Equipments
3.3 Methods
3.3.1 Skin preparation
3.3.2 MN array fabrication
3.3.2.1 MN array preparation
3.3.2.2 Preparation of the MN + EP array
3.3.3 Characterization of the microconduits created by the MN arrays
3.3.4 In vitro skin permeation studies
3.3.4.1 Skin permeation using MN
3.3.4.2 Skin permeation using EP
3.3.4.3 Skin permeation using SN
3.3.4.4 Skin permeation using the combination of MN and EP
3.3.4.5 Skin permeation using the combination of MN and SN
3.3.4.6 Skin permeation using the combination of EP and SN
3.3.4.7 Skin permeation using the combination of MN, EP, and SN
3.3.5 Quantitative analysis
3.3.6 Confocal laser scanning microscopy (CLSM) studies
3.3.7 Histological examination of the skin
3.3.8 Mathematics of skin permeation
3.3.9 Statistical analysis
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3.1 Materials
1. Fluorescein isothiocyanate (FITC)-dextrans; FD-4, FD-20, and FD-40 (Sigma
Aldrich®, St. Louis, MO, USA)
2. Di-Sodium hydrogen orthophosphate 12-water; Na2HPO4.12H2O (Ajax Finechem,
Australia)
3. Potassium dihydrogen orthophosphate; KH2PO4 (Ajax Finechem, Australia)
4. Sodium chloride; NaCl (QReC Chemical, New Zealand)
5. Potassium chloride; KCl (Ajax Finechem, Australia)
6. Sodium hydroxide; NaOH (Ajax Finechem, Australia)
7. Hydrochloric acid 37%; HCl (QReC Chemical, New Zealand)
8. Methyl salicylate; C6H4 (HO) COOCH3 9. Pig skin, Adult (Large white, Nakhon Pathom, Thailand)
3.2 Equipments
1. Acupuncture needle; 0.25×30 mm (DongBang Acupuncture, Inc., Boryeong,
Korea)
2. Silicone sheet; thickness 2 mm
3. Copper wire; surface area 0.5 mm2
4. Pulse generator (ECM 830 Electro Cell Manipulator; BTX, San Diego, CA,
U.S.A.)
5. An ultrasonic transducer (Vibra-cell™, VCX130 PB, Sonics and Materials, Inc.,
Newtown, CT, USA)
6. Inverted Zeiss LSM 510 META microscope (Carl Zeiss, Jena, Germany)
7. Centrifuge (Sorvall® Biofuge Stratos)
8. Digital thickness gauge (Type 25 mm – 0.001/1" – .00005, Sylvac, Switzerland)
9. Suction Catheter (ch/fr 8; diam 2.67 mm; 50 cm, Suzhou Yudu Medical, China)
10. Fluorescence spectrophotometer (RF 5300PC; Shimadzu, Kyoto, Japan)
11. Thermo-regulated water bath
12. Franz diffusion cell; volume of receiver 6 ml
13. Micropipette (20-200 L, 100-1,000 L, 1-5 mL) and micropipette tip
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14. Magnetic stirrer and Magnetic bar
15. Analytical balance (Model CP224S and CP3202S, SARTORIUS, Germany)
16. pH meter (Mettler Toledo)
17. Parafilm (BEMIS, WI, USA)
18. Vortex mixer (VX100, Labnet)
19. Kimwipes disposable wipers (Kimberly-Clark Professional, Australia)
20. Beaker; 50, 100, 250, 1000, 2000 mL (PYREX, USA)
21. Cylinder (PYREX, USA)
22. Microcentrifuge tube 1.7 mL; Costar tubes (CORNING; Corning Incorporated,
NY, USA)
23. Centrifuge tube 15 mL; RUNLA
24. Refrigerator
25. Stopwatch
26. Adhesive tape
27. Medical scissor
28. Fine forceps
29. Freezer/ Refrigerator –20°C
30. Silicone tube
31. Aluminium foil
32. Thermometer
33. Dropper
34. Electric clipper
35. Watch glass
36. Paraffin sheet
37. Glass slide and cover slip
38. Gloves
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3.3 Methods
3.3.1 Skin preparation
Porcine skin was used as an animal skin model in this study because it is a
good model of human skin with regard to hair sparseness and physical properties
(Godin and Touitou, 2007). Porcine skin was obtained from a local slaughterhouse
immediately after the animal was killed (Nakhon Pathom, Thailand). The excess
subcutaneous fat was removed, and the skin was carefully trimmed to obtain a
thickness of approximately 2.0–2.5 mm. The trimmed-skin thickness was measured
using Vernier caliper.
3.3.2 MN array fabrication
In this study, the MN arrays were classified into the following two
categories depending on the enhancement method used: MN array and MN + EP
array. The MN arrays used in the study were not associated with electric current, i.e.
MN and MN + SN. In contrast, the latter, the MN + EP array was used in the
following experimental groups: EP, MN + EP, and MN + EP+ SN.
3.3.2.1 MN array preparation
The MN arrays were prepared using nine acupuncture needles
(0.25×30 mm, DongBang Acupuncture, Inc., Boryeong, Korea). First, each MN was
cut into 5 mm lengths and used to puncture a silicone sheet (15×15×2 mm), allowing
1,000 μm of the needle tips to be exposed out of the silicone sheet. The opposite end
of the acupuncture needle was bent to obtain a perpendicular shape to anchor the
needle. After piercing nine needles, with 4-mm center-to-center spacing, the MN
array was fixed on the silicone sheet using an adhesive tape to ensure that the needles
remain stationary. A schematic diagram of the MN array and a photograph of a home-
made MN array are shown in Figure 9.
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Figure 9 A schematic diagram of the MN array (a), a homemade MN array fabricated
by nine acupuncture needles (b), a photograph of a needle tip protruding from a
silicone sheet (c), and a MN array on a fingertip showing a very small patch when
compared with a fingertip (d).
3.3.2.2 Preparation of the MN + EP array
To determine the combinatorial effect of MN and EP, the array
was prepared as described above. However, before fixing the needles on the silicone
sheet with an adhesive tape, copper wires, i.e. positive and negative line, were
inserted under a perpendicular part of the needle, so the MN + EP array could be used
as an electrode in the EP experiments.
To determine the effect of EP alone and eliminate the effect of
MN, all of the needle tips were cut and filed until dull; these needles are referred to as
blunted-MN and were used to prepare the blunted-MN arrays following a method
similar to that used to construct the MN + EP array. A schematic diagram of the MN
+ EP array and a photograph of the MN + EP array are displayed in Figure 10.
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Figure 10 A schematic diagram of the MN + EP array (a) and a homemade MN + EP
array which each of needle was systematically connected by copper wire (b).
3.3.3 Characterization of the microconduits created by the MN arrays
The micropores created by the MN arrays were observed using a scanning
electron microscopy (SEM, Camscan MX2000, England). The MN-treated skin was
dried and then covered with a thin layer of gold prior to observation at an accelerating
voltage of 15 kV.
3.3.4 In vitro skin permeation studies
In vitro permeation studies of FD-4 (2.5 mg/mL) were performed using a
Franz diffusion cell apparatus. The diffusion cell has an average diffusion area of
2.022 cm2, and the receptor compartment has an approximate volume of 6 mL. Before
starting the experiments, the receptor compartment was filled with phosphate buffered
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saline (PBS, pH 7.4) and maintained at 32°C using a water bath. The solution in the
receptor compartment was continuously stirred at 400 rpm using a magnetic stirrer.
Each piece of treated porcine skin was placed on the receptor compartment by
providing stratum corneum contact with the donor compartment. The donor and
receptor compartments were then fixed using a clamp. The top of the donor
compartment was filled with the model drug solution (1 mL). Moreover, the untreated
porcine skin was used as a control.
To investigate the cumulative permeation profiles, 500 μL of the model
drug solution in the receptor compartment was sampled at 0.08, 0.25, 0.50, 1, 2, 4, 6,
8, 12, 16, 20, and 24 h. After sampling, the solution was immediately replaced with
the same volume of fresh PBS to maintain the sink condition. For better comparisons,
the in vitro skin permeation studies were carefully performed in eight parallel
sessions: (1) passive delivery, (2) MN alone, (3) EP alone, (4) SN alone, (5) MN +
EP, (6) MN + SN, (7) EP + SN, and (8) MN + EP + SN.
3.3.4.1 Skin permeation using MN
To determine the effect of the insertion force on model drug
permeation, each skin was pierced using a homemade MN array with different forces
(2.5, 5.0, 10, 20, and 30 N). Before starting experiments, a plastic container with
smooth lid was filled with water to obtain the weight of 0.25, 0.5, 1, 2, and 3 kg.
According to Newton’s theory of gravitation (g = 9.8 m/s2), these can be converted to
2.5, 5, 10, 20, and 30 N, respectively. Before the permeation study, the plastic
container with desired weight was carefully placed on a homemade MN patch and left
it on the skin for 2 min. After that it was lifted from MN patch and MN patch was
removed from the skin. Untreated skin was used as a control.
3.3.4.2 Skin permeation using EP
EP was applied using a square wave pulse generator (ECM 830
Electro Cell Manipulator; BTX, San Diego, CA, U.S.A.). A homemade patch
consisting of nine-blunted acupuncture needles connected with copper wire (blunted
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MN + EP array) was used as an electrode in order to eliminate the effect of needle.
The skins were treated with pulse voltages of 50, 100, 200, and 300 V. The pulse
duration and number of pulses were fixed at 1 ms and 99, respectively.
3.3.4.3 Skin permeation using SN
A low ultrasonic frequency of 20 kHz was generated using an
ultrasonic transducer (Vibra-cell, VCX130 PB, Sonics and Materials, Inc., Newtown,
CT, U.S.A.) and continuously applied. The radiating diameter of the transducer is 6
mm. Before starting the experiment, the donor compartment of the Franz diffusion
cell was filled with the model drug solution (1 mL). The ultrasonic probe was
positioned 3 mm above the skin inside the donor compartment during the ultrasound
application. The intensities applied were 1.7, 3.9, and 6.1 W/cm2.
3.3.4.4 Skin permeation using the combination of MN and
EP
A homemade patch consisting of nine-acupuncture needles
connected with copper wire (MN + EP array) was used as an electrode to evaluate the
effect of pulse voltage on FD-4 permeation in MN + EP-treated skin. The skin was
punctured using the MN + EP array with a force of 10 N for 2 min. Next, various
voltages of electric current (50, 100, 200, and 300 V) were transferred to the skin
using a pulse generator following conditions similar to those described above. An
illustration of the in vitro skin permeation study using MN and EP is shown in Figure
11 (a).
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Figure 11 Illustration of the in vitro skin permeation study with MN + EP (a) and MN
+ SN (b).
3.3.4.5 Skin permeation using the combination of MN and
SN
First, the MN array was used to create aqueous microconduits
within the skin using 10 N for 2 min. The MN array was removed, and the skin was
immediately placed on the Franz diffusion cell. Before starting the experiment, the
diffusion cell was filled with the FD-4 solution (1 mL). The probe was placed 3 mm
above the MN-treated skin, and ultrasonic energy was continuously applied to the
skin using the following intensities: 1.7, 3.9, and 6.1 W/cm2. An illustration of the in
vitro skin permeation study using MN and SN is displayed in Figure 11 (b).
3.3.4.6 Skin permeation using the combination of EP and
SN
The blunted MN + EP array was used as an electrode in this
experiment. EP was transferred to the skin using a 300 V pulse voltage. Pulse duration
was fixed at 1 ms, and pulse number was fixed at 99. Next, the blunted-MN array was
removed, and the skin was immediately placed on the Franz diffusion cell. The probe
was placed 3 mm above the EP-treated skin. Ultrasonic energy was then applied
through the FD-4 solution in the diffusion cell using 6.1 W/cm2 for 2 min.
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3.3.4.7 Skin permeation using the combination of MN, EP,
and SN
The MN + EP array was used as an electrode to evaluate the
efficacy of this combination method. First, the skin was punctured with the MN + EP
array using 10 N force. Next, electrical current was applied to the skin using a pulse
generator. All electrical parameters, including pulse voltage, pulse time, and the
number of pulses, were selected from the optimal protocol. The MN + EP-treated skin
was mounted, and the FD-4 solution was instantly added to the diffusion cell.
Ultrasonic energy was then applied using the optimized conditions. An illustration of
this method is described in Figure 11 (a) (the first step) and Figure 11 (b) (the second
step).
In order to further study the effect of MW on the
macromolecular delivery through the skin treated with MN + EP + SN, the skin
permeation of 625 μM of FD-4, FD-20, and FD-40 were investigated.
3.3.5 Quantitative analysis
The concentration of model drug in the receiver compartment was
determined using a spectrofluorophotometer (RF-1501; Shimadzu, Kyoto, Japan). The
fluorescent intensity was measured at the wavelength of 495 nm for excitation and
515 nm for emission. The concentrations were calculated from the fluorescent
intensities using a calibration curve. All experiments were performed 4–6 times.
3.3.6 Confocal laser scanning microscopy (CLSM) studies
Before the observations, the treated skins were collected at the specified
time following the permeation studies. The skins were washed with PBS (pH 7.4) and
then wiped with tissue paper to eliminate the excess dye outside the skin. The treated
skin was placed on the modified cover slip, which was constructed as a reservoir by
spreading silicone glue around the rim of cover slip, so that methyl salicylate used as
an immersion oil can be added in the modified cover slip. After that, the treated skin
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was scanned using an inverted Zeiss LSM 510 META microscope (Carl Zeiss, Jena,
Germany).
3.3.7 Histological examination of the skin
Histological alterations in skins treated with various physical enhancement
methods were evaluated. Each skin sample was fixed in a 10% formaldehyde solution
for at least 3 days. The skin samples were dehydrated using ethanol and embedded in
paraffin wax. The skins were vertically cut along the surface and stained with
hematoxylin and eosin. The skin samples were then observed using light microscopy.
3.3.8 Mathematics of skin permeation
Fick’s first law of diffusion was employed to predict the rate at which
substances penetrate the skin using different physical skin enhancement methods. The
law assumes that the rate of transport of a drug passing through a unit area is
proportional to the concentration gradient according to the following formula:
= −
where J is the rate of transfer per unit area, C is the concentration of the diffusing
substance, x is the space coordinate, and D is the diffusion coefficient. Using the in
vitro permeation studies, the steady-state flux (J) of the drug can be calculated from
the slope of the linear portion of the plot of the cumulative amount permeated per unit
area against time.
3.3.9 Statistical analysis
In vitro drug permeation measurements were collected from 4 to 6
experiments. The values are expressed as the means ± standard error (S.E.). The
statistical significance of the differences in the amount of model drug permeated into
the skin between the groups was examined using one-way ANOVA followed by
Student’s t-test. The significance level is set at p<0.05.
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CHAPTER 4
RESULTS AND DISCUSSION
4.1 In vitro skin permeation studies using MN
4.1.1 MN array fabrication
4.1.2 Evaluation of the MN arrays using SEM
4.1.3 Effect of MN insertion force on skin permeation
4.2 In vitro skin permeation studies using EP
4.2.1 Effect of pulse voltage
4.2.2 Skin permeation using the combination of MN and EP
4.3 In vitro skin permeation studies using SN
4.3.1 Effect of ultrasound intensity
4.3.2 Skin permeation using the combination of MN and SN
4.3.3 Skin permeation using the combination of EP and SN
4.4 In vitro skin permeation studies using MN + EP + SN
4.4.1 Skin permeation of FD-4
4.4.2 Effect of MW on skin permeation
4.5 CLSM studies
4.6 Skin histology
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4.1 In vitro skin permeation studies using MN
4.1.1 MN array fabrication
The stratum corneum, the skin’s outermost layer, is a barrier that prevents
molecular transport across the skin (Menon, Cleary, and Lane, 2012). Therapeutic
agents, such as peptides, proteins, and oligonucleotides, are difficult to deliver by
conventional methods or topical delivery. However, advances in physical
enhancement methods in the last decade has led to the development of powerful
strategies to overcome the skin’s barrier function (Naik, Kalia, and Guy, 2000). MN
are micron-sized needles that can perforate the skin in a minimally invasive and
painless manner, thereby creating aqueous transport pathways within the skin referred
to as microchannels (Henry et al., 1998; Milewski, Brogden, and Stinchcomb, 2010;
Noh et al., 2010). Moreover, these microchannels have no limitation regarding the
size of molecules that can pass. In terms of size, the microchannels are in the range of
microns, while the macromolecules delivered are typically nanometers in size (Kumar
and Banga, 2012).
One of factors that challenge to development of MN-assisted delivery is
skin elasticity (Martanto, Jason S Moore, et al., 2006; Verbaan et al., 2008). If the
needle is not long enough to overcome the skin elasticity, it would provide an
unsuccessful result. To date many research groups have reported the effect of MN
length on skin permeability. The length has been varied between 25 and 2,000 μm
(Tuan-Mahmood et al., 2013). Their results demonstrated that the longer MN, the
higher skin permeability would be achieved. Yan et al. (2010) evaluated the effect of
MN length on the skin permeation of acyclovir. Their results showed that the
permeation of acyclovir after pretreatment with 100–300 μm MN was very low
compared to control. In contrast, if the MN was lengthened up to 600 μm, the
permeation would be more effective. Corresponding to the results obtained from
Verbaan et al. (2007), 300 μm MN did not pierce human skin in vitro. While the
needles with a length of 550, 700, and 900 μm were able to pierce the skin, leading to
an elevated transepidermal water loss (TEWL) level in human dermatomed skin
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(Verbaan et al., 2007). Furthermore, 1,100 μm-long MN showed the highest skin
permeation (Yan et al., 2010). Teo et al. (2005) observed skin permeation of calcein
after pretreatment with 130 μm needle in vitro. However, this similar size of MN
could not have an effectiveness to deliver insulin in vivo. They discussed that the
length and tip sharpness have to be improved for systemic drug delivery (Teo et al.,
2005). Martanto et al. (2006) have reported that only one third of the length of hollow
MN can pierce the skin. MN was inserted to 1,080 μm from the skin surface, however
it could penetrate the skin only 100–300 μm (Martanto, Jason S Moore, et al., 2006).
Though MN was relatively long, the tip of MN remained in viable epidermis. This
could be implied that MN can breach the stratum corneum without stimulating nerves
locating in dermis layer. In clinical study, the relatively long MN, i.e. 620 μm, can
successfully deliver naltrexone from its patch into systemic circulation (Wermeling et
al., 2008). Furthermore, the two commercial products associated with MN have
relatively long needles ranging from 1,100 μm to 1,500 μm. One of them is Soluvia®
(Becton, Dickinson and Company). This product has 1.5 mm-long MN attached to 0.1
mL syringe that is prefilled with influenza vaccine. Another is AdminPatch
(nanoBioSciences, Sunnyvale, California). The length of the MN is 1,500 μm and
1,200 μm with 31 and 43 individual MN on each patch.
Above all, to assure that the length of MN will be long enough to penetrate
stratum corneum and provide an efficiency to deliver macromolecules, MN arrays
were therefore fabricated by providing the length of 1,000 μm (Figure 9 (b) and
Figure 10 (b)).
4.1.2 Evaluation of the MN arrays using SEM
SEM imaging was used to visualize the surface of the MN-treated skin to
ensure that the MN array can effectively breach the skin and create a hole. Figure 12
(a) displays the micropores created by the MN array on the skin surface after piercing
with 10 N force, and Figure 12 (b) shows an individual hole with a diameter of 100
μm created by MN.
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These images demonstrate that MN successfully punctures and creates
obvious micropores on the skin, indicative of the stratum corneum barrier disruption
(Park, Allen, and Prausnitz, 2005; Coulman et al., 2009; Kumar and Banga, 2012;
Kaur et al., 2014). This finding could imply that these micropores can serve as a
significant pathway for the transportation of molecules across the skin.
Figure 12 Scanning electron microscope (SEM) images of pores created by MN array
(a) and an individual hole created in porcine skin by MN (b).
4.1.3 Effect of MN insertion force on skin permeation
Insertion force is one of the factors influencing drug delivery and skin
permeability through MN-treated skin (Milewski, Brogden, and Stinchcomb, 2010). A
proper insertion force helps MN to overcome the resistance of the skin, determined by
viscoelasticity of the skin, especially, stratum corneum (Cheung, Han, and Das,
2014). Before starting the in vitro permeation studies using combination enhancement
methods, the application forces were first optimized.
Based on in vitro permeation studies (Figure 13), FD-4 can be successfully
delivered using a homemade MN array. In contrast, FD-4 was not detected in the
receptor compartment after passive delivery. This could be due to its MW larger than
500 Da and log partition coefficient; -2 (Sakai et al., 1997). As previously reported, a
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MN array with different dimensions overcame the stratum corneum barrier and
improved FD-4 transport (Wu, Todo, and Sugibayashi, 2007; Yan, Todo, and
Sugibayashi, 2010; Liu et al., 2014). In addition, it was believed that convection
generated by MN played a key role in the transport of FD-4 (Zhang, Qiu, and Gao,
2014).
The total cumulative amount of FD-4 permeated MN-treated skin was
significantly higher than the control group. As insertion force increased from 2.5 to 10
N , FD-4 permeated significantly increased. These results could be attributed to the
fact that different insertion forces create microconduits with different diameters and
depths (Martanto, Jason S Moore, et al., 2006; Wei-Ze et al., 2010). These results
agreed with Cheung et al. (2014) who investigated the effect of 1,400 μm-long MN to
promote insulin (MW = 5.8 kDa) delivery in in vitro porcine skin. The MN arrays
were inserted into a skin sample by an in-house device. Their results showed that after
4 h, the amount of insulin permeated increased from 3 μg to 25 μg when the insertion
force increased from 60.5 N to 69.1 N (Cheung, Han, and Das, 2014). However up to
30 N insertion force, FD-4 permeated was not significantly enhanced with 10, 20, and
30 N insertion force. This result indicated that the 10 N insertion force was strong
enough to overcome the resistance of skin, and higher insertion forces would not
affect the property of MN to puncture skin.
The slope of the linear portion of the line plotted from the steady-state
values of the cumulative amount of FD-4 permeated and time (h) represents the
permeation rate or the steady-state flux (Bhatt, Koland, and Shama, 2013). Based on
the data, an increase in the insertion force improves FD-4 flux (Table 3). As a result, a
10 N insertion force was therefore applied in further experiments.
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Table 3 The rate of FD-4 permeation across skins treated with MN using various
insertion forces.
Insertion force (N) Flux (μg/cm2/h-1) R2
2.5 0.482 0.141 0.996 0.002
5.0 1.370 0.362 0.997 0.002
10.0 2.358 0.551 0.995 0.002
20.0 2.440 0.860 0.995 0.003
30.0 2.579 0.684 0.996 0.002
4.2 In vitro skin permeation studies using EP
4.2.1 Effect of pulse voltage
Pulse voltages significantly influence the amount of drug delivered through
EP-treated skins (Sung et al., 2003; Denet, Vanbever, and Préat, 2004). Firstly, in
order to ascertain that the blunted-MN array did not cause some holes or scratches on
the skin, the skin permeation of FD-4 was evaluated using only the blunted-MN array,
without applying electric current. The force used was 10 N which was the same force
as MN + EP protocol. The result revealed that FD-4 could not be detected in the
receiver compartment after 24 h (data not shown). This could be suggested that the
blunted-MN array can be used as electrode for EP application, without causing MN
effect.
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Figure 13 In vitro permeation profiles of FD-4 in porcine skin pierced with MN array
using various insertion forces; no pretreatment (), 2.5 N (), 5.0 N (), 10.0 N
(), 20.0 N (), 30.0 N ().
To determine the effect of pulse voltage on FD-4 transdermal delivery,
pulse duration was fixed at 1 ms with 100 ms pulse intervals, and number of pulses
were set at 99 pulses. The 50, 100, 200, and 300 V were transferred using a blunted-
MN array as an electrode. The results revealed that EP (200 – 300 V) significantly
enhanced the transdermal delivery of FD-4 compared with passive diffusion. This
finding is possibly due to the fact that EP pretreatment promotes enhanced
macromolecular transport through permeabilized skin by creating new aqueous
pathways in lipid bilayer membranes (Prausnitz, 1999; Lombry, Dujardin, and Préat,
2000; Denet, Vanbever, and Préat, 2004).
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FD-4 permeated was undetectable at voltages below 200 V, which could
serve as the voltage threshold of porcine skin under this protocol (data not shown).
The FD-4 flux (μg/cm2/h-1) in EP-treated skin after the application of 200 V and 300
V was 0.151 ± 0.047 (R2 = 0.975) and 0.216 ± 0.085 (R2 = 0.975), respectively.
Nevertheless, the effect of pulse voltages was still not clear in this experiment because
the penetrant was relatively large molecules, i.e. low skin permeability. This effect
would distinctly demonstrate when used the combined methods, i.e. MN + EP. Unlike
previous reports, the flux of calcein, a small molecule (623 Da), obviously increased
with an increase in voltage (Prausnitz et al., 1993). The similar result could be
observed in in vitro skin permeation study for metoprolol (267 Da) (Vanbever and
Préat, 1995).
Figure 14 In vitro permeation profiles of FD-4 across porcine skin after treatment
with MN + EP using various pulse voltages: 0 V or MN alone (), 50 V (), 100 V
(), 200 V (), and 300 V ().
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In order to further study whether increased pulse duration would probably
increase skin permeation of FD-4. Pulse duration was set at 300 ms and pulse voltage
was constant at 300 V. The number of pulses was set at 10 to avoid burning the skin.
The amount of FD-4 permeated across 300 ms EP-treated skin (10 pulses) could be
detected in the receiver compartment after 24 h (5.577 0.308 μg/cm2). In contrast to
EP-treated skin with 1 ms and 99 times of pulse number, the amount of FD-4
permeated could be detected after 4 h (4.803 0.375 μg/cm2). This could be implied
that FD-4 could penetrate faster with short duration protocol compared with long
duration. Additionally, considering to the safety, application of pulse shorter than 1
ms could reduce or diminish sensation (Prausnitz, 1999). As this guide, 1 ms pulse
duration was used in the further experiments.
4.2.2 Skin permeation using the combination of MN and EP
The effect of pulse voltages on MN-treated skin, i.e. the MN + EP method,
was also investigated by maintaining a constant insertion force of 10 N. FD-4 skin
permeability significantly increased as pulse voltages increased from 100 V to 300 V,
with the exception of 50 V and 100 V (Figure 14). The flux of FD-4 in MN + EP-
treated skin was dramatically enhanced compared with MN alone (Table 4). Though
the higher cumulative amount permeated attributed to an increase in voltage, the
highest voltage that skin can be tolerated was 300 V, as the skin was burned at 400 V.
As a result, the combination of 10 N insertion force and 300 V was chosen for the
next experiment.
As mentioned above, the effect of pulse voltage was clearly demonstrated
when EP was employed in combination with MN. The total cumulative amount of
permeated FD-4 increased with increasing applied voltages via the MN + EP array.
This result may be attributed to increased voltages that result in increased transport
pathways (Vanbever and Préat, 1995; Zewert et al., 1995; Prausnitz, 1996; Sung et al.,
2003).
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Table 4 The rate of FD-4 permeation across skins treated with MN followed by EP
using various pulse voltages
Pulse voltage (V) Flux (μg/cm2/h-1) R2
0a 2.358 0.551 0.995 0.002
50 5.829 0.454 0.994 0.004
100 5.588 0.737 0.996 0.003
200 6.190 1.493 0.997 0.002
300 7.582 2.226 0.996 0.002
a) MN alone
4.3 In vitro skin permeation studies using SN
4.3.1 Effect of ultrasound intensity
SN increases the skin permeability of several molecules by disturbing the
stratum corneum barrier's function. The primary mechanism associated with this
phenomenon is cavitation. Cavitation is dependent on various parameters, such as
ultrasound frequency, intensity, and application time (Tezel et al., 2001; Boucaud et
al., 2002; Polat et al., 2011).
In literature review, the frequency that has been widely used in
transdermal drug delivery are LFS and HFS, however LFS was preferred to use for
evaluation in this study for many reasons. They have been reported that LFS has more
effectiveness to deliver a wide range of macromolecules including DNA and proteins
into the skin than HFS (Polat et al., 2011). This is because the bubbles generated
during HFS were much smaller size compared with those for LFS. The larger bubbles
can cause more disruption when they burst (Han and Das, 2013). Another reason
associated with the equipment (an ultrasonic transducer) that cannot adjust the
frequency. According to the principle of ultrasound generation, the amplified electric
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signal sent to ultrasound horn is converted into a mechanical wave by piezoelectric
crystals. After that the mechanical wave will pass the tip of transducer through the
medium in which drug molecules contained. The frequency of ultrasound wave is
determined by piezoelectric crystal that cannot be adjustable by the equipment. As a
result, LFS (20 kHz) was therefore used for entire studies.
In the preliminary study, the effect of application time at 1, 2, and 5 min
was also investigated. The intensity was fixed at 1.7 W/cm2. The results demonstrated
that the skin permeation of FD-4 across SN-treated skin was significantly increased
compared to passive diffusion. However, there was no significant difference in skin
permeability among three conditions. The fluxes of FD-4 (μg/cm2/h-1) permeated
across SN-treated skin for 1, 2, and 5 min were 0.449 0.130 (R2 = 0.995), 0.449
0.205 (R2 = 0.990), and 0.571 0.285 (R2 = 0.993), respectively. Though the
ultrasonic energy applied for 1 min showed a minimum effective application time, it
was expected that the longer application time would provide the higher skin
permeability (Boucaud et al., 2002; Herwadkar et al., 2012; Aldwaikat and Alarjah,
2015). However, it should not be too long to cause skin damage. It was observed that
during 5 min of application, the increasing temperature was occurred in the coupling
medium. Thus, to avoid an increasing temperature in coupling medium that causes
skin damage during ultrasound application, the application time of 2 min was chosen
for the further experiments.
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Figure 15 In vitro permeation profiles of FD-4 across porcine skin after treatment
with SN using various intensities: 0 W/cm2 (), 1.7 W/cm2 (), 3.9 W/cm2 (), and
6.1 W/cm2 ().
To evaluate the effect of ultrasound intensity (1.7, 3.9, and 6.1 W/cm2) on
skin permeation of FD-4, LFS (20 kHz) was applied, and the application time was
maintained at a constant of 2 min during the experiments. After 2 min of continuous
SN application, the amount of FD-4 permeated through the SN-treated skin was
significantly increased compared with passive delivery. It should be noted that LFS,
i.e. 20 kHz, can improve the transdermal delivery of hydrophilic macromolecules.
The results demonstrated that there was no significant difference in the cumulative
amount of FD-4 permeated after the application of all ultrasound intensities (Figure
15). The FD-4 flux (μg/cm2/h-1) after the application of 1.7, 3.9, and 6.1 W/cm2 was
0.335 ± 0.086 (R2 = 0.996), 0.178 ± 0.040 (R2 = 0.984), and 0.169 ± 0.040 (R2 =
0.967), respectively. This could be suggested that the range of intensities used in this
study (1.7 – 6.1 W/cm2) did not affect FD-4 permeated through SN-treated skin.
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4.3.2 Skin permeation using the combination of MN and SN
The effect of MN pretreatment on skin permeation of FD-4 following the
application of various SN intensities is displayed in Figure 16. The results showed
that the SN and MN combination dramatically enhanced FD-4 permeated compared
with MN alone; however, no statistically significant differences among all of the
intensities used were observed. The FD-4 fluxes (μg/cm2/h-1) across MN-treated skin
after treatment with 1.7, 3.9, and 6.1 W/cm2 were 3.235 ± 0.970 (R2 = 0.995), 4.772 ±
1.579 (R2 = 0.995), and 3.554 ± 1.121 (R2 = 0.996), respectively. These results were
in accordance with Han and Das (2013). They investigated the synergistic effect of
solid MN pretreatment (1,200 μm and 1,500 μm in length) and SN (20 kHz, 12 - 18
W for 10 min). Their results demonstrated the permeability of BSA across porcine ear
skin after treatment with both lengths of MN followed by SN was significantly higher
than those of SN alone. These could be explained that the cavitation generated during
SN application could affect with both the stratum corneum layer and the interface of
MN-pretreated area, which was underlying layer, leading to the higher skin
permeation (Han and Das, 2013).
To compare skin permeation of FD-4 in the next experiments, an intensity
of 6.1 W/cm2 was used as the high ultrasound intensity.
4.3.3 Skin permeation using the combination of EP and SN
To evaluate the skin permeation using EP + SN, the high voltage, i.e. 300
V, and the high intensity, i.e. 6.1 W/cm2, were performed. The total cumulative
amount of FD-4 permeated (μg/cm2) using EP, SN, and EP + SN after 24 h was 9.234
1.782, 16.891 5.641, and 16.337 3.118, respectively. The flux of FD-4
(μg/cm2/h-1) permeated using EP, SN, and EP + SN was 0.216 ± 0.085 (R2 = 0.975),
0.169 ± 0.040 (R2 = 0.967), and 0.485 ± 0.140 (R2 = 0.993), respectively.
The results demonstrated that the total cumulative amount of FD-4
permeated following SN and EP + SN was not different after 24 h. In addition, the
amount of FD-4 permeated after EP application was lower than that of SN, and EP +
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Figure 16 In vitro permeation profiles of FD-4 across MN-treated skin after the
application of SN with various intensities: 0 W/cm2 or MN alone (), 1.7 W/cm2 (),
3.9 W/cm2 (), 6.1 W/cm2 ().
SN. These results suggested that SN applied after EP showed more important role
than EP in the skin permeation of EP + SN.
4.4 In vitro skin permeation studies using MN + EP + SN
4.4.1 Skin permeation of FD-4
In previous studies, the protocols used were carefully optimized and then
selected to compare the skin permeability of FD-4. In vitro permeation profile of FD-
4 following treatment with several enhancement methods, i.e. MN, EP, SN, MN + EP,
MN + SN, EP + SN, and MN + EP + SN, is presented in Figure 17. These results
demonstrated that FD-4 did not permeate using passive delivery (no treatment);
however, the other techniques significantly enhanced FD-4 skin permeability.
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The total amount of FD-4 permeated through MN + EP-treated skin over
24 h was 20-fold higher than EP-treated skin and 3.6-fold higher than MN-treated
skin. Similarly, the total amount of FD-4 permeated through MN + SN-treated skin
was 6.3-fold and 2.1-fold higher than skin treated with SN or MN alone, respectively.
The highest cumulative amount of permeated FD-4 was obtained when these three
techniques were combined (MN + EP + SN). The total amount of FD-4 was 1.8-fold
and 3.2-fold higher than MN + EP and MN + SN, respectively. The flux obtained
from MN + EP was significantly higher than MN or EP alone (Table 5). Likewise,
MN + SN contributed to increase flux compared with MN or SN alone. The highest
flux was observed with the combination of the three strategies, i.e. MN + EP + SN.
In the comparative studies, the amount of FD-4 permeated the skin treated
with either MN or EP alone was minimal. As expected, the MN + EP method
displayed a synergistic effect on FD-4 skin permeation. These results are consistent
with the studies of Yan et al.(2010), who reported that the in-skin EP technique
increased FD-4 permeability compared with MN alone and conventional EP (Yan,
Todo, and Sugibayashi, 2010). Given that the stratum corneum displays increased
electrical resistance compared with the underlying skin and deeper tissues, an electric
current applied to the skin will be concentrated in the stratum corneum (Denet,
Vanbever, and Préat, 2004). To improve the electric field inside the skin, MN, which
can perforate the skin and bypass the stratum corneum barrier, was employed in
combination with EP to allow the electric current to reach the underlying skin. These
results obtained from in vitro permeation studies suggested that each MN could serve
as an electrode for EP, thereby resulting in the formation of an electric field inside the
skin. Interestingly, the microconduits created by the MN + EP array are larger and
deeper compared with MN alone (see Figure 23 (b)). This finding might be attributed
to the fact that decreased skin resistance with the MN + EP array could enhance the
explosion of skin following EP application at the site of each needle tip.
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Figure 17 In vitro permeation profiles of FD-4 across porcine skin following
treatment with various enhancement methods: no pretreatment (), MN (), EP (),
SN (), MN + EP (), MN + SN (), EP + SN (), MN + EP + SN ().
The MN + SN strategy enhanced the transport of FD-4 compared with
each technique alone. The synergistic outcome could be attributed to the creation of
microconduits in the skin that could serve as a shunt to deliver macromolecules. Then,
the collapse of acoustic cavitation generated by the sonophoretic probe could provide
energy to the media containing FD-4 that promotes FD-4 diffusion into these channels
(Chen, Wei, and Iliescu, 2010). In previous report, ultrasonic energy results in the
creation of 1–2 μm diameter micropores on the skin. Using SEM, these pores were
observed on the surface after LFS application, and they could be an outcome of
cavitation occurring from bubbles at interface area (Machet et al., 1998; Han and Das,
2013; Park et al., 2014). Based on these data, the combined method may serve as a
useful technique for macromolecule delivery; the size of the microconduits created by
MN appeared to increase after 2 min of SN application (see Figure 23 (c)). When EP
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and SN were employed in combination, the total cumulative amount of permeated
FD-4 remained low. It should be noted that MN significantly affected skin permeation
of FD-4.
The best result was obtained using all of the enhancement methods in
combination, i.e. MN + EP + SN. As discussed above, MN can effectively bypass the
stratum corneum barrier. EP aided in the enlargement of the microconduits produced
by MN + EP array. Finally, these microconduits were slightly further expanded
following SN. For these reasons, the total amount of permeated FD-4 in MN + EP +
SN-treated skin over 24 h was the highest compared with pair wise combinations or
individual techniques.
Table 5 The permeation rate of FD-4 across skins treated with different enhancement
techniques
Enhancement method Flux (μg/cm2/h-1) R2
MN a 2.358 0.551 0.995 0.002
EP b 0.216 0.085 0.975 0.017
SN c 0.169 0.040 0.967 0.037
MN a + EP b 7.582 2.226 0.996 0.002
MN a + SN c 3.554 1.121 0.996 0.005
EP b + SN c 0.485 0.140 0.993 0.004
MN a + EP b + SN c 16.585 2.349 0.996 0.002
a) 10 N, b) 300 V, c) 6.1 W/cm2
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4.4.2 Effect of MW on skin permeation
In order to study the effect of MW on the macromolecular delivery
through the skin treated with MN + EP + SN, the skin permeation of FD-4, FD-20,
and FD-40 were investigated. The results showed that the cumulative amount of FD
permeated can be ranked as follow; FD-4 > FD-20 > FD-40 (Figure 18). This
suggested that the higher MW of penetrant contributed to the lower skin permeation.
The flux (μg/cm2/h-1) of FD-40 (1.950 0.328, R2 = 0.992) and FD-20 (4.329
0.745, R2 = 0.994) were dramatically lower than that of FD-4 (16.585 2.349, R2 =
0.996).
This finding was in accordance with the previous reports. Wu et al. (2006)
demonstrated that the permeability coefficient of FDs through the MN-treated skin
decreased with an increased in MW of penetrant (Wu, Todo, and Sugibayashi, 2006).
In addition, the research work from Zhang et al. (2014) showed that the cumulative
amount permeated of four hydrophilic peptides, i.e. oxytocin (1,007.2 Da), acetyl
hexapeptides-3 (889 Da), hexapeptide (498.6 Da), and tetrapeptide-3 (456.6 Da),
across porcine ear skin treated with 150 μm MN depended on their MW. The amount
permeated decreased with an increase in MW (Zhang, Qiu, and Gao, 2014).
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Figure 18 In vitro permeation profiles of FD with various MW across porcine skin:
FD-4 (), FD-20 (), and FD-40 ().
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4.5 CLSM studies
CLSM was employed to visualize model compound penetration by
providing controllable depth imaging and collecting sequential optical sections from
the entire treated skin without sectioning (Lombry, Dujardin, and Préat, 2000;
Alvarez-Román et al., 2004). In the control group (untreated skin), minimal
fluorescent signal was observed on the skin surface (Figure 19). The FD-4 permeated
through the skin following an hour of in vitro permeation studies was also observed
and recorded at increasing depths from the skin surface to compare the characteristics
of microconduits created by MN and the combined methods, i.e. MN + EP, MN + SN,
and MN + EP + SN (Figure 20).
In contrast to passive delivery, the fluorescence intensity was localized
around the microchannel where the stratum corneum was breached in MN-treated
skin. However, the fluorescence depth was not greater than 100 μm (Figure 20 (a)).
These observations agreed with Zhang et al. (2010), who confirmed the formation of
microconduits by CLSM images. The fluorescence signal of calcein could be seen
around the sites of microconduits created by MN with 150 μm length, however, at the
100 μm depth of the skin sample, the fluorescence signal and microconduits were
disappeared (Zhang, Qiu, and Gao, 2014).
As the combined strategies, fluorescence was observed up to a depth of
220 μm in the skins treated with MN + EP, MN + SN, and MN + EP + SN.
Furthermore, the hole size created by the combined techniques was greater than the
hole size produced by MN alone (Figures 20 (b)–(d)). Above all, CLSM images could
be suggested that MN can effectively facilitate FD-4 transport into the skin as a result
of the stratum corneum opening (Zhang et al., 2010).
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Figure 19 CLSM image of untreated skin (passive delivery) at different depth
representing fluorescent signal observed on the skin surface
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Figure 20 CLSM images of a microchannel created by MN (a), MN + EP (b), MN +
SN (c), and MN + EP + SN (d) at various depths
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4.8 Skin histology
Figure 21 (a)–(h) displays representative biological cross sections of
porcine skin treated with the following enhancement methods: (a) untreated skin, (b)
MN, (c) EP, (d) SN, (e) MN + EP, (f) MN + SN, (g) EP + SN, and (h) MN+ EP+ SN.
These histological images indicated that no noticeable damage or changes in skin
treated with all of the enhancement methods were observed. Both skin layers, i.e.
epidermis and dermis, remained intact in all of the skin treatment groups (Figure 21
(b)–(h)). These results were similar to the normal appearance of untreated porcine
skin (Figure 21 (a)).
These could be suggested that the application of all physical enhancement
methods in these studies demonstrated safety under the specified conditions (Dujardin
et al., 2002; Tezel and Mitragotri, 2003; Wolloch and Kost, 2010; Stahl, Wohlert, and
Kietzmann, 2012; Blagus et al., 2013). However, the in vivo safety in clinical use has
to be evaluated in further study.
Figure 21 Histological images of treated skin after H&E staining: control (no
pretreatment) (a), MN (b), EP (c), SN (d), MN + EP (e), MN + SN (f), EP + SN (g),
and MN + EP + SN (h)
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CHAPTER 5
CONCLUSIONS
In this study, three physical enhancement methods of interest, i.e. MN, EP,
and SN were first investigated individually. Thereafter, the MN array was used in
conjunction with the other enhancement strategies, i.e. EP and SN, to investigate the
effects of the combined techniques.
The homemade MN arrays with the length of 1,000 μm were successfully
fabricated using nine acupuncture needles. Additionally, they showed the potential to
deliver FD-4, a hydrophilic macromolecular compound, into the skin compared to
passive delivery. The amount of FD-4 permeated across in vitro porcine skin
depended on insertion force applied. An increasing insertion force resulted in an
increase in the amount of FD-4 permeated.
EP is the application of high voltage pulse in a very short duration to
generate transient aqueous transport pathway in lipid bilayer membranes. On the
conditions that the homemade MN array served as an electrode in this study, FD-4
can favorably permeated when the voltage used was higher than 200 V. Moreover, the
result demonstrated that an increase in pulse voltage led to an increased FD-4
permeated.
SN is the use of cavitation generated by ultrasound energy to disorganize
the stratum corneum barrier, resulting in an improved skin permeability of hydrophilic
compounds. The application of 20 kHz and intensities between 1.7 - 6.1 W/cm2 in
continuous mode for 2 min significantly enhanced FD-4 permeated through SN-
treated skin compared with passive delivery. Nonetheless, the range of intensities
applied in this study did not affect the skin permeation of FD-4.
The increased amount of FD-4 permeated using MN + EP went along with
an increased pulse voltage. The MN pretreatment followed by SN vividly facilitated
the transport of FD-4. Corresponding to the results obtained from in vitro permeation
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study using SN alone, the range of intensities did not show any influences on the
transport of FD-4. Interestingly, the amount of FD-4 permeated following EP + SN
method was lower than MN alone. This implied that MN played an important role in
FD-4 permeation.
The combination of all three methods, MN + EP + SN, notably promoted
FD-4 permeated as compared to either two combined methods or separate methods.
The images obtained from CLSM studies suggested that the two or three combined
methods dramatically enlarged the microchannels created by MN, resulting in an
improved FD-4 transport. However, the MW of penetrant affected the skin
permeation using this three combined method. The higher MW of FD was applied, the
lower skin permeability could be obtained. In addition, histological examination
suggested that all of methods used were considered as safe under specific conditions.
Finally, it could be concluded that MN + EP + SN could serve as an alternative
strategy for transdermal drug delivery that does not cause structural changes and skin
damage.
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BIBLIOGRAPHY
Aldwaikat, Mai, and Mohammed Alarjah. (2015). "Investigating the sonophoresis
effect on the permeation of diclofenac sodium using 3d skin equivalent."
Ultrasonics Sonochemistry 22 (January): 580–587.
Alexander, Amit. et al. (2012). "Approaches for breaking the barriers of drug
permeation through transdermal drug delivery." Journal of Controlled
Release 164, 1 (November): 26–40.
Alvarez-Román, R. et al. (2004). "Visualization of Skin penetration using confocal
laser scanning microscopy." European Journal of Pharmaceutics and
Biopharmaceutics 58, 2 (September): 301–316.
Ameri, Mahmoud, Xiaomei Wang, and Yuh-Fun Maa. (2010). "Effect of irradiation
on parathyroid hormone PTH(1-34) coated on a novel transdermal
microprojection delivery system to produce a sterile product--adhesive
compatibility." Journal of Pharmaceutical Sciences 99, 4 (April): 2123–
2134.
Arora, Anubhav, Mark R. Prausnitz, and Samir Mitragotri. (2008). "Micro-scale
devices for transdermal drug delivery." International Journal of
Pharmaceutics. 364, 2 (August): 227–236.
Barry, B W. (2001). "Novel mechanisms and devices to enable successful transdermal
drug delivery." European Journal of Pharmaceutical Sciences 14, 2
(September): 101–114.
Benson, Heather A. E. (2005). "Transdermal drug delivery: Penetration enhancement
techniques." Current Drug Delivery 2, 1 (January):23-33.
Bhatt, Bhavik, Marina Koland, and K Prasanna Shama. (2013). "Investigation of the
ex vivo and in vivo iontophoretic delivery of aceclofenac from topical gels
in albino rats." International Journal of Pharmaceutical Investigation
3, 2 (April): 105–111.
สำนกัหอ
สมุดกลาง
83
Blagus, Tanja. et al. (2013). "In vivo real-time monitoring system of electroporation
mediated control of transdermal and topical drug delivery." Journal of
Controlled Release 172, 3 (December): 862–871.
Boucaud, Alain. et al. (2002). "Effect of sonication parameters on transdermal
delivery of insulin to hairless rats." Journal of Controlled Release 81, 1-2
(May): 113–119.
Boucaud, A. et al. (2001). "In vitro study of low-frequency ultrasound-enhanced
transdermal transport of fentanyl and caffeine across human and hairless
rat skin." International Journal of Pharmaceutics 228, 1-2 (October):
69–77.
Chang, S L. et al. (2000). "The effect of electroporation on iontophoretic transdermal
delivery of calcium regulating hormones." Journal of Controlled Release
66, 2-3 (May): 127–133.
Chen, Bangtao, Jiashen Wei, and Ciprian Iliescu. (2010). "Sonophoretic enhanced
microneedles array (sema)—improving the efficiency of transdermal drug
delivery." Sensors and Actuators B: Chemical 145, 1 (March): 54–60.
Cheung, Karmen, Tao Han, and Diganta Bhusan Das. (2014). "Effect of force of
microneedle insertion on the permeability of insulin in skin." Journal of
Diabetes Science and Technology 8, 3 (January): 444–452.
Chu, Leonard Y., and Mark R. Prausnitz. (2011). "Separable arrowhead
microneedles." Journal of Controlled Release: Official Journal of the
Controlled Release Society 149, 3 (February): 242–249.
Coulman, Sion A. et al. (2009). "Microneedle mediated delivery of nanoparticles into
human skin." International Journal of Pharmaceutics 366, 1–2
(January): 190–200.
Daddona, P.E. et al. (2011). "Parathyroid hormone (1-34)-coated microneedle patch
system: clinical pharmacokinetics and pharmacodynamics for treatment of
osteoporosis." Pharmaceutical Research 28, 1 (January): 159–165.
สำนกัหอ
สมุดกลาง
84
Davis, Shawn P. et al. (2004). "Insertion of microneedles into skin: measurement and
prediction of insertion force and needle fracture force." Journal of
Biomechanics 37, 8 (August): 1155–1163.
Davis, Shawn P. et al. (2005). "Hollow metal microneedles for insulin delivery to
diabetic rats." IEEE Transactions on Bio-Medical Engineering 52, 5
(May): 909–915.
Denet, Anne-Rose, and Véronique Préat. (2003). "Transdermal delivery of timolol by
electroporation through human skin." Journal of Controlled Release 88,
2 (March): 253–262.
Denet, Anne-Rose, Rita Vanbever, and Véronique Préat. (2004). "Skin electroporation
for transdermal and topical delivery." Advanced Drug Delivery Reviews
56, 5 (March): 659–674.
Ding, Z. et al. (2009). "Microneedle arrays for the transcutaneous immunization of
diphtheria and influenza in BALB/c mice." Journal of Controlled
Release 136, 1 (May): 71–78.
Donnelly, Ryan F., Thakur Raghu Raj Singh, and A. David Woolfson. (2010).
"Microneedle-based drug delivery systems: Microfabrication, drug
delivery, and safety." Drug Delivery 17, 4 (May): 187-207.
Dujardin, Nathalie. et al. (2002). "In vivo assessment of skin electroporation using
square wave pulses." Journal of Controlled Release 79, 1-3 (February):
219–227.
Fang, J. et al. (1999). "Effect of low frequency ultrasound on the in vitro percutaneous
absorption of clobetasol 17-propionate." International Journal of
Pharmaceutics 191, 1 (November): 33–42.
Garland, Martin J. et al. (2012). "Dissolving polymeric microneedle arrays for
electrically assisted transdermal drug delivery." Journal of Controlled
Release 159, 1 (April): 52–59.
สำนกัหอ
สมุดกลาง
85
Gill, Harvinder S., and Mark R. Prausnitz. et al. (2007). "Coated microneedles for
transdermal delivery." Journal of Controlled Release 117, 2 (February):
227–237.
Gill, Harvinder S. et al. (2008). "Effect of microneedle design on pain in human
subjects." The Clinical Journal of Pain 24, 7 (September): 585–594.
Godin, Biana, and Elka Touitou. (2007). "Transdermal skin delivery: predictions for
humans from in vivo, ex vivo and animal models." Advanced Drug
Delivery Reviews 59, 11 (September): 1152–1161.
Gonzalez-Gonzalez, Emilio. et al. (2010). "Silencing of reporter gene expression in
skin using sirnas and expression of plasmid DNA delivered by a soluble
protrusion array device (PAD)." Molecular Therapy 18, 9 (September):
1667–1674.
Guo, Siqi. et al. (2011). "Electro-gene transfer to skin using a noninvasive
multielectrode array." Journal of Controlled Release: Official Journal
of the Controlled Release Society 151, 3 (May): 256–262.
Hadgraft, J. (2001). "Skin, the final frontier." International Journal of
Pharmaceutics 224, 1-2 (August): 1–18.
Hadgraft, Jonathan, and Majella E. Lane. (2005). "Skin permeation: The years of
enlightenment. International Journal of Pharmaceutics 305, 1-2
(November): 2-12.
Han, Tao, and Diganta Bhusan Das. (2013). "Permeability enhancement for
transdermal delivery of large molecule using low-frequency sonophoresis
combined with microneedles." Journal of Pharmaceutical Sciences 102,
10 (October): 3614–3622.
Henry, S. et al. (1998). "Microfabricated microneedles: A novel approach to
transdermal drug delivery." Journal of Pharmaceutical Sciences 87, 8
(August): 922–925.
สำนกัหอ
สมุดกลาง
86
Herwadkar, Anushree, and Ajay K. Banga. (2012). "Peptide and protein transdermal
drug delivery." Drug Discovery Today: Technologies 9, 2 (Summer):
e147–e154.
Herwadkar, Anushree. et al. (2012). "Low frequency sonophoresis mediated
transdermal and intradermal delivery of ketoprofen." International
Journal of Pharmaceutics 423, 2 (February): 289–296.
Kalluri, Haripriya, and Ajay K. Banga. (2011a). "Formation and closure of
microchannels in skin following microporation." Pharmaceutical
Research 28, 1 (January): 82-94.
_________(2011b). "Transdermal delivery of proteins." AAPS PharmSciTech 12, 1
(March): 431–441.
Kaur, Monika. et al. (2014). "Microneedle-assisted delivery of verapamil
hydrochloride and amlodipine besylate." Theme Issue:Dermal and
Transdermal Drug Delivery 86, 2 (February): 284–291.
Kim, Yeu-Chun, Jung-Hwan Park, and Mark R. Prausnitz. (2012). "Microneedles for
drug and vaccine delivery." Advanced Drug Delivery Reviews 64, 14
(November): 1547–1568.
Kumar, Vijay, and Ajay K Banga. (2012). "Modulated iontophoretic delivery of small
and large molecules through microchannels." International Journal of
Pharmaceutics 434, 1-2 (September): 106–114.
Lee, Jeong W., Jung-Hwan Park, and Mark R. Prausnitz. (2008). "Dissolving
microneedles for transdermal drug delivery." Biomaterials 29, 13 (May):
2113–2124.
Liu, Shu. et al. (2014). "Transdermal delivery of relatively high molecular weight
drugs using novel self-dissolving microneedle arrays fabricated from
hyaluronic acid and their characteristics and safety after application to the
skin." European Journal of Pharmaceutics and Biopharmaceutics 86,
2 (February): 267–276.
สำนกัหอ
สมุดกลาง
87
Lombry, C, N Dujardin, and V Préat. (2000). "Transdermal delivery of
macromolecules using skin electroporation." Pharmaceutical Research
17, 1 (January): 32–37.
Machet, L. et al. (1998). "In vitro phonophoresis of mannitol, oestradiol and
hydrocortisone across human and hairless mouse skin." International
Journal of Pharmaceutics 165, 2 (May): 169–174.
Martanto, Wijaya. et al. (2004). "Transdermal delivery of insulin using microneedles
in vivo." Pharmaceutical Research 21, 6 (June): 947–952.
Martanto, Wijaya. et al. (2006). "Microinfusion using hollow microneedles."
Pharmaceutical Research 23, 1 (January): 104–113.
Martanto, Wijaya. et al. (2006). "Mechanism of fluid infusion during microneedle
insertion and retraction." Journal of Controlled Release 112, 3 (May):
357–361.
McAllister, Devin V. et al. (2003). "Microfabricated needles for transdermal delivery
of macromolecules and nanoparticles: Fabrication methods and transport
studies." Proceedings of the National Academy of Sciences of the
United States of America 100, 24 (November): 13755–13760.
McGrath, Marie G. et al. (2011). "Determination of parameters for successful spray
coating of silicon microneedle arrays." International Journal of
Pharmaceutics 415, 1-2 (August): 140–149.
Medi, Babu M, and Jagdish Singh. (2003). "Electronically facilitated transdermal
delivery of human parathyroid hormone (1-34)." International Journal of
Pharmaceutics 263, 1-2 (September): 25–33.
Medi, Babu M., and Jagdish Singh. (2006). "Skin Targeted DNA Vaccine Delivery
Using Electroporation in Rabbits II. Safety." International Journal of
Pharmaceutics 308, 1-2 (February): 61–68.
Menon, Gopinathan K., Gary W. Cleary, and Majella E. Lane. (2012). "The structure
and function of the stratum corneum." International Journal of
Pharmaceutics 435, 1 (October): 3–9.
สำนกัหอ
สมุดกลาง
88
Merino, Gustavo. et al. (2003). "Frequency and thermal effects on the enhancement of
transdermal transport by sonophoresis." Journal of Controlled Release
88, 1 (February): 85–94.
Milewski, Mikolaj, Nicole K Brogden, and Audra L Stinchcomb. (2010). "Current
aspects of formulation efforts and pore lifetime related to microneedle
treatment of skin." Expert Opinion on Drug Delivery 7, 5 (May): 617–
629.
Morimoto, Yasunori. et al. (2005). "Elucidation of the transport pathway in hairless
rat skin enhanced by low-frequency sonophoresis based on the solute-
water transport relationship and confocal microscopy." Journal of
Controlled Release 103, 3 (April): 587–597.
Murthy, S. Narasimha, Srinivasa M. Sammeta, and C. Bowers. (2010).
"Magnetophoresis for enhancing transdermal drug delivery: mechanistic
studies and patch design." Journal of Controlled Release 148, 2
(December): 197–203.
Naik, Aarti, Yogeshvar N. Kalia, and Richard H. Guy. (2000). "Transdermal drug
delivery: overcoming the skin’s barrier function." Pharmaceutical
Science & Technology Today 3, 9 (September): 318–326.
Noh, Young-Wook. et al. (2010). "In vitro characterization of the invasiveness of
polymer microneedle against skin." International Journal of
Pharmaceutics 397, 1-2 (September): 201–205.
Oh, Jae-Ho. et al. (2008). "Influence of the delivery systems using a microneedle
array on the permeation of a hydrophilic molecule, calcein." European
Journal of Pharmaceutics and Biopharmaceutics 69, 3 (August): 1040–
1045.
Park, Donghee. et al. (2014). "Sonophoresis in Transdermal Drug Deliverys."
Ultrasonics 54, 1 (January): 56–65.
สำนกัหอ
สมุดกลาง
89
Park, Jung-Hwan, Mark G. Allen, and Mark R. Prausnitz. (2005). "Biodegradable
polymer microneedles: Fabrication, mechanics and transdermal drug
delivery." Journal of Controlled Release 104, 1 (May): 51–66.
Pearton, Marc. et al. (2008). "Gene delivery to the epidermal cells of human skin
explants using microfabricated microneedles and hydrogel formulations."
Pharmaceutical Research 25, 2 (February): 407–416.
Pearton, Marc. et al. (2012). "Microneedle delivery of plasmid DNA to living human
skin: Formulation coating, skin insertion and gene expression." Journal of
Controlled Release 160, 3 (June): 561–569.
Polat, Baris E. et al. (2011). "Ultrasound-mediated transdermal drug delivery:
Mechanisms, scope, and emerging trends." Journal of Controlled
Release 152, 3 (June): 330–348.
Prausnitz, Mark R. (1999). "A practical assessment of transdermal drug delivery by
skin electroporation." Advanced Drug Delivery Reviews 35, 1 (January):
61-76.
Prausnitz, Mark R. (1996). "Do high-voltage pulses cause changes in skin structure?"
Journal of Controlled Release 40, 3 (July): 321–326.
Prausnitz, Mark R. (2004). "Microneedles for transdermal drug delivery." Advanced
Drug Delivery Reviews 56, 5 (March): 581–587.
Prausnitz, Mark R, and Robert Langer. (2008). "Transdermal Drug Delivery." Nat
Biotech 26, 11 (November): 1261–1268.
Prausnitz, M. R. et al. (1993). "Electroporation of mammalian skin: A mechanism to
enhance transdermal drug delivery." Proceedings of the National
Academy of Sciences 90, 22 (November): 10504–10508.
Qiu, Yuqin. et al. (2008). "Enhancement of skin permeation of docetaxel: A novel
approach combining microneedle and elastic liposomes." Journal of
Controlled Release 129, 2 (July): 144–150.
สำนกัหอ
สมุดกลาง
90
Saliba, Susan. et al. (2007). "Phonophoresis and the absorption of dexamethasone in
the presence of an occlusive dressing." Journal of Athletic Training 42, 3
(July-September): 349–354.
Schoellhammer, Carl M., Daniel Blankschtein, and Robert Langer. (2014). "Skin
permeabilization for transdermal drug delivery: Recent advances and
future prospects." Expert Opinion on Drug Delivery 11, 3 (March): 393–
407.
Seto, Jennifer E. et al. (2010). "Effects of ultrasound and sodium lauryl sulfate on the
transdermal delivery of hydrophilic permeants: Comparative in vitro
studies with full-thickness and split-thickness pig and human skin."
Journal of Controlled Release 145, 1 (July): 26–32.
Stahl, Jessica, Mareike Wohlert, and Manfred Kietzmann. (2012). "Microneedle
pretreatment enhances the percutaneous permeation of hydrophilic
compounds with high melting points." BMC Pharmacology and
Toxicology 13, 1 (December): 1–7.
Sung, K C. et al. (2003). "Transdermal delivery of nalbuphine and its prodrugs by
electroporation." European Journal of Pharmaceutical Sciences 18, 1
(January): 63–70.
Tachibana, K., and S. Tachibana. (1993). "Use of ultrasound to enhance the local
anesthetic effect of topically applied aqueous lidocaine." Anesthesiology
78, 6 (June): 1091–1096.
Tang, Hua, Daniel Blankschtein, and Robert Langer. (2002). "Effects of low-
frequency ultrasound on the transdermal permeation of mannitol:
Comparative studies with in vivo and in vitro skin. Journal of
Pharmaceutical Sciences 91, 8 (August): 1776–1794.
Tang, Hua. et al. (2002). "An investigation of the role of cavitation in low-frequency
ultrasound-mediated transdermal drug transport." Pharmaceutical
Research 19, 8 (August): 1160–1169.
สำนกัหอ
สมุดกลาง
91
Teo, Melissa Ai Ling. et al. (2005). "In vitro and in vivo characterization of MEMS
microneedles." Biomedical Microdevices 7, 1 (March): 47–52.
Tezel, Ahmet, and Samir Mitragotri. (2003). "Interactions of inertial cavitation
bubbles with stratum corneum lipid bilayers during low-frequency
sonophoresis." Biophysical Journal 85, 6 (December): 3502–3512.
Tezel, A. et al. (2001). "Frequency dependence of sonophoresis." Pharmaceutical
Research 18, 12 (December): 1694–1700.
Tokudome, Yoshihiro, and Kenji Sugibayashi. (2003). "The effects of calcium
chloride and sodium chloride on the electroporation-mediated skin
permeation of fluorescein isothiocyanate (FITC)-dextrans in vitro."
Biological & Pharmaceutical Bulletin 26, 10 (October): 1508–1510.
Tuan-Mahmood, Tuan-Mazlelaa. et al. (2013). "Microneedles for intradermal and
transdermal drug delivery." European Journal of Pharmaceutical
Sciences 50, 5 (December): 623–637.
Van, Damme P. et al. (2009). "Safety and efficacy of a novel microneedle device for
dose sparing intradermal influenza vaccination in healthy adults." Vaccine
27, 3 (January): 454–459.
Vanbever, Rita, and Véronique Préat. (1995). "Factors affecting transdermal delivery
of metoprolol by electroporation." Bioelectrochemistry and
Bioenergetics 38, 1 (August): 223–228.
Vanbever, R. et al. (1998). "Transdermal delivery of fentanyl: rapid onset of analgesia
using skin electroporation." Journal of Controlled Release 50, 1-3
(January): 225–235.
Vanbever, R., M. A. Leroy, and V. Préat. (1998). "Transdermal permeation of neutral
molecules by skin electroporation." Journal of Controlled Release 54, 3
(August): 243–250.
Verbaan, F. J. et al. (2007). "Assembled microneedle arrays enhance the transport of
compounds varying over a large range of molecular weight across human
สำนกัหอ
สมุดกลาง
92
dermatomed skin." Journal of Controlled Release 117, 2 (February):
238–245.
Verbaan, F J. et al. (2008). "Improved piercing of microneedle arrays in dermatomed
human skin by an impact insertion method." Journal of Controlled
Release 128, 1 (May): 80–88.
Wei-Ze, Li. et al. (2010). "Super-short solid silicon microneedles for transdermal drug
delivery applications." International Journal of Pharmaceutics 389, 1–2
(April): 122–129.
Wermeling, Daniel P. et al. (2008). "Microneedles permit transdermal delivery of a
skin-impermeant medication to humans." Proceedings of the National
Academy of Sciences 105, 6 (February): 2058–2063.
Wolloch, Lior, and Joseph Kost. (2010). "The importance of microjet vs shock wave
formation in sonophoresis." Journal of Controlled Release 148, 2
(December): 204–211.
Wonglertnirant, Nanthida. et al. (2010). "Macromolecular delivery into skin using a
hollow microneedle." Biological & Pharmaceutical Bulletin 33, 12:
1988–1993.
Wong, Tin Wui. (2014). "Electrical, magnetic, photomechanical and cavitational
waves to overcome skin barrier for transdermal drug delivery." Journal of
Controlled Release 193, (November): 257–269.
Wu, Xue-Ming, Hiroaki Todo, and Kenji Sugibayashi. (2006). "Effects of
pretreatment of needle puncture and sandpaper abrasion on the in vitro
skin permeation of fluorescein isothiocyanate (FITC)-dextran."
International Journal of Pharmaceutics 316, 1-2 (June): 102–108.
Wu, Xue-Ming, Hiroaki Todo, and Kenji Sugibayashi. (2007). "Enhancement of skin
permeation of high molecular compounds by a combination of
microneedle pretreatment and iontophoresis." Journal of Controlled
Release 118, 2 (April): 189–195.
สำนกัหอ
สมุดกลาง
93
Yamashita, Fumiyoshi, and Mitsuru Hashida. (2003). "Mechanistic and empirical
modeling of skin permeation of drugs." Advanced Drug Delivery
Reviews 55, 9 (September): 1185–1199.
Yang, Jae-Heon. et al. (2005). "Anti-inflammatory effects by transdermal application
of triamcinolone acetonide gel using phonophoresis in rats." International
Journal of Pharmaceutics 302, 1-2 (September): 39–46.
Yan, Guang. et al. (2010). "Evaluation needle length and density of microneedle
arrays in the pretreatment of skin for transdermal drug delivery.
International Journal of Pharmaceutics 391, 1-2 (May): 7–12.
Yan, Keshu, Hiroaki Todo, and Kenji Sugibayashi. (2010). "Transdermal drug
delivery by in-skin electroporation using a microneedle array."
International Journal of Pharmaceutics 397, 1-2 (September): 77–83.
Zewert, T. E. et al. (1995). "Transdermal transport of DNA antisense oligonucleotides
by electroporation. Biochemical and Biophysical Research
Communications 212, 2 (July): 286–292.
Zhang, Suohui, Yuqin Qiu, and Yunhua Gao. (2014). "Enhanced delivery of
hydrophilic peptides in vitro by transdermal microneedle pretreatment."
Drug Delivery System and Pharmaceutical Technology 4, 1 (February):
100–104.
Zhang, Wei. et al. (2010). "Penetration and distribution of PLGA nanoparticles in the
human skin treated with microneedles." International Journal of
Pharmaceutics 402, 1-2 (December): 205–212.
Zorec, Barbara. et al. (2013). "Skin electroporation for transdermal drug delivery: the
influence of the order of different square wave electric pulses."
International Journal of Pharmaceutics 457, 1 (November): 214–223.
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APPENDIX
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APPENDIX A
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Standard curve for the in vitro skin permeation study
1. Determination of the amount of FD-4 permeated
Standard : FD-4
Method : Fluorescence spectrophotometry
Detector : The excitation wavelength at 495 nm
The emission wavelength at 515 nm
Concentration (μg/mL) : 0.215, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0
Figure 22 Calibration curve of FD-4 for the in vitro skin permeation study
y = 121.24x - 26.061R² = 0.9998
0
200
400
600
800
1000
0 1 2 3 4 5 6
Fluo
resc
ence
inte
nsity
Conc. (μg/mL)
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2. Determination of the amount of FD-20 permeated
Standard : FD-20
Method : Fluorescence spectrophotometry
Detector : The excitation wavelength at 495 nm
The emission wavelength at 515 nm
Concentration (μg/mL) : 0.15, 0.215, 1.0, 2.0, 3.0
Figure 23 Calibration curve of FD-20 for the in vitro skin permeation study
y = 297.91x - 25.537R² = 0.999
0
200
400
600
800
1000
0 1 2 3 4
Fluo
resc
ence
inte
nsity
Conc. (μg/mL)
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3. Determination of the amount of FD-40 permeated
Standard : FD-40
Method : Fluorescence spectrophotometry
Detector : The excitation wavelength at 495 nm
The emission wavelength at 515 nm
Concentration (μg/mL) : 0.1, 0.15, 0.215, 1.0, 2.0, 3.0, 4.0
Figure 24 Calibration curve of FD-40 for the in vitro skin permeation study
y = 230x + 2.1419R² = 0.999
0
200
400
600
800
1000
0 1 2 3 4 5
Fluo
resc
ence
inte
nsity
Conc. (μg/mL)
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APPENDIX B
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In vitro skin permeation studies
1. In vitro skin permeation studies using MN
Table 6 The cumulative amount of FD-4 permeated using MN with various insertion
forces
Time (h)
Cumulative amount of FD-4 permeated (μg/cm2)
Passive diffusion 2.5 N 5.0 N 10 N 20 N 30 N
AVG S.E. AVG S.E. AVG S.E. AVG S.E. AVG S.E. AVG S.E.
0.083 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
0.25 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
0.5 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.732 1.936
2 N/A N/A N/A N/A N/A N/A N/A N/A 3.820 3.590 3.016 3.906
4 N/A N/A N/A N/A 6.570 1.050 8.480 1.870 11.050 4.100 9.646 4.225
6 N/A N/A N/A N/A 8.240 0.850 13.300 2.970 17.400 6.800 14.611 6.181
8 N/A N/A 5.920 0.860 10.070 1.680 18.960 4.370 24.040 8.990 19.909 7.604
12 N/A N/A 7.470 1.400 14.500 3.370 29.360 6.900 33.340 12.230 30.847 9.298
16 N/A N/A 10.150 1.450 19.250 5.330 37.790 8.850 43.180 15.180 42.229 11.719
20 N/A N/A 12.380 2.050 24.240 4.920 45.350 10.610 49.610 17.360 50.922 13.988
24 N/A N/A 14.480 2.350 30.030 6.610 51.390 12.380 55.810 18.690 58.378 14.701
* N/A ; the amount of FD-4 permeated was below the limit of detection.
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2. In vitro skin permeation studies using EP
2.1 Effect of pulse voltage
Table 7 The cumulative amount of FD-4 permeated using EP with various pulse
voltages
Time
(h)
Cumulative amount of FD-4 permeated (μg/cm2) Passive
diffusion 50 V 100 V 200 V 300 V
AVG S.E. AVG S.E. AVG S.E. AVG S.E. AVG S.E.
0.083 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
0.25 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
0.5 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
4 N/A N/A N/A N/A N/A N/A N/A N/A 4.803 0.375
6 N/A N/A N/A N/A N/A N/A N/A N/A 5.340 0.508
8 N/A N/A N/A N/A N/A N/A N/A N/A 5.674 0.811
12 N/A N/A N/A N/A N/A N/A 5.509 0.037 6.245 1.052
16 N/A N/A N/A N/A N/A N/A 6.148 0.352 7.293 1.041
20 N/A N/A N/A N/A N/A N/A 6.778 0.594 8.106 1.145
24 N/A N/A N/A N/A N/A N/A 7.316 0.587 9.234 1.782
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2.2 Effect of pulse voltage on skin permeation using MN + EP
Table 8 The cumulative amount of FD-4 permeated using MN + EP with various
pulse voltages
Time
(h)
Cumulative amount of FD-4 permeated (μg/cm2)
MN alone MN + 50 V MN + 100 V MN + 200 V MN + 300 V
AVG S.E. AVG S.E. AVG S.E. AVG S.E. AVG S.E.
0.083 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
0.25 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
0.5 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
1 N/A N/A 6.261 0.627 5.893 1.437 5.780 0.497 5.707 0.587
2 N/A N/A 12.436 3.235 10.078 2.363 9.206 1.248 8.952 2.476
4 8.480 1.870 24.684 8.368 20.685 3.775 23.154 5.517 19.902 3.951
6 13.300 2.970 37.092 10.417 33.416 4.789 35.485 9.387 32.538 5.152
8 18.960 4.370 50.035 11.689 46.263 5.479 50.273 15.316 50.222 8.318
12 29.360 6.900 75.186 15.804 68.840 8.099 73.328 16.298 77.444 16.309
16 37.790 8.850 87.392 19.741 86.382 10.618 101.872 31.452 107.005 26.189
20 45.350 10.610 106.066 24.147 104.472 12.395 122.676 31.855 144.640 31.950
24 51.390 12.380 116.687 25.79 117.709 14.543 141.795 31.869 184.929 38.603
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3. In vitro skin permeation studies using SN
3.1 Effect of ultrasound intensity
Table 9 The cumulative amount of FD-4 permeated using SN with various ultrasound
intensities
Time (h) Cumulative amount of FD-4 permeated (μg/cm2)
Passive diffusion 1.7 W/cm2 3.9 W/cm2 6.1 W/cm2
AVG S.E. AVG S.E. AVG S.E. AVG S.E.
0.083 N/A N/A N/A N/A 5.393 0.348 6.950 2.738
0.25 N/A N/A 5.851 1.289 6.163 0.939 7.602 1.926
0.5 N/A N/A 6.631 1.209 6.293 0.473 7.898 1.824
1 N/A N/A 7.046 1.151 6.811 0.523 8.384 2.469
2 N/A N/A 7.767 1.112 7.459 0.683 9.304 2.359
4 N/A N/A 8.492 1.075 8.013 0.434 10.030 1.829
6 N/A N/A 9.464 1.285 8.506 0.366 11.410 2.993
8 N/A N/A 10.070 1.095 9.146 0.358 12.083 1.433
12 N/A N/A 11.377 0.702 9.758 0.488 13.425 1.819
16 N/A N/A 12.460 0.762 10.319 0.412 14.500 3.344
20 N/A N/A 13.805 1.381 10.697 0.312 15.849 4.607
24 N/A N/A 15.910 2.302 10.824 0.457 16.891 5.641
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3.2 Effect of ultrasound intensity on skin permeation using MN + SN
Table 10 The cumulative amount of FD-4 permeated using MN + SN with various
ultrasound intensities
Time (h)
Cumulative amount of FD-4 permeated (μg/cm2)
MN alone MN + 1.7 W/cm2 MN + 3.9 W/cm2 MN + 6.1 W/cm2
AVG S.E. AVG S.E. AVG S.E. AVG S.E.
0.083 N/A N/A 6.032 0.520 3.016 3.484 5.575 0.369
0.25 N/A N/A 6.955 0.791 3.436 3.978 6.182 0.545
0.5 N/A N/A 7.914 1.022 3.557 4.111 6.249 1.245
1 N/A N/A 9.828 1.574 5.494 3.922 8.182 1.509
2 N/A N/A 13.721 3.213 9.930 3.461 14.633 3.727
4 8.480 1.870 21.512 6.673 18.734 7.016 28.496 10.757
6 13.300 2.970 30.140 4.800 28.487 12.134 40.120 16.301
8 18.960 4.370 37.609 6.471 39.357 16.667 49.451 20.777
12 29.360 6.900 49.74 11.666 56.653 21.529 64.411 26.802
16 37.790 8.850 61.603 13.804 73.666 25.220 77.929 28.563
20 45.350 10.610 73.714 18.014 87.991 25.962 92.848 29.009
24 51.390 12.380 88.040 20.255 101.558 26.430 106.732 32.218
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3.3 Skin permeation using the combination of EP + SN
Table 11 The cumulative amount of FD-4 permeated using EP, SN, and EP + SN
Time (h)
Cumulative amount of FD-4 permeated (μg/cm2)
EP 300 V SN 6.1 W/cm2 EP + SN
AVG S.E. AVG S.E. AVG S.E.
0.083 N/A N/A 6.95 2.738 N/A N/A
0.25 N/A N/A 7.602 1.926 5.851 0.292
0.5 N/A N/A 7.898 1.824 6.379 0.248
1 N/A N/A 8.384 2.469 6.894 0.215
2 N/A N/A 9.304 2.359 7.658 0.117
4 4.803 0.375 10.030 1.829 8.467 0.239
6 5.340 0.508 11.410 2.993 9.674 0.318
8 5.674 0.811 12.083 1.433 10.695 0.454
12 6.245 1.052 13.425 1.819 12.295 0.983
16 7.293 1.041 14.500 3.344 13.666 1.722
20 8.106 1.145 15.849 4.607 14.763 2.476
24 9.234 1.782 16.891 5.641 16.337 3.118
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4. In vitro skin permeation studies using MN + EP + SN
4.1 Skin permeation of FD-4
Table 12 The cumulative amount of FD-4 permeated using all enhancement methods
Time (h)
Cumulative amount of FD-4 permeated (μg/cm2) Passive
diffusion MN EP SN MN + EP MN + SN EP + SN MN + EP + SN
AVG S.E. AVG S.E. AVG S.E. AVG S.E. AVG S.E. AVG S.E. AVG S.E. AVG S.E.
0.083 N/A N/A N/A N/A N/A N/A 6.95 2.738 N/A N/A 5.575 0.369 N/A N/A N/A N/A
0.25 N/A N/A N/A N/A N/A N/A 7.602 1.926 N/A N/A 6.182 0.545 5.851 0.292 N/A N/A
0.5 N/A N/A N/A N/A N/A N/A 7.898 1.824 N/A N/A 6.249 1.245 6.379 0.248 3.631 3.384
1 N/A N/A N/A N/A N/A N/A 8.384 2.469 5.707 0.587 8.182 1.509 6.894 0.215 7.020 4.855
2 N/A N/A N/A N/A N/A N/A 9.304 2.359 8.952 2.476 14.633 3.727 7.658 0.117 18.954 14.340
4 N/A N/A 8.480 1.870 4.803 0.375 10.030 1.829 19.902 3.951 28.496 10.757 8.467 0.239 49.442 32.646
6 N/A N/A 13.300 2.970 5.340 0.508 11.410 2.993 32.538 5.152 40.120 16.301 9.674 0.318 79.769 38.523
8 N/A N/A 18.960 4.370 5.674 0.811 12.083 1.433 50.222 8.318 49.451 20.777 10.695 0.454 116.351 44.329
12 N/A N/A 29.360 6.900 6.245 1.052 13.425 1.819 77.444 16.309 64.411 26.802 12.295 0.983 168.106 38.707
16 N/A N/A 37.790 8.850 7.293 1.041 14.500 3.344 107.005 26.189 77.929 28.563 13.666 1.722 244.412 16.097
20 N/A N/A 45.350 10.610 8.106 1.145 15.849 4.607 144.640 31.950 92.848 29.009 14.763 2.476 286.584 29.503
24 N/A N/A 51.390 12.380 9.234 1.782 16.891 5.641 184.929 38.603 106.732 32.218 16.337 3.118 342.776 42.218
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4.2 Skin permeation of FD-4, FD-20, and FD-40
Table 13 The cumulative amount of FD permeated
Time (h)
Cumulative amount of FD permeated (μg/cm2)
FD-4 FD-20 FD-40
AVG S.E. AVG S.E. AVG S.E.
0.083 N/A N/A 4.121 1.735 1.927 2.475
0.25 N/A N/A 4.593 1.521 2.206 2.223
0.5 3.631 3.384 4.491 1.231 2.179 2.213
1 7.020 4.855 5.717 1.667 2.333 2.201
2 18.954 14.340 8.353 2.272 2.234 2.133
4 49.442 32.646 16.158 4.054 2.647 2.394
6 79.769 38.523 22.569 5.686 3.650 2.438
8 116.351 44.329 29.547 6.598 5.101 2.685
12 168.106 38.707 56.176 11.326 14.196 4.531
16 244.412 16.097 67.626 12.875 18.734 5.175
20 286.584 29.503 88.324 18.329 28.358 6.438
24 342.776 42.218 101.708 16.684 38.622 7.708
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CLSM studies
1. MN-treated skin
Figure 25 CLSM image of MN-treated skin at different depth representing
fluorescent signal observed on the skin surface
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2. MN + EP-treated skin
Figure 26 CLSM image of MN + EP-treated skin at different depth representing
fluorescent signal observed on the skin surface
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3. MN + SN-treated skin
Figure 27 CLSM image of MN + SN-treated skin at different depth representing
fluorescent signal observed on the skin surface
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4. MN + EP + SN-treated skin
Figure 28 CLSM image of MN + EP + SN-treated skin at different depth representing
fluorescent signal observed on the skin surface
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BIOGRAPHY
Name Maleenart Petchsangsai, Miss.
Date of Birth April 28, 1982
Place of Birth Bangkok, Thailand
Nationality/ Religion Thai/Buddhism
E-mail address [email protected]
Education
2010 - 2014 Doctor of Philosophy, Ph.D. (Pharmaceutical
Technology), Silpakorn University, Nakhon Pathom,
Thailand
2005 - 2007 Master of Pharmacy (Pharmaceutical Technology)
Silpakorn University, Nakhon Pathom, Thailand.
2000 - 2005 Bachelor of Pharmacy (Pharmaceutical Technology)
Silpakorn University, Nakhon Pathom, Thailand.
1997 - 2000 Mahidolwittayanusorn School, Nakhon Pathom,
Thailand
1994 - 1997 Sarawittaya School, Bangkok, Thailand.
Scholarships
2011 - 2013 The Royal Golden Jubilee Ph.D. Program from
Thailand Research Funds
2005 - 2007 Thailand Graduate Institute of Science and Technology
Scholarship (TGIST) from National Nanotechnology
Center (NANOTEC)
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Certificate
2004 “Emerging Science and Profession in Pharmacy” the
20th FAPA congress
Presentations
Poster presentations
1. Praneet Opanasopit, Suwannee Panomsuk, Tanasait Ngawhirunpat, Anurak
Khuntapol, Monthinee Kongsawatsak, Maleenart Petchsangsai, Suwipa
Tangsombatpaiboon, Saowaluk Chuchauy. Development of Isosorbide Dinitrate
Pressure Sensitive Adhesive for transdermal Delivery Systems. FAPA 20th
Congress, November 30 - December 3, 2004, Bangkok, Thailand.
2. Maleenart Petchsangsai, Praneet Opanasopit, Tanasait Ngawhirunpat, Theerasak
Rojanarata, Auayporn Apirakaramwong, Uracha Ruktanonchai, Warayuth Sajomsang,
Supawan Tantayanon. Development of a novel gene delivery carrier using
methylated N-(4-N,N-dimethylaminobenzyl) chitosans. Proceedings of the 23th
Annual Research Conference in Pharmaceutical Sciencs & JSPS 1st Medicinal
Chemistry Seminar of Asia/Africa Science Platform Program, December 14 - 15,
2006, Bangkok, Thailand.
3. Praneet Opanasopit, Maleenart Petchsangsai, Tanasait Ngawhirunpat, Theerasak
Rojanarata, Auayporn Apirakaramwong, Uracha Ruktanonchai, Warayuth Sajomsang,
Supawan Tantayanon. Development of a novel gene carrier using water soluble
chitosan derivatives. NSTDA Annual Conference, March 28-30, 2007, Thailand
Science Park, Pathumthani, Thailand.
4. Maleenart Petchsangsai, Praneet Opanasopit, Tanasait Ngawhirunpat, Theerasak
Rojanatara, Auayporn Apirakaramwong, Uracha Ruktanonchai, Warayuth Sajomsang
Development of a novel gene carrier using methylated N-(4-N,N-
dimethylaminobenzyl) chitosans. Innovation in drug delivery from biomaterials to
devices, September 30 – October 3, 2007, Naples, Italy.
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5. Maleenart Petchsangsai, Praneet Opanasopit, Theerasak Rojanarata, Tanasait
Ngawhirunpat Using hollow microneedle for in vitro transdermal delivery of
bovine serum albumin-fluorescein isothiocyanate conjugate. Chiangmai
international conference on biomaterials & applications (CMICBA 2011), August 9 -
10, 2011, Chiangmai, Thailand.
Oral presentations
1. Maleenart Petchsangsai, Warayuth Sajomsang, Pattarapond Gonil, Uracha
Ruktanonchai. Physicochemical properties of nanocomplexes between diclofenac
sodium and chitosan derivatives as a new drug carrier : effect of degree of
substitution. Asian Aerosol Conference AAC09, November 24 - 27, 2009, Bangkok,
Thailand.
2. Maleenart Petchsangsai, Warayuth Sajomsang, Pattarapond Gonil, Uracha
Ruktanonchai. Physicochemical study of nanocomplexes between diclofenac
sodium and three types of chitosan derivatives as a new drug carrier. 4th
International Symposium on Biomedical Engineering, IEEE ISBME 2009, December
14 - 18, 2009, Plaza Athenee Bangkok - A Royal Meridien Hotel, Bangkok, Thailand.
Proceeding
1. Maleenart Petchsangsai, Nantida Wonglertnirant, Theerasak Rojanarata, Praneet
Opanasopit, Tanasait Ngawhirunpat (2012). Microneedles-mediated transdermal
delivery. World Academy of Science, Engineering and Technology, 69, 505-508.
Publications
1. Theerasak Rojanarata, Maleenart Petchsangsai, Praneet Opanasopit, Tanasait
Ngawhirunpat, Uracha Rungsardthong Ruktanonchai, Warayuth Sajomsang,
Supawan Tantayanon (2008). Methylated N-(4-N,N-dimethylaminobenzyl) chitosan
for novel effective gene carriers. European Journal of Pharmaceutics and
Biopharmaceutics, 70, 207–214.
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2. Praneet Opanasopit, Maleenart Petchsangsai, Theerasak Rojanarata, Tanasait
Ngawhirunpat, Warayuth Sajomsang, Uracha Rungsardthong Ruktanonchai (2009).
Methylated N-(4-N,N-dimethylaminobenzyl) chitosan as effective gene carriers:
Effect of degree of substitution. Carbohydrate Polymers, 75, 143149.
3. Maleenart Petchsangsai, Nantida Wonglertnirant, Theerasak Rojanarata, Praneet
Opanasopit, Tanasait Ngawhirunpat (2011). Application of hollow microneedle for
transdermal delivery of bovine serum albumin-fluorescein isothiocyanate conjugate.
Advanced Materials Research, 338, 365-368.
4. Maleenart Petchsangsai, Warayuth Sajomsang, Pattarapond Gonil, Onanong
Nuchuchua, Boonsong Sutapun, Satit Puttipipatkhachorn, Uracha Rungsardthong
Ruktanonchai (2011). A water-soluble methylated N-(4-N,N-dimethylaminocinnamyl)
chitosan chloride as novel mucoadhesive polymeric nanocomplexplat form for
sustained-release drug delivery. Carbohydrate Polymers, 83, 1263–1273.
5. Warayuth Sajomsang, Pattarapond Gonil, Uracha Rungsardthong Ruktanonchai,
Maleenart Petchsangsai, Praneet Opanasopit, Satit Puttipipatkhachorn (2013).
Effects of molecular weight and pyridinium moiety on water-soluble chitosan
derivatives for mediated gene delivery. Carbohydrate Polymers, 91, 508– 517.
6. Maleenart Petchsangsai, Theerasak Rojanarata, Praneet Opanasopit, and Tanasait
Ngawhirunpat (2014). The Combination of Microneedles with Electroporation and
Sonophoresis to Enhance Hydrophilic Macromolecule Skin Penetration. Biological
and Pharmaceutical Bulletin, 37(8). 1373–1382.
Patent
June 2007 Trimethylated N-(4-N,N-dimethylaminobenzyl) chitosan and synthetic
method (Praneet Opanasopit, Uracha Ruktanonchai, Warayuth
Sajomsang, Maleenart Petchsangsai)
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Relevant courses
2004 Senior project
“Development of isosorbide dinitrate pressure sensitive adhesive for
transdermal delivery systems”
2005 – 2007 Thesis (Master degree)
“Development of a novel gene carrier using water soluble chitosan
derivatives”
Work experiences
Full-time employment
July 2007 - Mar 2008 Thammasat University Hospital: Clinical pharmacist
Apr 2008 - Aug 2008 Life Sci-Cosmetics Research Center: R&D Manager in
Clinical Research Assessment of Cosmetic product
Sep 2008 - Oct 2011 National Nanotechnology Center: Research assistant
(Nano Delivery System Laboratory)
Research collaboration
Jan 2010 - Apr 2010 School of Optometry, Indiana University,
Bloomington, IN, USA.
Jan 2014 - Aug 2014 University of Colorado Anschutz Medical Campus,
Aurora, CO, USA.
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