SKIN PENETRATION ENHANCEMENT OF HYDROPHILIC … · ELECTROPORATION AND SONOPHORESIS. THESIS ADVISORS : ASSOC. PROF. TANASAIT NGAWHIRUNPAT, Ph.D., AND ASSOC. PROF. PRANEET OPANASOPIT,

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

สำนกัหอ

สมุดกลาง

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



การเพิม่การซึมผ่านผวิหนงัของสารโมเลกลุใหญ่ที่ชอบนํา้โดยใช้ไมโครนีเดิลส์ร่วมกบั อเิลก็โทรพอเรชันและซอนอฟอรีซิส

โดย นางสาวมาลนีาฎ เพชรแสงใส

วทิยานิพนธ์นีเ้ป็นส่วนหนึง่ของการศึกษาตามหลกัสูตรปริญญาเภสัชศาสตรดุษฎีบัณฑติ

สาขาวชิาเทคโนโลยเีภสัชกรรม บัณฑติวทิยาลยั มหาวทิยาลยัศิลปากร

ปีการศึกษา 2557 ลขิสิทธิ์ของบัณฑติวทิยาลยั มหาวทิยาลยัศิลปากร



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.) ............../................./.............



iv

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. ...........................



v

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. .............................



vi

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.



vii

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



viii

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



ix

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



x

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


27 CLSM image of MN + SN-treated skin at different depth representing


28 CLSM image of MN + EP + SN-treated skin at different depth

representing fluorescent signal observed on the skin surface....... 112



xi

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



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



xii

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)



1

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



2

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,



3

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



4

(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



5

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



6

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



7

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

of hydrophilic macromolecules through the skin barrier. สำนกัหอ


8

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



9

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



10

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)



11

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.



12

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).



13

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.



14

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



15

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)



16

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



17

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).



18

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



19

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).



20

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).



21

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).



22

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



23

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



24

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



25

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



26

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



27

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).



28

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.



29

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



30

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



31

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



32

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).



33

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



34

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



35

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.



36

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



37

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.



38

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



39

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



40

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-



41

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).



42

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).



43

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.



44

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-



45

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).



46

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



47

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



48

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



49

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.


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.



50

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.



51

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



52

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



53

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).



54

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.



55

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



56

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.



57

CHAPTER 4

RESULTS AND DISCUSSION

4.1 In vitro skin permeation studies using MN


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



58

4.1 In vitro skin permeation studies using MN


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



59

(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.



60

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



61

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.



62

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.



63

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).



64

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 ().



65

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).



66

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



67

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.



68

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.



69

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 +



70

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.



71

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.



72

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



73

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



74

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).



75

Figure 18 In vitro permeation profiles of FD with various MW across porcine skin:

FD-4 (), FD-20 (), and FD-40 ().



76

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).



77

Figure 19 CLSM image of untreated skin (passive delivery) at different depth

representing fluorescent signal observed on the skin surface



78

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



79

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)



80

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



81

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.



82

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.



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.



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.



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.



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.



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


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.



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


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.



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.



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.



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



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.



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.



APPENDIX



APPENDIX A



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)




Standard : FD-20




Concentration (μg/mL) : 0.15, 0.215, 1.0, 2.0, 3.0


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)




Standard : FD-40




Concentration (μg/mL) : 0.1, 0.15, 0.215, 1.0, 2.0, 3.0, 4.0


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)



APPENDIX B



100

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



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.



101

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



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



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



102

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)


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.




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



103

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



104

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)


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



105

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)


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



106

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



107

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



APPENDIX C



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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



110

2. MN + EP-treated skin

Figure 26 CLSM image of MN + EP-treated skin at different depth representing




111

3. MN + SN-treated skin

Figure 27 CLSM image of MN + SN-treated skin at different depth representing




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4. MN + EP + SN-treated skin

Figure 28 CLSM image of MN + EP + SN-treated skin at different depth representing




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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.



115

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.



116

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)



117

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.



Documents

SKIN PENETRATION ENHANCEMENT OF HYDROPHILIC … · ELECTROPORATION AND SONOPHORESIS. THESIS ADVISORS : ASSOC. PROF. TANASAIT NGAWHIRUNPAT, Ph.D., AND ASSOC. PROF. PRANEET OPANASOPIT,