Controlled Drug Delivery via Carbon Nanotube

By Chia-Hsuan Wu

M.S., National Taiwan Normal University, 2004

B.S., National Taiwan Normal University, 2001

A Dissertation Submitted in Partial Fulfillment of the

Requirements for the Degree of Doctor of Philosophy

in the School of Engineering at Brown University

Providence, Rhode Island

May 2012

© Copyright

by

Chia-Hsuan Wu

2012

iii

This dissertation by Chia-Hsuan Wu is accepted in its present form

by the School of Engineering as satisfying the

dissertation requirement for the degree of

Doctor of Philosophy.

Recommended to the Graduate Council

Date_____________ _________________________________

Date_____________ ________________________________

Date_____________ _______________________________

Date_____________ ________________________________

Date_____________ ________________________________

Approved by the Graduate Council

Date_____________ _________________________________

Professor Jimmy Xu, Advisor

Professor John Marshall, Reader

Professor Anubhav Tripathi, Reader

Peter M. Weber,

Dean of the Graduate School

Professor Robert Hurt, Reader

Dr. Harold J. Wanebo, Reader

iv

Curriculum Vitae of Chia-Hsuan Wu

Chia-Hsuan Wu was born in Taichung, Taiwan (Republic of China) on November

17th

, 1978. She received the degrees Bachelor of Science, National Taiwan Normal

University, 2001 and Master of Science, National Taiwan Normal University, 2004. In

year 2004, after obtaining her Master degree, she worked full time in National Taiwan

Normal University as research assistant focusing on utilizing nanomaterials in biomedical

applications. In year 2005, she began her doctoral studies at Brown University’s

Laboratory of Emerging Technologies under the supervision of Professor Jimmy Xu.

Her doctoral work spanned Biomedical Engineering and Chemistry resulting in scientific

contributions to both of the fundamental scientific disciplines.

Chia-Hsuan Wu’s publications are listed below:

C.H. Wu, C. Cao, J.H. Kim, C.H. Hsu, W.D. Bowen, H.J. Wanebo, J.Xu & J.

Marshall, Trojan-Horse Nanotube On-Command Drug Delivery and Kill of

Pancreatic Cancer Cells. Science (in preparation)

J.H. Kim, G. Withey, C.H. Wu & J.Xu, Carbon Nanotube Array for Addressable

Nano-Bioelectronic Transducers. IEEE Sensors Journal, 11, 1274-1283 (2011)

C.C. Chen, Y.P. Lin, C.W. Wang, H.C. Tzeng, C.H. Wu, Y.C. Chen, C.P. Chen, L.C.

Chen & Y.C. Wu, DNA-Gold Nanorod Conjugates for Remote Control of

v

Localized Gene Expression by Near Infrared Irradiation. J. Am. Chem. Soc., 128,

3709-3715 (2006).

C.Y. Lai, C.H. Wu, C.C. Chen & P.C. Li, Quantitative Relations of Acoustic Inertial

Cavitation with Sonoporation and Cell Viability. Ultrasound Med. Biol., 32,

1931-1941 (2006).

C.C. Chen, Y.C. Liu, C.H. Wu, C.C. Yeh, M.T. Su & Y.C. Wu, Preparation of

Fluorescent Silica Nanotubes and their Application in Gene Delivery. Adv. Mater.,

17, 404-407 (2005).

vi

Acknowledgements

I would like to thank my supervisor Professor Jimmy Xu for his support and mentoring

throughout my time in his lab. He always wanted us to be creative and carefully

seeking for opportunities in exploring new technologies. Under his supervision, I was

trained to think out of the box and therefore we were able to develop the revolutionary

Trojan-Horse carbon nanotube system.

I want to salute all my co-workers and friends in the lab. On the science front, I want to

acknowledge Chih-Hsun Hsu, Jin Ho Kim, Hongsik Park, Gary Withey, Aijun Yin,

Gustavo Fernandes and Lyuba Kuznetsova for their contribution to different aspects of

this work.

vii

Contents

Chapter 1 Introduction 1

1.1 Preface........................................................................................................... 1

1.2 Background................................................................................................. 4

1.2.1 Nanotechnology for Drug Delivery ………………………………………….. 4

1.2.2 Carbon Nanotubes for Drug Delivery ……………………………………….. 5

1.3 Thesis Overview.......................................................................................... 8

Chapter 2 Single Walled Carbon Nanotube as Biomolecule

Delivery Vehicle

10

2.1 Background……………………………………………………………….. 10

2.2 Conjugating Biomolecules to Single Walled Carbon Nanotubes ………... 12

2.2.1 SWNT surface functionalization …..………………………………………….. 12

2.2.2 siRNA conjugated to CNT with enzyme-cleavable disulfide bond ……….. 14

2.3 Conjugate Biocompatibility, Delivery, and Characterization ………….. 16

2.4 Preservation of the Delivered Biomolecule’s Bioactivity ……………… 20

Chapter 3 Trojan-Horse Multiwalled Carbon Nanotube

on-command Delivery of Chemotherapeutic Drugs

23

3.1 Background…………………………………………….………………….. 23

3.1.1 Nanotechnology for Drug Delivery .………………………………………….. 23

3.1.2 Trojan Horse Multiwalled Carbon Nanotubes …………………………….. 25

3.1.3 Chemotherapy of Pancreatic Cancer ……………………………………….. 26

3.2 Loading and On-Command Release of Drugs by Trojan-Horse CNTs .... 27

3.2.1 Synthesis of CNTs by anodic aluminum oxide (AAO) template …..……….. 27

viii

3.2.2 Drug Loading using Temperature Sensitive Gelatin …..…………………. 30

3.3 On-Command Drug Release of Trojan-Horse CNTs by Induction Heating 34

3.3.1 Induction Heating of Trojan-Horse CNTs…………………………………….. 34

3.3.2 Payload Release upon the Exposure to A.C. Magnetic Field …………….. 35

3.3.3 Chemo-Sensitization Effects of C6 Ceramide with Taxol on

Pancreatic Cancer……………………………………………….

35

3.3.4 Internalization of Trojan-Horse CNTs and on-command therapeutic

treatment to pancreatic cancer cells in vitro ……………………….

38

3.4 Discussion and Conclusion ……………………………………………… 41

Chapter 4 Future Horizons 45

4.1 RNAi Neural Delivery via SWNT ……………………………………….. 45

4.2 Trojan-Horse CNT for in vivo application …………………………...... 48

Chapter 5 Conclusions 50

Appendix Induction Heating of Trojan-Horse CNTs 53

xiii

List of Figures

2-1 SWNTs coated with ssDNA or PL-PEG-NH2 (PL=phospholipid,

PEG=poly(ethylene glycol)) and then conjugated with (a) a fluorescent

label or (b) a thiol-siRNA (via a disulfide linker). A structure model of

DNA-wrapped carbon nanotube on the right (c) atomic force microscopy

(AFM) image showing the structural properties of a

SWNT-ssDNA-AlexaFluor594 conjugate; green arrow shows the height

of SWNT, and blue arrow shows the periodic structure with an identical

pitch width (d) bundled SWNT as control (e) Soluble SWNT modified

with green fluorescent tag under UV light (AFM done by Dr. Hongsik

Park).………………………………………………………………………. 16

2-2 (a-c) Confocal fluorescence images of HeLa cells after incubation with

SWNT-PL-PEG- AlexaFluor 594 conjugates. Cell membranes stained

green with fluorescent wheat germ agglutinin. SWNT conjugates in red.

The fact that the red signal is confined within the green cell membranes

indicates that the SWNT conjugates were internalized by the HeLa cells.

(b) a series of confocal images (z-stack) taken at different focal planes

(starting at the top and moving down through the cells). (c) the control

cells incubated with free AlexaFlour594 (no uptake seen).. .……………... 17

2-3 Detecting SWNTs in living 293T cells using characteristic Raman

signatures of SWNTs. (a) Raman spectra were collected from control cells

(black), SWNT conjugates (green), and cells incubated with SWNT

conjugates (red). Note the G band of the SWNTs at 1592cm-1. (b) the

live cell image from the Raman spectrometer microscope (the red cross

indicates where the Raman spectra were collected).………………………. 19

2-4 Confocal fluorescence images of HEK293 cells after incubation with

SWNT-PL-PEG- AlexaFluor 488 conjugates. Cell nucleus stained blue

with DAPI. SWNT conjugates in green. The fact that the green signal

is located in perinuclear space indicates that the SWNT conjugates were

internalized by the HEK293 cells.……..………………………………....... 20

2-5 Epifluorescence microscopy siRNA assay for (a) untreated control

293T-EGFP cells and (b) 293T-EGFP cells incubated with

SWNT-PL-PEG-siRNA conjugates. (b) shows weaker fluorescence than

(a) due to the silencing of EGFP by the released siRNA…...…………....... 22

3-1 The fabrication process of template synthesized multiwalled carbon

nanotubes. (a) the Anodic Aluminum Oxide (AAO) nanopore array

template, a photo of an AAO template (with a ruler for scale), an AFM

image of the AAO surface, and SEM images of different pore sizes and

periodicities, (b) CNT array formation, a photo of a CNT array (with a

ruler for scale), and an SEM image of a CNT array (Fabrication team in

Xu Lab).………........................................................................................... 29

xiv

3-2 Loading and on-command release of Trojan-Horse nanotubes. (a)

Schematic illustration of filling QDs/gelatin/drug into nanotubes. The

payload is loaded into the nanotubes array by the vacuum suction at the

back side of template followed by the release of Trojan-Horse carbon

nanotube from template by chemical dissolution of AAO template.

Finally, the payload is released from the Trojan-Horse nanotubes by

exposing nanotubes to the a.c. magnetic field (b) (left) TEM image

showing the high loading yield of QDs into nanotubes. (right) A magnified

TEM micrograph showing the QDs with diameter ~ 4nm are inside the

nanotubes. (c) QD loaded nanotube array was immersed in water and

subjected to the application of a.c. magnetic field for 30 min. Thereafter,

the solution was drawn under epifluorescence microscope. (left) Without

a.c. magnetic field, no luminescence was observed. (right) With a.c.

magnetic field on, the significant green luminescent signal, QD

luminescence emission wavelength band, was observed in the solution,

suggesting successful demonstration of on-command release of

QD-gelatin via the induction heating of CNT.…………………………… 32

3-3 Cell permeable short-chain C6 ceramide sensitizes Taxol induced

pancreatic cancer cell death in vitro. (a) MTT assay was utilized to test

L3.6 pancreatic cancer cell viability after a combination of different

concentration of Taxol and C6 ceramide treatment for 48 hours. Though

C6 ceramide by itself had almost no effects on L 3.6 cell viability, (b) it

dramatically enhanced Taxol induced L 3.6 cell death (cell death was

reflected by reduced cell viability/MTT OD). Note that a relative high

concentration of C6 ceramide (5-10 µg/ml) is needed to reach the

chemo-sensitization effect. (c) The chemo-sensitization effect of C6

ceramide was also seen in two additional pancreatic cancer cell lines

PANC-1 and (d) MIA PaCa-2 cells. The experiments in this figure were

repeated at least three times and similar results were obtained.

*P<0.05…………………………………………………………………….. 37

3-4 Internalization of Trojan-Horse carbon nanotubes and on-command

therapeutic treatment to pancreatic cancer cells in vitro. (a) A confocal

fluorescence image in plan-view of L3.6 pancreatic cancer cells taken

after the incubation with TexasRed-labelled Trojan-Horse carbon

nanotubes for overnight. The Trojan-Horse carbon nanotubes (red signal)

reach the perinuclear space indicates the nanotubes were internalized by

the pancreatic cancer. Cell nuclei were stained in blue with Hoechst 33342

(Invitrogen). (b) As all imaging slices recorded along a whole cell in the z

direction, the three-dimensional imaging of intracellular distribution of

TexasRed-labelled CNTs was demonstrated. From the cross-sectional

views of cells both vertically (b) and horizontally (c), TexasRed-labelled

CNTs were confirmed to be internalized inside pancreatic cancer cells

(a-c). (d) They were subjected to a.c. magnetic field for on-command drug

(C6 ceramide+Taxol) release. The cell morphology of pancreatic cancer

cells (L 3.6) were observed at different conditions. Significant cell death

xv

was observed only when Trojan-horse CNTs were subjected to a.c.

magnetic field. (e) Cell viability study by an in vitro cytotoxicity assay

(MTT) 48 hours after treatment. With a.c. magnetic field on, the

Trojan-Horse CNTs resulted in over 70% cell death with the release of

Taxol and C6 ceramide. (f) Histone-DNA ELISA assay was also

performed to test L3.6 cells apoptosis at different conditions after 36

hours treatments, note that only the group 6’ (CNT+Taxol(0.03)+C6

Ceramide(0.1)) with on-command a.c. magnetic field on had increased

Histone-DNA ELISA OD (indicator of cell apoptosis), which is

comparable with group received exogenous C6 ceramide plus Taxol. (g) L

3.6 cell apoptosis was also reflected by increased cleavage of caspase-3.

Phosphorylation (Ser 473) and total level of Akt was also tested………….

4-1 Internalization of SNWT by neurons in vitro. (a) A bright field image of

neurons. (b) A confocal fluorescence image in plan-view of neurons taken

after the incubation with TexasRed-labelled SWNT for overnight. The

SWNT (red signal) reach the perinuclear space indicates the nanotubes

were internalized by the neurons. Cell nuclei were stained in blue with

DAPI. (c) As all imaging slices recorded along a whole cell in the z

direction, the three-dimensional imaging of intracellular distribution of

red SWNT was demonstrated. From the cross-sectional views of cells

both vertically (c) and horizontally (d), red SWNTs were confirmed to be

internalized inside neurons (b-d)…………………………………………... 47

4-2 The proposed simultaneously drug encapsulation and surface

functionalization of Trojan-Horse carbon nantubes to achieve specific

targeting of cells and subsequently on-demand drug release……………… 48

40

1

CHAPTER 1

INTRODUCTION

1.1 Preface

Having witnessed the advancements and developments in nanotechnology over the past

decade, it‟s always a natural question being raised: “What can Nano do for drug delivery

in the next decade?” Although nanotechnology itself is indeed not mature at the current

stage, it opens up tremendous opportunities by teaming up with various scientific

disciplines and therefore promotes such rapid advancements.

Conventional target drug delivery is usually made achievable by chemical

functionalization of the polymer microsphere and liposomes to improve efficacy and side

effects. Such drug delivery vehicles commonly suffer the uncontrolled release of drugs

that limit their clinical applications.

In this dissertation, we dedicated using carbon nanotubes as the drug delivery vehicle in

order to develop systems that have high therapeutic efficiency and yet have minimum

harm to the health cells. Our first task was to develop an effective interface for nano-bio

2

medicine by carbon nanotubes. Two nanotube systems, namely single walled carbon

nanotubes (SWNTs) and multi walled carbon nanotubes (MWNTs) were included in this

study. Our first goal was to chemically functionalize the CNTs and make them water

soluble since CNTs are intrinsically hydrophobic. Two different approaches were

carried out to accomplish the tasks. CNTs were non-covalently functionalized with

different amphiphilic macromolecules that render them hydrophilic, e.g. amine-

terminated polyethylene glycol phospholipids (PL-PEG-NH2) and single-strand DNA.

The covalent functionalization method was achieved by strong acid treatment to generate

carboxylic acid groups. Both types of chemical functionalization increase the CNT‟s

dispersibility in aqueous solutions.

Our second task was to develop methods for reliable and controllable release of the drug

by both proposed systems. In the SWNT system, by incorporating an enzymatic

cleavable disulfide bond, we show that therapeutic cargos are only released inside the

cytosol upon cellular internalization. In the MWNT system, we encapsulate the

therapeutic drugs to perform the controlled release in vitro by an external stimulus using

a.c. magnetic field, which has the advantage of better penetration depth, easy

accessibility and the perfect nano-vial for drug protection.

3

As mentioned in the beginning, the beauty of the Nano-Drug Delivery research is the

interdisciplinary nature of it. This dissertation essentially covers materials science,

physics, chemistry, genetics and molecular pharmacology. We wish the reported

research using two carbon nanotube systems will spark ideas on more innovative

developments of nano drug delivery.

4

1.2 Background

1.2.1 Nanotechnology for Drug Delivery

Since Nobel Prize winner Paul Ehrlich proposed the “magic bullet” concept for effective

drug delivery, in the past century, people have been dedicated to develop such an ideal

drug delivery system that selectively delivers to target sites in the body and release drug

cargo in a controlled manner1. Nanotechnology sheds light on the realization of this

ideal drug delivery system applicable to various disease treatments. Conventional drug

delivery systems often result in many undesired side effects to normal cells because it

circulates through the whole body. By taking the advantage of the multivalency of

nanomaterials, the use of nanomaterials allows much better localization of therapies to

the targeted cells with reduced side effects. Through the chemical functionalization of

nanomaterials, the therapeutic agents are able to reach target with enhanced drug

absorption, and respond to changes in pH, enzyme catalysis, and temperature in the

biological environment2-4

.

5

1.2.2 Carbon Nanotubes for Drug Delivery

The carbon nanotube (CNT) is a tubular structure which essentially is a rolled up form of

graphite sheet. People classify CNTs into two general categories, based on the number of

graphite sheet(s): single-walled nanotubes (SWNTs), which consist of single layer of

graphene and multiwalled nanotubes (MWNTs), which are in the form of several

graphite sheets. Typically, SWNTs have diameters from 0.4 to 2.0 nm and MWNTs

varies from 2–100 nm in diameter. Nanotubes exhibit many remarkable properties that

make it a great candidate for drug delivery. Below is listed a few unique properties we

specificially utilized in this thesis.

a. Biocompatibility: CNTs are composed of carbon that essentially has higher

biocompatibility than inorganic CdSe quantum dots. Moreover, with suitable

chemical functionalization, CNTs are more biocompatible than pristine CNTs.

b. High aspect ratio: Literature demonstrated that the rate of internalization of

nanomaterials is highly increased when their aspect ratio are high5. Also, they

would possess a prolonged circulation time as a drug delivery carrier.

c. Ease of chemical functionalization: An example is given in Chapter 2 that we

conjugated SWNTs with amine-terminated single-stranded DNA (ssDNA)

6

resulting in multiple amine terminals immobilized on SWNTs.

d. Semi-metallic property: In Chapter 3, we utilized the semi-metallic property to

achieve the on command drug release by using induction heating. When a

conductor is placed in an a.c. magnetic field, swirls of current (Lenz‟s Law) in the

conductor will be induced, and the induced current subsequently results in

resistive heating, also called induction heating. The same happens to a carbon

nanotube with a drug substance loaded inside.

e. Hollow structure for template synthesized CNTs: Hollow nanostructures as

drug delivery carriers have many exceptional properties. Such protective

enclosure prevents the degradation and leakage of drug during circulation.

f. High surface area: The high surface area enables the multivalency of CNTs

which is crucial to the target drug delivery with high efficacy and low side effects.

g. Size controllable: In Xu lab, the physical dimension of template synthesized

CNTs can be precisely controlled by the fabrication process over a wide range.

One can customize the size in order to optimize the internalization rate,

circulation time, conductivity and drug loading capacity.

h. Unique Raman signatures: SWNT produces unique resonance-enhanced Raman

7

bands at 150–300 cm–1

, 1590–1600 cm–1

, and ~ 2600 cm–1

. As demonstrated in

Chapter 2, we monitored the SWNTs-ssDNA conjugate in the live cells using the

Raman technique.

Please note that we utilized all the above mentioned unique properties to conduct our

research in this dissertation in order to achieve a novel and yet robust method for drug

delivery.

8

1.3 Thesis Overview

Overall, this dissertation describes the development of new platforms for drug delivery

application using carbon nanotubes (CNT). Two CNT systems are studied:

(1) Single-walled carbon nanotube (SWNT) and

(2) Multi-walled carbon nanotube (MWNT).

In the SWNT system, SWNTs are non-covalently functionalized with (PL-PEG-NH2) or

single-strand DNA that render them more hydrophilic and individually dispersed.

Therapeutic molecules, such as short interfering RNA (siRNA) and peptides, are

covalently conjugated onto SWNT with controlled release chemistry. In the MWNT

system, we successfully encapsulated the therapeutic drugs and achieved control release

in vitro by the external stimulus using a.c. magnetic field.

In Chapter 2, well dispersed single walled carbon nanotube and its capacity for

intracellular siRNA and therapeutic peptide delivery are reported. In Chapter 3, we

demonstrate the controlled loading and unloading of drugs from template synthesized

CNTs that resembles Trojan-Horse in function. With the application of induction heating

to CNTs, the on command drug release was achieved. Chapter 4 summarizes some

9

ongoing projects and proposes some future prospects of CNT drug delivery technology

developments. Chapter 5 summarizes all the chapters.

10

CHAPTER 2

Single Walled Carbon Nanotube as

Biomolecule Delivery Vehicle

2.1 Background

Single-walled carbon nanotubes (SWNT) are of both therapeutic and diagnostic value in

biomedical applications. SWNT with the appropriate surface modification can be easily

internalized by cells, which makes them an ideal vehicle for the delivery of various

therapeutic biomolecules, e.g. DNA, si RNA, drugs, peptides, and proteins. Moreover,

by utilizing SWNT intrinsic Raman spectroscopic signatures, we can easily track SWNT

in cells, even for a long time without fluorescent bleaching problems. We utilized these

significant advantages of SWNT for further disease treatment.

11

Non-covalent functionalization of SWNT

It is found that the surface chemistry of nanotubes is critical to their in vivo behavior6. In

the SWNT system, carbon nanotbues were non-covalently functionalized with amine-

terminated polyethylene glycol phospholipids (PL-PEG-NH2)7 or amine-terminated

single-strand DNA8 that render them more hydrophilic and individually dispersed. The

amine functional group allows further covalent conjugation of all variety of biomolecules.

Polyethylene glycol (PEG) is well known for its low reticuloendothelial system (RES)

uptake, relatively fast clearance from organs and excretion from the body. Herein,

amine-terminated polyethylene glycol phospholipids (PL-PEG-NH2) were coated onto

the nanotube‟s side-wall surface by physical adsorption. PEG functionalization enables

prolonged circulation in bloodstream. Another method to non-covalently

functionalization is using single-stranded DNA (ssDNA) as coating surfactant for SWNT.

Aromatic nucleotide bases in ssDNA may be exposed to form π-stacking interactions

with the side-wall of carbon nanotubes.

12

Enzyme catalysis

The task was to develop the methods for reliable and controlled release of the drug.

Since SWNT was known to be internalized by cells by endocytosis pathway which

involves the engulfment of SWNT conjugate into endosomal or lysosomal compartment

of cells. To prevent further degradation inside endosomes/lysosome, active release of

therapeutic cargos was needed. By utilizing the degradation nature of enzyme in the

endosomes/lysosomes, enzymatic cleavable disulfide bond was incorporated into the

SWNT conjugate to achieve intracellular delivery.

2.2 Conjugating Biomolecules to Single Walled Carbon Nanotubes

2.2.1 SWNT surface functionalization:

The mixture of Hipco SWNTs and 1,2-Distearoyl-sn-Glycero

-3-Phosphoethanolamine-N-[Amino(Polyethylene Glycol)2000] (Avanti Polar Lipids)

were sonicated for 3 hours as described7,8

. The phospholipid molecule contains amine

group (PL-PEG-NH2). The concentration of PL-PEG is around 0.1-1 mg/mL.

13

Afterwards, the solution were filtered by a membrane filter (Millipore, pore size 100 nm)

to remove excess phospholipids, rinsed thoroughly with water. The CNT solution was

then centrifuged at 24,000 g for 6 h to remove impurities and bundled nanotubes as

precipitate. The supernatant was collected as the nanotubes present in the supernatant are

expected to be short with tube lengths mainly in the 50-300 nm range. Both AFM and

Raman spectroscopy was utilized to characterize the purity of SWNTs in the final

solution of SWNTs.

Adsorption of amine-terminated polyethylene glycol phospholipids (PL-PEG-NH2) or

amine-terminated single-stranded DNA (ssDNA) to SWNTs results in multiple amine

terminals immobilized on SWNTs (Figure 2.1) that can be used to conjugate a wide

variety of biomolecules to SWNTs. In order to visualize the modified SWNTs, a

fluorescent tag, AlexaFluor 594, was covalently conjugated to both SWNT-PL-PEG and

SWNT-ssDNA. Atomic force microscopy (AFM) was then used to characterize the

structural properties of the SWNT-ssDNA-AlexaFluor594 conjugates (average length ~

150 nm, average diameter ~ 1-2 nm, Figure 2.1c). AFM measurements show that the

SWNT-ssDNA has a uniform periodic structure with a regular pitch.

14

2.2.2 siRNA conjugated to CNT with enzyme-cleavable disulfide bond

In order to controllably release genetic material into cells, a biocleavable SWNT-siRNA

conjugate was constructed. siRNA was bound to SWNTs with a linker containing an

enzyme-cleavable disulfide bond (Figure 2.1b). This linker is a heterobifunctional cross-

linker sulfosuccinimidyl 6-(3-[2- pyridyldithio] propionamido) hexanoate (sulfo-LC-

SPDP, Thermo Fisher Scientific Inc.) used to link an amine-functionalized CNT to a

thiol-containing siRNA. This bond is cleaved by cell enzymes in the cytosol, thereby

releasing and delivering siRNA to the cell.

15

NH―

(a) (b)

Fluorescent Label Linker siRNA

(c)

PL-PEG ׃

NH2

WRAPPED WITH:

OR

ssDNA ׃ single strand DNA terminated with NH2

H ~ 1.5 nm

(d) nm

Cross section

(e) Bundled Soluble Bundled Soluble

Individual SWNT

Bundled SWNT

nm

16

Figure 2.1 SWNTs coated with ssDNA or PL-PEG-NH2 (PL=phospholipid,

PEG=poly(ethylene glycol)) and then conjugated with (a) a fluorescent label or

(b) a thiol-siRNA (via a disulfide linker). A structure model of DNA-wrapped

carbon nanotube on the right (c) atomic force microscopy (AFM) image showing

the structural properties of a SWNT-ssDNA-AlexaFluor594 conjugate; green

arrow shows the height of SWNT, and blue arrow shows the periodic structure

with an identical pitch width (d) bundled SWNT as control (e) Soluble SWNT

modified with green fluorescent tag under UV light (AFM done by Dr. Hongsik

Park).

2.3 Conjugate Biocompatibility, Delivery, and Characterization:

Fluorescently Labeled CNT Internalized by Cells in vitro:

In order to determine the biocompatibility and cell uptake rates of our SWNT-PL-PEG-

AlexaFluor594 and SWNT-ssDNA-AlexaFluor594 conjugates, the conjugates were

incubated with HeLa and 293T cells for 4 h at 37 °C. After incubation, all cells were

washed twice and placed in fresh culture medium. Confocal microscopy and Raman

spectroscopy were used to detect the internalization of SWNT conjugates. In all cases,

confocal microscopy revealed appreciable fluorescence from the SWNTs in the cell

interior. The results from the HeLa cells incubated with SWNT-PL-PEG-AlexaFluor594

are shown in Figure 2.2a. The focal plane slices taken at the top, middle, and bottom of

17

the cell confirm that the signal is indeed coming only from the interior of the cell (Figure

2.2b).

Figure 2.2: (a-c) Confocal fluorescence images of HeLa cells after incubation

with SWNT-PL-PEG- AlexaFluor 594 conjugates. Cell membranes stained green

with fluorescent wheat germ agglutinin. SWNT conjugates in red. The fact that

the red signal is confined within the green cell membranes indicates that the

SWNT conjugates were internalized by the HeLa cells. (b) a series of confocal

bottom

top (c)

(a) (b) Control No fluorophore uptake

18

images (z-stack) taken at different focal planes (starting at the top and moving

down through the cells). (c) the control cells incubated with free AlexaFlour594

(no uptake seen).

In order to confirm that it was indeed the internalization of SWNT conjugates that were

detected (and not free AlexaFluor594 in the conjugate solution), we took advantage of the

unique Raman signal of SWNTs. SWNTs produce strong, resonance-enhanced Raman

bands at 150–300 cm–1

, 1590–1600 cm–1

, and ~ 2600 cm–1

. The SWNT conjugates were

incubated with 293T cells for ~ 4 h. The cells were then washed and placed in fresh PBS

buffer. Raman spectroscopy clearly revealed the existence of SWNTs in the live 293T

cells, showing a high-intensity G-band. The band appears at 1592 cm–1

and is the

spectral signature of graphite and its derivatives (Figure2.3).

19

Figure 2.3: Detecting SWNTs in living 293T cells using characteristic Raman

signatures of SWNTs. (a) Raman spectra were collected from control cells (black),

SWNT conjugates (green), and cells incubated with SWNT conjugates (red). Note

the G band of the SWNTs at 1592cm-1

. (b) the live cell image from the Raman

spectrometer microscope (the red cross indicates where the Raman spectra were

collected).

SWNT G band

(a) (b)

0 500 1000 1500 2000 2500 3000

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

60000

Ra

ma

n I

nte

nsity

Raman Shift (cm-1)

Cell only

CNT @ Cell

CNT

20

2.4 Preservation of the Delivered Biomolecule’s Bioactivity

Having verified the delivery of modified SWNTs to living HEK293 (Human Embryonic

Kidney 293) cells (Figure 2.4), we next moved to controlled release of siRNA from the

modified SWNTs in live cells and investigating their post-delivery silencing efficacy.

Figure 2.4: Confocal fluorescence images of HEK293 cells after incubation with

SWNT-PL-PEG- AlexaFluor 488 conjugates. Cell nucleus stained blue with

DAPI. SWNT conjugates in green. The fact that the green signal is located in

perinuclear space indicates that the SWNT conjugates were internalized by the

HEK293 cells.

21

Released siRNA Silence Target Gene:

PL-PEG was again adsorbed to SWNTs and siRNA was attached to the PEG-PL-SWNTs

with a disulfide bond that can be cleaved by Thioredoxin family. This means that the

siRNA will only be released when the conjugate reaches the cell cytosol. The 22mer-

siRNA sense sequence used against EGFP is 5‟-

Th.GCAAGCUGACCCUGAAGUUCAU (Dharmacon RNA Technologies) designed to

silence the EGFP gene in EGFP-transfected cells. EGFP- transfected 293T cells were

incubated with SWNT-PL-PEG-siRNA conjugates for 24 h. Epifluorescence

microscopy differentiated the significantly weaker fluorescence of the conjugate-treated

cells (Figure 2.5b) from the fluorescence of the untreated cells (Figure 2.5a), indicating

that the PEG-PL-SWNTs were able to effectively release the siRNA once inside the

cytosol. The silencing shown in Figure 2.5b confirms the preserved bioactivity of the

siRNA post-delivery. In other words, our loading, delivery, and release methods do not

compromise the native activity of the siRNA.

22

Figure 2.5: Epifluorescence microscopy siRNA assay for (a) untreated control

293T-EGFP cells and (b) 293T-EGFP cells incubated with SWNT-PL-PEG-

siRNA conjugates. (b) shows weaker fluorescence than (a) due to the silencing of

EGFP by the released siRNA.

(a)

(b)

23

CHAPTER 3

Trojan-Horse Multiwalled Carbon Nanotube

on-command Delivery of Chemotherapeutic Drugs

3.1 Background

3.1.1 Nanotechnology for Drug Delivery

Selective delivery of a therapeutic agent to a target site in the body which is released in a

controlled manner is a much sought-after method of delivery. Through advancements in

nanotechnology, functionalized nanomaterials are shown to be able to deliver therapeutic

agents to target cells with enhanced drug absorption, and respond to different biology

environmental stimuli of the target site.

Furthermore, external stimuli such as laser, magnetic field, and electrical field offer

active means to controlled release9-11

. In cancer therapy, electromagnetic field induced

heating by magnetic nanoparticle or mechanical force generated by magnetic micro disks

24

are utilized for physical destruction of cancer cells12-13

. These physical destruction

methods lack of the ability of carrying therapeutic agent into the target and limited their

therapeutic applications. In addition, even if one could chemically attach payloads on the

outside of the nanostructure, it would cause loss and degradation of the drug and allows

the drug to damage the surrounding biological environments during transport. It remains

challenging to encapsulate toxic compounds inside a nanostructure such that the

nanostructure is non-toxic to cells until being subjected to an external release command.

Hence, we propose a „Trojan-Horse‟ carbon nanotubes delivery vehicle for intracellular

delivery of drugs and on-command release. A various types of drug payload can be

encapsulated into the inner cavity of nanotubes together with temperature sensitive

hydrogel. The Trojan-Horse nanotube can precisely perform on-command drug release

through inductive heating in an a.c. or pulsed magnetic field. In this chapter, we show

successful loading of hydrogel/drugs into multiwalled carbon nanotubes, and also

effective on-command release of payload.

25

3.1.2 Trojan Horse Multiwalled Carbon Nanotubes

Carbon nanotubes (CNTs) as drug delivery carriers have many exceptional

physiochemical properties. They are hollow structures, easily internalized by a cell; they

have excellent biocompatibility, high surface area, high aspect ratio and metallic or semi-

metallic behaviour14

. Additionally, the outer surface of the CNT can be chemically

functionalized by target moieties and diagnosis agents. Meanwhile, the drugs can be

encapsulated into the inner cavity of the nanotubes and it the CNT shells prevent those

drugs from premature degradation and interaction with non-target sites. To date, it has

proven challenging to engineer a drug carrier with high loading efficiency while being

able to perform high unloading capacity15. Although fullerenes, metal halides and small

molecules have shown to be possible to be encapsulated into carbon nanotubes16,17

, these

encapsulations usually require high temperature molten-phase loading. In addition, the

selections of cargo are limited only to the molecules with low surface tension which can

be drawn into nanotubes by capillary force or through van der Waals force, which make

it inherently difficult to release the agents from CNTs. Controlled loading and release of

the therapeutic agent becomes unattainable. Development of externally triggered drug

26

release by exploitation of the intrinsic properties of CNTs is imperative for effective drug

release methods18,19

.

Differing from the usual CNTs whose interior space is difficult or even impossible to

access; the CNTs used here were grown in the highly ordered and uniform anodic

aluminum oxide (AAO) nanopore array template by chemical vapor deposition (CVD) 20

.

These CNTs embedded in the template after growth can be opened on both ends with

relatively simple mechanical and chemical treatments, thereby providing an easy

accessibility to the interior for en-mass loading a variety of molecules.

3.1.3 Chemotherapy of Pancreatic Cancer

Pancreatic cancer is a malignant neoplasm with a particularly poor prognosis21

.

Conventional treatments with the chemotherapeutic cytotoxic drugs cause side effects

and often fails to produce an adequate response due to the acquisition of chemo-

resistance. Recent studies demonstrate that cell-permeable ceramides (C6 ceramide)

synergistically increase the efficacy of chemotherapeutic agents such as Taxol (paclitaxel)

and doxorubicin22-24

, and may be a useful adjunct to treating chemo-resistant forms of

cancer. However, the use of ceramide in vivo is limited by its inherent lipid

27

hydrophobicity and physicochemical properties25

. Similarly, Taxol has low aqueous

solubility26

. Therefore, a pharmaceutically more suitable intravenous administration

method is needed.

Here, we report an innovative drug delivery system based on carbon nanotubes (CNTs)

that resembles the Trojan-Horse in function, which can encapsulate toxic compounds in a

hydrogel gelatin within the nanotube. Using this novel approach, we demonstrate

delivery of a low dose combination of ceramide plus Taxol to multi-drug resistance

pancreatic cancer cell lines, that is precisely released on-command by inductive heating

of the nanotubes with an external a.c. or pulsed magnetic field.

3.2 Loading and On-Command Release of Drugs by Trojan-Horse

CNTs

3.2.1 Synthesis of CNTs by anodic aluminum oxide (AAO) template

Differing from the usual CNTs grown by catalytic CVD whose interior space is difficult

or even impossible to access, the CNTs used here were grown in the highly ordered and

uniform anodic aluminum oxide (AAO) nanopore array template by chemical vapor

28

deposition (CVD). We have developed nanofabrication processes for 3D arrays of

carbon nanotubes (CNTs). The nanotube fabrication processes rely upon the highly

ordered, highly uniform anodic aluminum oxide (AAO) nanopore array template

developed by our lab (Figure 3.1). The carbon nanotubes are formed on the inner walls

of the template pores by chemical vapor deposition (CVD, Figure 3.1b). We can control

both the diameters and the spacing of the nanotubes by varying the template pore

diameters and periodicity (Figure 3.1a). The lengths of the CNTs exposed from the

template can be controlled by the wet-etch time (Figure 3.1b). Wet-etching removes the

top surface of the AAO template (but leaves the CNTs untouched). These CNTs

embedded in the template after growth can be opened on both ends with relatively simple

mechanical and chemical treatments, thereby providing an easy accessibility to the

interior for en-mass loading a variety of molecules. The uniformity and tunability of

these nanotube arrays distinguishes them as ideal platforms for the proposed project as

they provide us with millions of identical CNT with the same inner cavity volume and

length for systematic study of drug delivery.

29

Figure 3.1: The fabrication process of template synthesized multiwalled carbon

nanotubes. (a) the Anodic Aluminum Oxide (AAO) nanopore array template, a

photo of an AAO template (with a ruler for scale), an AFM image of the AAO

surface, and SEM images of different pore sizes and periodicities, (b) CNT array

formation, a photo of a CNT array (with a ruler for scale), and an SEM image of

a CNT array (Fabrication team in Xu Lab).

(b)

(a)

30

3.2.2 Drug Loading using Temperature Sensitive Gelatin

Taxol/C6 ceramide loading into CNT arrays. The drug loading in our case was by

applying the mixture of Taxol/C6 ceramide and gelatin (0.2 g/mL, gelatin from porcine

skin, Sigma-Alderich) solution on top of vertical aligned nanotube array and vacuum

suction at the bottom. After loading, the individual CNTs were released from alumina

template by 0.1 M NaOH. The solution was then filtered through filter paper (Millipore,

pore size 50nm) to remove excess NaOH, and wash thoroughly with water. The outer

surface of the CNT was then chemically functionalized to improve its solubility and

compatibility with the biological milieu. To this end, the released CNTs were first

treated with diluted nitric acid to reduce their length to 200-1000 nm. Afterwards, CNTs

were further sonicated with amine-terminated phospholipid-polyethylene glycol (PL-

PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-

2000], Avanti Polar Lipids Inc. ) for 1h. The PL-PEG concentration used was 0.1-1

mg/mL. The mixture was then filtered through filter paper and wash thoroughly with

water or buffer. The PEG-functionalized CNTs were well soluble in buffer solution.

31

The PEG-functionalized CNTs were dispersed onto a copper grid coated with a lacy film

as a sample specimen of transmission electron microscopy (TEM, JEOL JEM-2010F

operated at 200 kV). These CNTs were 60 nm in outer diameter and 40 nm in the inner

diameter. Leica TCS SP2 AOBS spectral confocal microscope was used for the study of

internalization of Trojan-Horse carbon nanotubes.

The drug loading in our case was by applying the molecules solution on top of vertical

aligned nanotube array and vacuum suction at the bottom (Fig. 3.2a). To assess

encapsulation we loaded aqueous Quantum Dot (QD, CdSe/ZnS nanocrystals) into CNT

array, which allowed us to verify the encapsulation of the nano size particles into CNT

cavity under transmission electron microscopy (TEM). After loading, the individual

CNTs were released by dissolving the alumina template for TEM characterization. The

TEM images of these QD-loaded CNTs demonstrate the successful encapsulation as

shown in Fig. 3.2b where QDs are inside the CNT whose interior radius is ~40nm.

32

Figure 3.2: Loading and on-command release of Trojan-Horse nanotubes. (a)

Schematic illustration of filling QDs/gelatin/drug into nanotubes. The payload is

loaded into the nanotubes array by the vacuum suction at the back side of

template followed by the release of Trojan-Horse carbon nanotube from template

by chemical dissolution of AAO template. Finally, the payload is released from

the Trojan-Horse nanotubes by exposing nanotubes to the a.c. magnetic field (b)

(left) TEM image showing the high loading yield of QDs into nanotubes. (right) A

(a)

(b)

a.c. Magnetic Field

(c)

33

magnified TEM micrograph showing the QDs with diameter ~ 4nm are inside the

nanotubes. (c) QD loaded nanotube array was immersed in water and subjected

to the application of a.c. magnetic field for 30 min. Thereafter, the solution was

drawn under epifluorescence microscope. (left) Without a.c. magnetic field, no

luminescence was observed. (right) With a.c. magnetic field on, the significant

green luminescent signal, QD luminescence emission wavelength band, was

observed in the solution, suggesting successful demonstration of on-command

release of QD-gelatin via the induction heating of CNT.

34

3.3 On-Command Drug Release of Trojan-Horse CNTs by Induction

Heating

3.3.1 Induction Heating of Trojan-Horse CNTs

Template-synthesized carbon nanotubes are intrinsically conductive yet have enough

resistivity to make them excellent vehicles for on-command drug delivery via external

induction heating stimulation. When a conductor is placed in an a.c. magnetic field,

swirls of current (Lenz‟s Law) in the conductor will be induced to generate an opposed

magnetic field and the induced current subsequently results in the resistive heating called

induction heating. The same happens to a carbon nanotube with a drug substance loaded

inside. The extent of heating generated in each nanotube is controlled by the applied

magnetic field strength, frequency, and by the tube‟s conductivity and physical

dimension, the former two parameters being field and application adjustable, whereas the

latter two are controllable by our fabrication process.

*Please see the Appendix A.1. for detailed discussion of induction heating theory.

35

3.3.2 Payload Release upon the Exposure to A.C. Magnetic Field

By incorporating a temperature sensitive hydrogel mixed with the drugs of interest, we

can use inductive heating to cause the hydrogel to undergo a gel-sol phase transformation

resulting the release of drug payload out of CNTs. To prove the feasibility of this

Trojan-Horse nanotube system, we tested the response of QD loaded nanotube array

subjected to the application of a.c. magnetic field (25 kHz) in water. As shown in Fig.

3.2c, green luminescent light was only observed when induction heating was applied,

indicative of QD release.

3.3.3 Chemo-Sensitization Effects of C6 Ceramide with Taxol on Pancreatic Cancer

The Trojan-Horse nanotube system could be particularly beneficial for the delivery of

drugs used in cancer chemotherapy that are insoluble or extremely cytotoxic to non-

cancer cells at the therapeutic dose. For example, Paclitaxel, an antimicrotubule agent, is

a commonly used anticancer drug consisting of a mixture of Cremophor EL and ethanol

to overcome poor water solubility. Its application has also been limited due to side-

effects such as hypersensitivity reactions and the acquisition of chemo-resistance27,28

.

Similarly, although recent studies suggest that C6 ceramide can augment the antitumor

36

effect of known chemotherapeutic agents such as Taxol29

, the chemotherapeutic utility of

ceramide is limited because of its insolubility. As a model to demonstrate the utility and

efficacy of CNTs as a new delivery system we chose to focus on the delivery of Taxol

and C6 ceremide for the treatment of pancreatic cancer, an aggressive malignancy with a

five-year mortality of 97–98%. We tested the chemo-sensitization effects of C6

ceramide with Taxol on three different pancreatic cancer cell lines tested, L 3.6, PANC-1

and MIA PaCa-2 cells. Even at high doses, either Taxol or C6 ceramide alone had a

limited effect in killing all three pancreatic cancer cells lines, whereas the combination of

these two agents at a low dose potently produced cell death (Fig. 3.3). These results

provide strong evidence that C6 ceramide dramatically sensitizes pancreatic cells to

Taxol-induced apoptosis and therefore could be a useful adjuvant.

37

Figure 3.3: Cell permeable short-chain C6 ceramide sensitizes Taxol induced

pancreatic cancer cell death in vitro. (a) MTT assay was utilized to test L3.6

pancreatic cancer cell viability after a combination of different concentration of

Taxol and C6 ceramide treatment for 48 hours. Though C6 ceramide by itself had

almost no effects on L 3.6 cell viability, (b) it dramatically enhanced Taxol

induced L 3.6 cell death (cell death was reflected by reduced cell viability/MTT

OD). Note that a relative high concentration of C6 ceramide (5-10 µg/ml) is

needed to reach the chemo-sensitization effect. (c) The chemo-sensitization effect

of C6 ceramide was also seen in two additional pancreatic cancer cell lines

PANC-1 and (d) MIA PaCa-2 cells. The experiments in this figure were repeated

at least three times and similar results were obtained. *P<0.05

(a) (b)

(c) (d)

38

3.3.4 Internalization of Trojan-Horse CNTs and on-command therapeutic

treatment to pancreatic cancer cells in vitro

In order to examine the cellular uptake of CNTs, CNTs were fluorescently labeled. First,

CNTs were non-covalently functionalized with amine-terminated polyethylene glycol

phospholipids (PL-PEG-NH2) that renders them hydrophilic. Afterwards, TexasRed-X

succinimidyl ester (Invitrogen) was conjugated onto amino-terminated CNT for

fluorescent labelling. Pancreatic cancer cells (L 3.6) were incubated with these

TexasRed-labelled CNTs for overnight and, thereafter, washed, followed by observation

under confocal fluorescence microscopy. With all imaging slices recorded along a whole

cell in the z direction, the three-dimensional imaging revealed intracellular distribution of

TexasRed-labelled CNTs. From the planar (Fig. 3.4a) and cross-sectional (Fig. 3.4b,c)

views of cells, TexasRed-labelled CNTs were confirmed to be internalized inside

pancreatic cancer cells (L 3.6).

We next wanted to test the on-command release of the encapsulated mixture of gelatin

and chemotherapeutic drugs, Taxol and C6 ceramide, in pancreatic cancer cells. The

release resulted in cell death as reflected by reduced cell viability (Fig. 3.4d and 3.4e),

39

apoptosis (Fig. 3.4f and 3.4g) and Akt inhibition (Fig. 3.4g), which is similar to the cell

fate when exogenously adding Taxol/C6 ceramide directly. Herein, MTT assay, Western

blots, stripping and re-probing were done by Dr. Cong Cao and were described

previously30

Application of an a.c. magnetic field for 30 minutes resulted in over 70% cell death with

the release of Taxol and C6 ceramide. Note that the drug concentration loaded into

Trojan-Horse CNTs in this test was 100 times lower (Taxol: 0.03 µg/ml /C6 ceramide:

0.1 µg/ml) than the concentration required for typical exogenously treatment (Taxol: 3

µg/ml /C6 ceramide: 10 µg/ml) while the Trojan-Horse resulted in comparable the cell

killing rate as exogenously treatment. Such high yield may due to the effective target

drug delivery of this system and indicates the benefits of low side effect using our

Trojan-Horse CNTs as drug delivery vehicle. The cell morphology corresponding to

different conditions was also shown in Fig. 3.4d.

40

Figure 3.4: Internalization of Trojan-Horse carbon nanotubes and on-command

therapeutic treatment to pancreatic cancer cells in vitro. (a) A confocal

fluorescence image in plan-view of L3.6 pancreatic cancer cells taken after the

incubation with TexasRed-labelled Trojan-Horse carbon nanotubes for overnight.

The Trojan-Horse carbon nanotubes (red signal) reach the perinuclear space

(c)

(a) (b)

(d)

(f)

(g)

(e)

41

indicates the nanotubes were internalized by the pancreatic cancer. Cell nuclei

were stained in blue with Hoechst 33342 (Invitrogen). (b) As all imaging slices

recorded along a whole cell in the z direction, the three-dimensional imaging of

intracellular distribution of TexasRed-labelled CNTs was demonstrated. From

the cross-sectional views of cells both vertically (b) and horizontally (c),

TexasRed-labelled CNTs were confirmed to be internalized inside pancreatic

cancer cells (a-c). (d) They were subjected to a.c. magnetic field for on-command

drug (C6 ceramide+Taxol) release. The cell morphology of pancreatic cancer

cells (L 3.6) were observed at different conditions. Significant cell death was

observed only when Trojan-horse CNTs were subjected to a.c. magnetic field. (e)

Cell viability study by an in vitro cytotoxicity assay (MTT) 48 hours after

treatment. With a.c. magnetic field on, the Trojan-Horse CNTs resulted in over

70% cell death with the release of Taxol and C6 ceramide. (f) Histone-DNA

ELISA assay was also performed to test L3.6 cells apoptosis at different

conditions after 36 hours treatments, note that only the group 6’

(CNT+Taxol(0.03)+C6 Ceramide(0.1)) with on-command a.c. magnetic field on

had increased Histone-DNA ELISA OD (indicator of cell apoptosis), which is

comparable with group received exogenous C6 ceramide plus Taxol. (g) L 3.6

cell apoptosis was also reflected by increased cleavage of caspase-3.

Phosphorylation (Ser 473) and total level of Akt was also tested.

3.4 Discussion and Conclusion

Just as importantly, cell viability study by an in vitro cytotoxicity assay (MTT) for the

therapeutic treatment by Taxol/C6 ceramide-loaded CNTs showed that the cancer cells

remain intact in the presence of the Taxol/C6 ceramide-loaded but dormant nanotubes

(Fig. 3.4e).

42

No noticeable drop in cell viability was observed in cells incubated with the empty-CNTs

control sample, indicating that the strength and frequency of the a.c. magnetic field used

proved to be safe to cells, showing 98% survivability (Fig. 3.4b). The potential leakage

of encapsulated drugs was tested by comparing the survivability of two groups of cells,

with and without applying a.c. magnetic fields, both having been treated with Taxol/C6

ceramide-loaded CNTs. Without a.c. magnetic field, the Taxol/C6 ceramide loaded

Trojan-Horse nanotubes were shown to be harmless to cells with a 97.9% survivability

even at the incubation condition for 3 days. We rationalize the inductive heating not

only causes the hydrogel to expand but also changes its state from gel to liquid along

with its surface tension.

Currently technology of remote drug release is mainly focusing on the near infrared laser

triggered release of gold nanorod31

or other carbon-based nanomaterial32

carriers with

millimeter laser spot size and penetration depth less than 1 millimeter. The technology

of using induction heating and carbon nanotubes demonstrated here is remarkably

influential. As indicated earlier, the induction power and penetration depth (tens of

centimeters) are controllable by the applied frequency, magnetic field strength. The area

of application is four orders of magnitudes larger than the laser (~102 cm

2 versus 10

-2

43

cm2

of laser). Moreover, unlike the high energy laser, induction heating is harmless to

health.

The use of Trojan-Horse carbon nanotubes as therapeutic drug delivery carrier could be

advantageous in many aspects. Pancreatic cancer cells treated with Taxol-loaded carbon

nanotubes remain intact without any triggered release of Taxol, whereas massive cell

death was observed upon applying inductive heating to the nanotubes and triggered

release of Taxol. The chemical inertness and safe encapsulation of the nanotubes

reduces or removes the concern of side effects common and often lethal to most

chemotherapies. The result indicated that not only the released drugs remained

therapeutic active, but also much less amount of drug were needed, two orders lower

dosage than the free drug usually used, to effectively kill cancer cells. The amount of the

induction heating required is not destructive to cells.

The on-command drug release system using Trojan-Horse carbon nanotubes reported

here are both scientifically intriguing and technologically significant, especially in the

light of the high therapeutic effect to this most aggressive pancreatic cancer, and the low

side effects demonstrated as well. Although we focused here on the delivery of

chemotherapeutic drugs, this Trojan-Horse delivery system could be extended to other

44

cargo such as plasmids, siRNAs, growth factors, and even metallic and atomic

substances. We hope that this report of the Trojan-Horse carbon nanotube on-command

delivery strategy could serve as a seed and one more stimulation for creative

developments of chemotherapies.

45

CHAPTER 4

Future Horizons

4.1 RNAi Neural Delivery via SWNT

RNA interference (RNAi), knocking down gene expression post-transcriptionally, is a

strategy to treat neurological disease by reducing toxic protein expression or minimize

the growth inhibitory signals. The potential of RNAi-based therapies for the treatment of

some neurological disease have been demonstrated in vivo33

. However, neurons are a

notoriously difficult cell type to transfect in vitro and in vivo33

. The key step for

successful transfection is to ensure the delivered plasmid DNA or siRNA escape from

endosomal or lysosomal compartment to their intracellular destinations. For example,

plasmid DNA should be orientated to the neuronal nucleus, while siRNA should be

translocated to the perinuclear region of the cytoplasm.

To observe if the neuron cells internalize SWNT, SWNT solutions were incubated with

neurons overnight. As shown in Figure 1, the neuron‟s nucleus were stained with DAPI

46

(blue), SWNT were labeled with red fluorescence. By utilizing confocal microcopy

scanning technique, when the focal plane slices taken from top to bottom of the neurons

confirm that the signal is indeed coming only from the interior of the neurons. With the

success of showing significant internalization of SWNT to neuron, we have a better

chance to delivery biomolecules, e.g. plasmid DNA and siRNA, to neurons for further

transfection.

Since we also have demonstrated the efficient siRNA delivery into HEK293T (Human

Embryonic Kidney cells) with SWNT as reported in Chapter 2, with similar intracellular

transfection route, we believe that the SWNT could overcome the intracellular barriers of

neuron cells as a potential delivery vehicle.

47

Figure 4.1: Internalization of SNWT by neurons in vitro. (a) A bright field image

of neurons. (b) A confocal fluorescence image in plan-view of neurons taken after

the incubation with TexasRed-labelled SWNT for overnight. The SWNT (red

signal) reach the perinuclear space indicates the nanotubes were internalized by

the neurons. Cell nuclei were stained in blue with DAPI. (c) As all imaging slices

recorded along a whole cell in the z direction, the three-dimensional imaging of

intracellular distribution of red SWNT was demonstrated. From the cross-

sectional views of cells both vertically (c) and horizontally (d), red SWNTs were

confirmed to be internalized inside neurons (b-d).

(a) (b)

(d)

(c)

48

4.2 Trojan-Horse CNT for in vivo application

Figure 4.2: The proposed simultaneously drug encapsulation and surface

functionalization of Trojan-Horse carbon nantubes to achieve specific targeting

of cells and subsequently on-demand drug release.

The Trojan-Horse delivery system reported in chapter 3 could be extended to other cargo

such as plasmids, siRNAs, growth factors, and even metallic and atomic substances.

First, the functionalization of the Trojan-Horse nanotubes by using antibody-antigen

chemistry can be achieved to target tumor cells. As shown in Figure 4.2, the amino

functional group of PEG polymer will be used to conjugate Trojan-Horse carbon

nanotubes with the specific antibody that recognizes cancer cell. Furthermore, on should

seek to maximize the induction heating power of Trojan-Horse carbon nanotube.

According to Lenz‟s law, swirls of currents in the conductor will be induced in order to

generate an opposed magnetic field when the conductor experiences a change in

49

magnetic field. By subjecting the Trojan-Horse nanotube (a good electron conductor) in

a.c. magnetic field, the induced Eddy current give rises to the induction heating. We

found that the power generated in each nanotube is controlled by the applied frequency

(Ѡ), magnetic field strength (B) and physical dimension of carbon nanotube, which is

controllable by our fabrication process.

Next, the efficacy of delivered drug and the toxicity study should be carried out to

evaluate the feasibility of Trojan-Horse carbon nanotubes. The statistic comparison on

the killing efficiency of cancer cell versus the viability of healthy cells can be perused.

Biodistribution and pharmacokinetics of Trojan-Horse CNT must be studied for future

clinical application. The novel drug delivery carrier, if feasible, would shed the light on

the therapeutics application for future generation.

50

CHAPTER 5

Conclusions

This dissertation focuses on the development of new drug delivery platforms using

carbon nanotubes. Two nanotube systems, namely single walled carbon nanotubes

(SWNTs) and multi walled carbon nanotubes (MWNTs) were employed to this study.

Extensive material characterizations were carried out to validate the successful loading of

therapeutic agents and the internalization of carbon nanotubes.

For SWNT system, in Chpater 2, the atomic force microscope (AFM) technique

indicated that the SWNTs were well dispersed after chemical functionalization. We then

further develop a method to efficiently conjugate CNTs with organic dyes and siRNA

reproducibly. We also successfully made the observation of the cellular internalization

of SWNT conjugate achievable by using Raman spectroscopy and Confocal microscopy.

The intracellular controlled release of siRNA delivery was carried out by incorporating

disulfide bond to the SWNT conjugate. The result showed a promising expression of

silencing effect on preserved bioactivity of EGFP gene in EGFP-transfected cells by our

51

SWCNT system post-delivery.

For the MWNT system, in Chapter 3, was prepared by filling the template synthesized

CNTs array with therapeutic agents. To visualize the successful encapsulation of drugs

into MWNTs, we used transmission electron microscope (TEM) to image quantum dots

(QDs) filled CNTs. High density of QDs was loaded into the nanotubes array. We

developed an innovative drug delivery system based on MWNTs that resembles the

Trojan-Horse in function, which can encapsulate toxic compounds in a hydrogel gelatin

within the nanotube. The mechanism relies on the induction heating power generated by

exposing CNTs to the a.c. magnetic field. Using this novel approach, we demonstrated

delivery of a low dose combination of ceramide plus Taxol to multi-drug resistance

pancreatic cancer cell lines, which was precisely released on-command by inductive

heating of the nanotubes with an external a.c. or pulsed magnetic field. The drug release

resulted in cell death as reflected by reduced cell viability which was confirmed by

apoptosis assay and Western blot analysis.

The door of opportunity for the development of carbon nanotubes as targeted

therapeutic nanomedicines has opened. The outcome of our systematic study of carbon

nanotube as drug delivery vehicle is intriguing and promising for drug delivery

52

application. Yet there are plenty of room for the development of nanomedicine using

carbon nanotubes as the drug delivery vehicle and we hope the dissertation will generate

broad interest in developing controlled and high-yield therapeutic platforms for more to

come.

53

APPENDIX

A.1. Induction Heating of Trojan-Horse CNTs

Induction heating is considered as a clean and efficient heating source that is widely used

as cooking, industrial welding and heat treatments. According to Lenz‟s law, swirls of

currents (called Eddy current) in the conductor will be induced in order to generate an

opposed magnetic field when the conductor experiences a change in magnetic field. By

exposing the conductor in a time-varying magnetic field (or called a.c. magnetic field),

the induced Eddy current give rises to the induction Joule heating. Multi-walled carbon

nanotube is intrinsically a good electron conductor that makes it an excellent candidate

for the on-demand drug delivery vehicle.

Starting with the Maxwell equation form of Faraday‟s Law,

describes that

the time varying magnetic field (B) creates the electric field (E).

It is known that the penetration depth δ (m),

√ 𝝇 is much larger (~millimetre) than

the physical dimension of CNTs (~200 nanometre). Where f is frequency of alternating

54

magnetic field, μ denotes as magnetic permeability and σ is the electrical conductivity

(S/m).

Therefore, we can consider the case is under quasi-statics condition where the

electromagnetic wave fully penetrates the CNTs.

Under such condition, one can estimate the power generated by the induced Eddy current

of a nanotube. Note that we simplify the case by assuming there is not magnetization of

the CNTs. Therefore, the

current density J 𝝇

𝝇 ,…………………………………Eq.(A.1)

As described earlier in Eq (A.1), 𝑱 𝝇𝑬 ⇒ 𝑰 𝑨𝝇 𝑩

We then derive the power generated from the induced Eddy current below:

𝐏 𝑰𝟐𝑹 𝑰𝟐 ∙ 𝝆 ∙𝒍

𝑨………………………………………………………………………………………….Eq.(A.2)

Where 𝒍 is the length of CNT, A is CNT surface area, 𝝆 is the resistivity of CNT and R is

the resistance of CNT.

By substituting Eq.(A.2) into Eq. (A.1), we now get the expression of the induced power

of individual CNT.

𝐏 𝐀𝝇𝟐 ∙ 𝟐 ∙ 𝑩𝟐 ∙ 𝒍 ∙ 𝝆 𝟐𝑩𝟐

𝝆∙ (𝑨 ∙ 𝒍)……………………………………………………………Eq.(A.3)

55

Now, we would have to estimate the resistance of the multiwalled CNTs. To simplify the

electron flow directions, three cases are discussed and listed in the graph below:

In Case 1, assume the electron is flowing along the axial direction:

{𝒍 𝑳

𝑨 𝝅(𝑹𝒐𝒖 𝟐 𝑹𝒊𝒏

𝟐 )

Applying the above relationship to Eq. (A.3), get the

𝐏 𝟐𝑩𝟐

𝝆∙ 𝑳 ∙ 𝝅(𝑹𝒐𝒖

𝟐 𝑹𝒊𝒏𝟐 )

In Case 2, assume the electron is flowing along the radial direction:

56

{𝒅𝒍 𝒅𝒓𝑨 𝟐𝝅𝒓𝑳

Similarly, by integrating from inner CNT wall to outer wall, we got

𝐏 𝟐𝑩𝟐

𝝆∙ (𝑨𝒍)

𝟐𝑩𝟐

𝝆∫ (𝟐𝝅𝒓𝑳) ∙ 𝒅𝒓𝑹𝒐𝒖

𝑹𝒊𝒏

𝟐𝑩𝟐

𝝆∙ 𝟐𝝅𝑳 ∙

𝟐∙ (𝑹𝒐𝒖

𝟐 𝑹𝒊𝒏𝟐 )

𝟐𝑩𝟐

𝝆∙ 𝝅𝑳 ∙ (𝑹𝒐𝒖

𝟐 𝑹𝒊𝒏𝟐 )

In Case 3, assume the electron is flowing in cyclic motion:

Where

{𝒍 (

𝑹𝒐𝒖 + 𝑹𝒊𝒏𝟐

)

𝑨 (𝑹𝒐𝒖 𝑹𝒊𝒏) ∙ 𝑳∙ 𝟐𝝅

Similarly, we got

𝐏 𝟐𝑩𝟐

𝝆∙ (𝑨𝒍)

𝟐𝑩𝟐

𝝆[𝑳 ∙ (𝑹𝒐𝒖 𝑹𝒊𝒏)] ∙ [𝟐𝝅 ∙ (

𝑹𝒐𝒖 + 𝑹𝒊𝒏𝟐

)]

𝝅𝑳 𝟐𝑩𝟐

𝝆(𝑹𝒐𝒖

𝟐 𝑹𝒊𝒏𝟐 )

57

Please note that all three cases indicate that the Joule heating power P (W/kg) generated

by induced Eddy current is followed the relationship below:

𝟐 𝟐(𝑹𝒐𝒖 𝟐 𝑹𝒊𝒏𝟐) 𝑳 𝝆⁄

Where B is peak flux density magnetic field (T), L is the length of nanotube (m), Ѡ is

the frequency (Hz), ρ denotes the resistivity of nanotube (Ωm), Rout and Rin are the

outer and inner radius of nanotube, respectively (m)

The extent of heating generated in each nanotube is controlled by the applied magnetic

field strength, frequency, and by the tube‟s conductivity and physical dimension, the

former two parameters being field and application adjustable, whereas the latter two are

controllable by our fabrication process.

58

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