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RICE UNIVERSITY
Cisplatin@US-tube Carbon Nanocapsules for Enhanced
Chemotherapeutic Delivery
by
Adem Guven
A THESIS SUBMITTED
IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE
Doctor of Philosophy
APPROVED, THESIS COMMITTEE
Lon J. Wilson, Committee Chair
Professor of Chemistry
Vicki L. Colvin, The Kenneth S. PitzerSchlumberger Professor of Chemistry and
Chemical & Biomolecular Engineering,
Vice Provost of Research
Enrique V. Barrera, Professor
Mechanical Engineering and Materials
Science
Michael T. Lewis, Associate Professor
Lester & Sue Smith Breast Cancer Center,
Baylor College of Medicine
HOUSTON, TEXAS
December 2013
ABSTRACT
Cisplatin@US-tube Carbon Nanocapsules for Enhanced
Chemotherapeutic Delivery
by
Adem Guven
The use of chemotherapeutic drugs in cancer therapy is often limited by
problems with administration such as insolubility, inefficient biodistribution, lack of
selectivelty, and inability of the drug to cross cellular barriers. To overcome these
limitations, various types of drug delivery systems have been explored, and recently,
carbon nanotube (CNT) materials have garnered special attention in the area. This
thesis details the preparation, characterization, and in vitro and in vivo testing of a
new, ultra-short single-walled carbon nanotube (US-tube)-based drug delivery
system for the treatment of cancer. In particular, the encapsulation of cisplatin
(CDDP), a widely-used anticancer drug, within US-tubes has been achieved by a
loading procedure that is reproducible, and the resulting CDDP@US-tube material
characterized by high-resolution transmission electron microscopy (HR-TEM),
energy-dispersive spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and
inductively-coupled optical emission spectroscopy (ICP-OES). Dialysis studies
performed in phosphate-buffered saline (PBS) at 37 °C have demonstrated that
CDDP release from CDDP@US-tubes can be controlled (retarded) by wrapping the
CDDP@US-tubes with Pluronic®-F108 surfactant. The anticancer activity of
Pluronic-wrapped CDDP@US-tubes (W-CDDP@US-tubes) has been evaluated
against two different breast cancer cell lines, MCF-7 and MDA-MB-231, and found to
exhibit enhanced cytotoxicity over free CDDP. Moreover, it has been shown that
CDDP release from W-CDDP@US-tubes nanocapsules can be stimulated remotely by
a radiofrequency (RF) field which disrupts the Pluronic coating to release CDDP. RFinduced release-dependent cytotoxicity of W-CDDP@US-tubes has been evaluated in
vitro against two different liver cancer cell lines, Hep3B and HepG2, and found to
exhibit superior cytotoxicity compared to W-CDDP@US-tubes not exposed to RF.
Finally, in vivo biodistribution and therapeutic efficacy of the CDDP@US-tube
material has been evaluated against three different breast cancer xenograft mouse
(SCID/Beige) models, and found to exhibit greater efficacy in suppressing tumor
growth than free CDDP for both a MCF-7 cell line xenograft model and a BCM-4272
patient-derived xenograft (PDX) model. The CDDP@US-tubes also demonstrated
prolonged circulation time compared to free CDDP which enhances permeability
and retention (EPR) effects resulting in significantly more CDDP accumulation in
tumors, as determined by Platinum (Pt) analysis via inductively-coupled plasma
mass-spectrometry (ICP-MS).
Acknowledgments
Although only my name appears on the cover of this thesis, many people
have contributed to its existence. I owe my gratitude to all of the people who have
made this journey nothing short of amazing.
First and foremost, I would like to express my sincere gratitude to my
advisor Prof. Lon J. Wilson for his constant assistance, encouragement, guidance and
the tremendous support he provided throughout my PhD studies. I couldn't have
asked for more from an advisor.
I also like to thank to Professor Vicki L. Colvin, Professor Enrique V. Barrera
and Professor Michael T. Lewis for their kindness to take time out of their busy
schedules to serve on my thesis committee.
I am particularly grateful to my collaborators Professor Michael. T. Lewis and
Dr. Gabriel J. Villarreal from Baylor College of Medicine, Professor Steven A. Curley
and Dr. Mustafa Raoof from The University of Texas M.D. Anderson Cancer Center.
Without their contribution, a substantial part of this thesis would have been blank
pages.
I have been very fortunate to be surrounded by the wonderful people in
Wilson Laboratory who made my years in the lab such a wonderful experience. I feel
obliged to thank you whom I have had opportunity to work: Ari Berlin, Ayrat
Gizzatov, Brandon Cisneros, Dr. Jeyarama Ananta, Justin Law, Dr. Lesa A. Tran,
Matthew Cheney, Matthew Makansi, Mayra Hernandez-Rivera, Meghan Jebb, Dr.
vi
Michael Matson, Nadia Lara, Dr. Scott Berger, Stuart Corr, Sue Friend, Tawana
Robinson, Rafael Lima, Dr. Richa Sethi, Sophia Phounsavath, Vazrik Keshishian, and
Yuri Mackeyev)! It has been a joy and a privilege to know you all.
In addition to those mentioned above, I am very thankful to many friends in
US, Turkey and other parts of the World who helped me with their various forms of
support throughout my graduate study and before.
Finally, and most importantly, I would like to thank my wife, Esra, for her
endless understanding, unconditional support and encouragement that allowed me
to finish this journey.
vii
Contents
Acknowledgments .................................................................................................................... v
Contents ..................................................................................................................................... vii
List of Figures ............................................................................................................................. x
List of Tables ............................................................................................................................ xv
Nomenclature ......................................................................................................................... xvi
Introduction ............................................................................................................................... 1
1.1. Overview................................................................................................................... 1
1.2. Organization of the thesis ........................................................................................ 2
Background and Literature Review .................................................................................. 4
2.1. Cancer Chemotherapy.............................................................................................. 4
2.2. Cisplatin .................................................................................................................... 6
2.3. Carbon nanotubes (CNTs) for biomedical applications ........................................... 8
2.4. CNTs as nanocapsules for drug delivery ................................................................ 10
Cisplatin@US-tube carbon nanocapsules for enhanced chemotherapeutic
delivery ...................................................................................................................................... 13
3.1. Introduction ............................................................................................................ 13
3.2. Materials and Methods .......................................................................................... 15
3.2.1. Preparation of US-tubes .................................................................................. 15
3.2.2. Encapsulation of CDDP within US-tubes.......................................................... 15
3.2.3. Characterization of CDDP@US-tubes .............................................................. 16
3.2.4. CDDP release studies from CDDP@US-tubes .................................................. 17
3.2.5. Cell viability studies ......................................................................................... 18
3.2.6. Cell internalization rate studies ....................................................................... 19
3.2.7. Transmission electron microscopy .................................................................. 19
3.2.8. Statistical evaluation........................................................................................ 20
viii
3.3. Results and Discussion ........................................................................................... 21
3.3.1. Encapsulation of CDDP within US-tubes.......................................................... 21
3.3.2. Release studies of CDDP from CDDP@US-tubes ............................................. 27
3.3.3. In vitro cytotoxicity studies .............................................................................. 29
3.4. Conclusions............................................................................................................. 34
Remotely-Triggered Cisplatin Release From Carbon Nanocapsules by
Radiofrequency Fields.......................................................................................................... 35
4.1. Introduction ............................................................................................................ 35
4.2. Methods ................................................................................................................. 39
4.2.1. Sample Preperation ......................................................................................... 39
4.2.2. Inductively-coupled plasma optical emission spectrometry (ICP-OES)........... 40
4.2.3. RF generator setup .......................................................................................... 41
4.2.4. CDDP release from W-CDDP@US-tubes.......................................................... 42
4.2.5. Fluorescence measurements ........................................................................... 42
4.2.6. Cell studies ....................................................................................................... 43
4.2.7. Statistical analysis ............................................................................................ 44
4.3. Results .................................................................................................................... 44
4.3.1. RF absorption by US-tubes .............................................................................. 44
4.3.2. Localized heating of US-tubes in a physiological environment ....................... 46
4.3.3. RF-triggered release of CDDP from US-tubes .................................................. 49
4.3.4. RF-triggered release and cytotoxicity .............................................................. 51
4.4. Discussion ............................................................................................................... 53
4.5. Conclusion .............................................................................................................. 57
In vivo Biodistribution and Anticancer Activity of Cisplatin Delivered by Ultrashort Carbon Nanotube Capsules ..................................................................................... 59
5.1. Introduction ............................................................................................................ 59
5.2. Materials and Methods .......................................................................................... 60
5.2.1. Sample preparation: ........................................................................................ 60
5.2.2. Establishment of xenografts and treatment ................................................... 61
5.2.3. Biodistribution and blood circulation studies ................................................. 62
ix
5.2.4. Inductively-coupled plasma-optical emission mass spectrometry (ICP-MS) .. 63
5.2.5. Statistical analysis ............................................................................................ 63
5.3. Results .................................................................................................................... 64
5.3.1. Tumor suppressing effect of CDDP@US-tubes on MCF-7 xenograft in
SCID/Beige mice ......................................................................................................... 64
5.3.2. Tumor suppressing effect of CDDP@US-tubes on MDA-MB-231 xenograft in
SCID/Beige mice ......................................................................................................... 69
5.3.3. Tumor suppressing effect of CDDP@US-tubes on the BCM-4272 PDX
xenograft in SCID/Beige mice .................................................................................... 73
5.3.4. Blood circulation and the biodistribution studies ........................................... 77
5.4. Discussion ............................................................................................................... 83
Conclusion and Future Perspective ................................................................................. 87
References ................................................................................................................................ 90
List of Figures
Figure 2.1 − Tumor resistance to cisplatin mediated by inadequate levels of
platinum reaching target DNA. Taken from.13 ............................................................... 8
Figure 2.2 − Preperation of US-tubes from SWCNTs. ................................................. 12
Figure 3.1 − Preparation and purification of CDDP@US-tubes.............................. 22
Figure 3.2 − Pt determination by ICP-OES analysis of each collected aliquot of
H2O wash solution for a typical CDDP@US-tube sample. The data are
expressed as mean ± SD of three independent experiment. Error bars may be
smaller than symbols............................................................................................................ 23
Figure 3.3 − HR-TEM images of a) bundled CDDP@US-tubes and b) bundled
empty US-tubes c) EDS spectrum of CDDP@US-tubes showing carbon and
nickel peaks (metal catalyst) from US-tubes, copper peaks (from the TEM Cugrid),
and
platinum
and
chloride
peaks
(from
CDDP).
...................................................................................................................................................... 25
Figure 3.4 − XPS spectrum of a) CDDP@US-tubes (survey), b) Pt 4f peaks for
various samples: i) metallic platinum (Pt0), ii) CDDP@US-tubes (Pt2+), iii)CDDP
(Pt2+) and iv) Na2PtCI6.6H2O (Pt4+)................................................................................... 27
Figure 3.5 − Comparative time release profiles of CDDP released from
CDDP@US-tubes and W-CDDP@US-tubes (Pluronic-F108-wrapped) in PBS at
37 °C. The data are expressed as mean ± SD of three independent experiments.
...................................................................................................................................................... 28
Figure 3.6 − Time and concentration dependency of cell viability studies for
MCF-7 and MDA-MB-231 cells incubated with CDDP, W-CDDP@US-tubes, and
W-US-tubes. The quantity of US-tubes at each CDDP concentration is
equivalent for the CDDP@US-tube and US-tube samples, with the
concentration being calculated from their wt % values obtained by ICP-OES
for Ni and Y which is contained within US-tube materials as the metal catalysts
for carbon nanotube growth. The data are expressed as mean ± SD for three
independent experiments. (*) and (#) indicate a statistically significant
difference between W-CDDP@US-tube data compared to free CDDP data (*p <
0.01, #p<0.05). ........................................................................................................................ 30
xi
Figure 3.7− Comparative internalization rate of CDDP and W-CDDP@US-tubes
into MCF-7 cells as a function of incubation time at 10 µM CDDP concentration.
The graph presents data for the average number of Pt2+ ions per cell for an
average of three trials. Error bars are standard errors for the three different
experiments. (ns=not significant, *p-value<0.05) ...................................................... 32
Figure 3.8 − (a, b) TEM images of a single MCF-7 cell from different angles. (c,
d, e, f)TEM images of MCF-7 cells incubated with W-CDDP@US-tubes at 10 µM
CDDP for 24 h. White arrows point to (a, b) ‘vesicularized’, and (c, d) ‘free’
CDDP@US-tube aggregates in the cytoplasm. Scale bar = 2 µm. ........................... 33
Figure 4.1 − Schematic describing synthesis, purification, and RF-triggered
disruption of Pluronic®-F108 and subsequent release of CDDP from WCDDP@US-tubes. .................................................................................................................... 39
Figure 4.2 − Heating rates of W-CDDP@US-tubes and W-US-tubes suspended in
0.17% pluronic F108 in comparison with filtrate alone as a function of
concentration. ......................................................................................................................... 45
Figure 4.3 − Panel A. Schematic demonstrating the principle of the
temperature-dependent Pluronic disruption assay. Panel B and C.
Temperature-dependent fluorescence quenching with a transition
temperature close to the melting point of Pluronic F108 is indicative of
Pluronic disruption from the w-CDDP-US-tube surface. Panel D. RF durationdependent quenching of fluorescence demonstrates localized heating of WCDDP@US-tubes in PBS. Water bath temperature controls (HT) fail to
demonstrate quenching of APC fluorescence suggesting lack of Pluronic
disruption due to bulk temperature. The RF-dependent quenching is therefore
not a result of non-thermal denaturation of APC. ...................................................... 47
Figure 4.4 − CDDP release from W-CDDP@US-tubes with or without RF. Panel
A. Release of CDDP from W-CDDP@US-tubes with or without RF exposure for
2.5 or 5 min is shown. In addition, bulk temperature controls are
demonstrated to evaluate the contribution of bulk ionic heating to CDDP
release from W-CDDP@US-tubes. Panel B. Relative areas-under-curve depicts
cumulative CDDP release over 168 h relative to untreated control. ................... 51
Figure 4.5 − Release-dependent cytotoxicity of W-CDDP@US-tubes. Hep3B
and HepG2 human liver cancer cells were plated in the bottom well of the
trans-well plate and treated with PBS, W-US-tubes or W-CDDP@US-tubes
followed with or without RF exposure for 3 min once, twice, or three times at
xii
24 h intervals. The cell viability was assessed 120 h after treating with WCDDP@US-tubes. .................................................................................................................... 52
Figure 5.1 − Suppression of tumor growth by intraperitoneal injection of
CDDP@US-tubes to an MCF-7 breast cancer xenograft. (A), Tumor growth of
MCF-7 tumor-bearing mice after receiving different treatments as indicated.
The mean body weight changes is also plotted in A. Fold increase in tumor
volume (B) and Kaplan-Meier survival curves (C) depicting the tumor
doubling times for different treatment groups. .......................................................... 66
Figure 5.2 − Platinum and yttrium concentration in tissue from mice treated
over a period of ~ 45 d with weekly saline, w-US-tubes, CDDP, CDDP@UStubes, and W-CDDP@US-tubes administration via i.p. injection. (A)
Concentration of platinum (from cisplatin) and (B) yttrium (from US-tubes) in
tissue (mean ± s.e., n = 8). Statistically significant different (p < 0.05) are
marked with symbols as indicated above (unpaired, two-sided t-test). ........... 68
Figure 5.3 − Suppression of tumor growth by intraperitoneal injection of
CDDP@US-tubes to an MDA-MB-231 breast cancer xenograft. (A), Tumor
growth of MDA-MB-231 tumor-bearing mice after receiving different
treatments as indicated. (* indicates p < 0.05, unpaired, two-sided t-test, n = 8;
error bars = s.e.) The mean body weight changes is also plotted in A. Fold
increase in tumor volume (B) and Kaplan-Meier survival curves (C) depicting
the tumor tribling times for different treatment groups. ........................................ 71
Figure 5.4 − Platinum and yttrium concentration in tissue from mice treated
over a period of ~ 45 d with weekly saline, w-US-tubes, CDDP, CDDP@UStubes, and W-CDDP@US-tubes administration via i.p. injection. (A)
Concentration of platinum (from cisplatin) and (B) yttrium (from US-tubes) in
tissue (mean ± s.e., n = 8). Statistically significant different (p<0.05) are
marked with symbols as indicated above (unpaired, two-sided t-test). ........... 72
Figure 5.5 − Suppression of tumor growth by intraperitoneal injection of
CDDP@US-tubes to an Patient-derived, 4272 transplant, xenograft. (A), Tumor
growth of Patient-derived tumor-bearing mice after receiving different
treatments as indicated. (unpaired, two-sided t-test, n=9; error bars= s.e.) The
mean body weight changes is also plotted in A. Fold increase in tumor volume
(B) and Kaplan-Meier survival curves (C) depicting the tumor doubling times
for different treatment groups. ......................................................................................... 75
xiii
Figure 5.6 − Platinum and yttrium concentration in tissue from mice treated
over a period of ~ 45 d with weekly saline, W-US-tubes, CDDP, CDDP@UStubes, and W-CDDP@US-tubes administration via i.p. injection. (A)
Concentration of platinum (from cisplatin) and (B) yttrium (from US-tubes) in
tissue (mean ± s.e., n = 8). Statistically significant different (p < 0.05) are
marked with symbols as indicated above (unpaired, two-sided t-test). ........... 77
Figure 5.7 − Blood circulation for CDDP, W-CDDP@US-tubes, CDDP@US-tubes
in MDA-MB-231 tumor-bearing mice as determined by measuring Pt (from
CDDP) and Y (from US-tubes) concentration in blood via ICP-MS at different
time points post injection (mean ± s.e., n = 3). CDDP@US-tubes showed
prolonged blood circulation compared to free CDDP. .............................................. 78
Figure 5.8 − Time-dependent in vivo biodistribution of intraperitoneally
injected CDDP, W-CDDP@US-tubes, and CDDP@US-tubes in MDA-MB-231
tumor bearing mice. (A) Concentration of platinum (from CDDP), (B)
Concentration of yttrium (from US-tubes) in tissue (mean ± s.e., n = 3).
Statistically significant different (p < 0.05) are marked with symbols as
indicated above (unpaired, two-sided t-test). ............................................................. 83
Figure 6.1 − Hydrolysis of the peptide sequence on a CDDP@US-tube by PSA
and/or MMP-2 . ....................................................................................................................... 89
xiv
List of Tables
Table 3.1 – Pt content by ICP-OES of CDDP@US-tubes under different loading
solvent and washing conditions. EtOH is ethyl alcohol and DMF is
dimethylformamide. Table presents the wt% Pt for an average of three trials
under each condition and the standard deviation. .................................................... 24
Nomenclature
°C
Degrees Celsius
ANOVA
One-Way Analysis of Variance
APC
Allophycocyanin
AUC
Area-Under-Curve
AMF
Alternating Magnetic Field
BCM
Baylor College of Medicine
BSA
Bovine Serum Albumin
CCD
Charge-Coupled Device
CDDP
Cisplatin (Cis-diamminedichloroplatinum(II))
CDDP@US-tube
CDDP within US-tube
CNT
Carbon Nanotube
CO2
Carbon Dioxide
DNA
Deoxyribonucleic acid
DMF
Dimethyformamide
DMSO
Dimethyl Sulfoxide
EDS
Energy Dispersive X-ray Spectroscopy
EDTA
Ethylenediaminetetraacetic Acid
EtOH
Ethanol
FBS
Fetal Bovine Serum
HNO3
Nitric Acid
HCIO3
Chloric Acid
xvii
HR-TEM
High-Resolution Transmission Electron
Microscopy
ICP-OES
Inductively-Coupled Plasma Optical Emission
Spectroscopy
ICP-MS
Inductively-Coupled Plasma Mass Spectrometry
MAbs
Monoclonal Antibodies
MEM
Minimal Essential Medium
MMP-2
Metalloproteinase-2
MRI
Magnetic Resonance Imaging
MTT
3-(4,5 dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide
MWCNT
Multi-Walled Carbon Nanotube
NIR
Near-Infrared
PBS
Phosphate Buffered Saline
PDX
Patient-Derived Xenograft
PSA
Prostate Specific Antigen
RES
Reticuloendothelial System
RF
Radiofrequency
RNA
Ribonucleic acid
RT
Room Temperature
SWCNT
Single-Walled Carbon Nanotube
SD
Standard Deviation
siRNA
Small Interfering RNA
xviii
TEM
Transmission Electron Microscopy
THF
Tetrahydrofuran
US-tube
Ultra-Short SWCNT
W-CDDP@US-tube
Pluronic® F108-wrapped CDDP@US-tubes
XPS
X-ray Photo Electron Spectroscopy
Chapter 1
Introduction
1.1. Overview
Although chemotherapy is one of the major approaches for the treatment of
cancer, a vast majority of existing chemotherapeutic (anticancer) drugs cannot be
employed to full clinical benefit due to problems associated with administration,
such as low water solubility, rapid elimination, nonspecific biodistribution, rapid
breakdown in vivo, insufficient accumulation in tumor tissue, inability to cross
cellular barriers, and inability to differentiate between healthy cells and cancer cells,
leading to systemic toxicity and adverse side effects that greatly limit the dosage
level. These limitations necessitate the development of more efficient ways to
administer anticancer drugs systemically, yet more selectively target tumor tissue,
thereby improving efficacy while minimizing undesirable side effects.
1
2
Over the last few years, with the advancement in nanotechnology, numerous
nanotechnology-based drug delivery systems have been developed in an effort to
maximize therapeutic effectiveness of conventional drug delivery, while limiting
undesirable side effects. Among different type of nanomaterials, carbon nanotubes
(CNTs) are of special interest in the area of drug delivery as potential drug delivery
agents due to their unique physical and chemical properties. This thesis details the
development of a new CNT-based drug delivery platform for the treatment of cancer
which is comprised of ultra-short single-walled carbon nanotubes (hereafter called
US-tubes) loaded with the cisplatin (CDDP) chemotherapeutic drug (CDDP@UStubes) that has demonstrated enhanced chemotherapeutic efficacy for breast cancer
both in vitro and in vivo.
1.2. Organization of the thesis

Chapter 1: Introduction and overview.

Chapter 2: Background and literature review. This chapter provides brief
background information about conventional cancer chemotherapy and
the cisplatin. It also reviews recent progress about the development and
use of CNTs-based drug delivery system.

Chapter 3: Cisplatin@US-tube carbon nanocapsules for enhanced
chemotherapeutic drug delivery. This chapter describes the synthesis,
characterization and in vitro anticancer activity evaluation of CDDP@UStubes in different breast cancer cell lines.
3

Chapter
4:
Remotely-triggered
cisplatin
release
from
carbon
nanocapsules by radiofrequency fields. This chapter demonstrates CDDP
release from CDDP@US-tubes nanocapsules as achieved by remote
radiofrequency (RF) field activation. Release-dependent in vitro
cytotoxicity data is also demonstrated for human hepatocellular
carcinoma cell lines.

Chapter 5: In vivo biodistribution and anticancer activity of cisplatin
delivered by ultra-short carbon nanotube capsules. This chapter is an
extension of Chapter 3 and provides the first in vivo biodistribution
behaviors and efficacy information for CDDP@US-tubes after the
systematic administration in three different breast cancer tumor models.

Chapter 6: Conclusion and future perspective. This chapter summarizes
the important conclusions of the thesis and proposes future research
directions.
4
Chapter 2
Background and Literature Review
2.1. Cancer Chemotherapy
Cancer is a multifactorial cell disease, characterized by cells with
unrestricted division potential, evasion of apoptosis, and invasive expansion to
other body parts through blood and lymph systems.1 It is known to be one of the
most devastating diseases, accounting for 7.6 million deaths in 2008 which is
expected to continue to rise to over 13.1 million by 2030.2 In 2012, nearly 577,190
Americans died of cancer. In other words, about one in every three women and one
in every two men in the United States will be affected by cancer in their lifetime.
Despite centuries of intensive research and progress, the development of effective
and favorable treatments for cancer continues as an elusive goal. Surgery, radiation,
and chemotherapy are the most common treatment modalities for cancer which are
performed on the basis of clinical and pathologic staging that is determined using
5
morphologic
diagnostic
tools,
such
as
conventional
radiological
and
histopathological examinations.3 In contrast to surgery, radiation therapy, and other
forms of “local" treatment, chemotherapy offers a type of “systemic" treatment
because chemotherapeutic (anticancer) drugs circulate throughout the body in the
bloodstream to reach cancer cells wherever they may have spread.
Despite some advances over the last few decades in conventional
chemotherapy, the efficacy of the vast majority of cancer therapeutics, while being
potent, is often limited for many reasons. A major problem limiting the success of
many anticancer agents is their inability to selectively target tumor cells and tissues.
Generally, chemotherapeutic agents target cells that are actively propagating, a
characteristic hallmark of cancer cells. Unfortunately, these agents are unable to
differentiate cancer cells from actively growing normal cells (e.g., cells in the blood,
mouth, intestines, hair, and particularly in bone marrow). Severe side effects occur
when chemotherapeutic agents suppress dividing immune and somatic cells in bone
marrow and peripheral blood lymphoid tissues, resulting in fatal side effects such as
suppression of bone marrow activity. Moreover, even if the anticancer drugs
selectively accumulate in the tumor interstitium, they can have limited efficacy
against numerous tumor types because cancer cells are able to develop mechanism
of resistance, which is a significant hurdle to overcome for successful chemotherapy.
In addition, rapid elimination and widespread distribution of drugs in the body
require high doses or continuous infusion in order to induce sufficient antitumor
effects, resulting in dose-limiting side effects. There are many chemotherapeutics
6
drugs available, and the choice of which one to use depends on the cancer type,
stage, and grade.
2.2. Cisplatin
Cisplatin, cis-diamminedichloroplatinum(II), (CDDP) is one of the most
potent, and widely-used, chemotherapeutic agents in cancer therapy for the
treatment of variety of solid tumors, including testicular, ovarian, bladder, cervical,
head and neck, oesophageal, small cell lung and breast cancer.4–6 The biological
activity of CDDP is derived from its interaction with DNA, with which it forms stable
adducts that interfere with the normal transcription and replication processes of the
cell which results in cell death.
Despite its success, the clinical use of CDDP faces a number of serious
problems. CDDP has limited bioavailability due to its reactive nature. Upon
administration, CDDP rapidly binds to albumin and other plasma proteins and
spontaneously degrades in the bloodstream, leading to inactivation of nearly 90% of
the injected dose,5 and it rapidly cleared from the blood by glomerular excretion.7 In
addition, the lack of selectivity of CDDP causes many dose-limiting severe adverse
reactions including renal toxicity from renal tubular damage, gastrointestinal
toxicity, peripheral neuropathy, asthenia (lack or loss of strength), and ototoxicity
(damage to the hearing or balance functions of the ear).8 Moreover, the resistance of
tumors to CDDP remains one of the main limitation of CDDP in the clinical. Certain
cancer types are intrinsically resistance to CDDP (e.g., colon cancer, non-small-cell
lung cancer), while others acquire resistance after exposure to the drug over time
7
(e.g., ovarian cancer, small cell lung cancer).9 CDDP resistance is known to be a
multifactorial phenomenon, involving reduced drug uptake, inactivation by thiol
containing species, increased repair/tolerance of platinum−DNA adducts, and
alterations in proteins involved in apoptosis. Among these, decreased intracellular
concentration of CDDP is generally considered the major cause of CDDP resistance
in cancer cells.6,10,11
Because CDDP is highly polar, it enters cells relatively slowly compared to
other small-molecule cancer drugs, using either transporters such as the copper
transporter CTR1 or by passive diffusion (Figure 2.1).12,13 The uptake of CDDP is
influenced by several factors, such as pH, sodium and potassium ion content, and the
presence of a reducing agent, transporters or gated channels.5,13 Loss of chloride
groups is required before CDDP binds to DNA. After CDDP is internalized within a
cell, it replaces one or both chloride groups with water molecules which is
facilitated by the low concentration of chloride that is found within cells, resulting in
the more reactive platinum species, [Pt(H2O)Cl(NH3)2]+ and [Pt(H2O)2(NH3)2]2+.
Binding of these activated species to thiol-containing peptides such as
metalothionein and tripeptide glutathione, consume the activated CDDP in the
cytoplasm before DNA binding occurs, thereby causing resistance.5,13 Because CDDP
is one of the most potent anticancer drug in the clinic, its potential use in cancer
therapy is very attractive. With the emerging trends and recent advances in
nanotechnology, it has become increasingly possible to address some of the
shortcomings associated with CDDP as an anticancer drug.
8
Figure 2.1 − Tumor resistance to cisplatin mediated by inadequate levels of
platinum reaching target DNA. Taken from.13
2.3. Carbon nanotubes (CNTs) for biomedical applications
In recent years, the application of nanotechnology in medicine, defined as
nanomedicine, has garnered tremendous attention for the purpose of cancer
diagnosis and therapy, and it is currently the subject of an intense research effort by
many scientists worldwide. Among diverse classes of nanomaterials, carbon
nanotubes (CNTs), an allotropic form of carbon which consists of graphite sheets
rolled up into tubular form, have been extensively explored in many fields.
Regardless whether they contain one (SWCNTs) or multiple (MWCNTs) graphene
9
sheets, CNTs exhibit many unique physical, mechanical and chemical properties that
make them potentially desirable for many application, including nanomedicine.14–18
In recent years, research toward applying CNTs for biomedical applications has
been progressing rapidly which has resulted in numerous publication in the field. In
particular, recent studies have shown that CNTs use in nanomedicine as a platform
holds great promise, especially for the detection and treatment of cancer due to
their many intrinsic unique properties such as high surface area, high aspect ratio,
high thermal conductivity, remarkable optical and electronic properties.19–26 Owing
to their high surface area, CNTs enable the engineering of surface modification for a
variety of therapeutics molecules either by specific adsorption or by covalent
bioconjugation.20–22,27–30 Due to their hollow cylindrical structure, CNTs provide
internal cavities which are capable of accommodating small molecules or ions.17
While the toxicological effects of CNTs have been widely debated in the literature,31–
34it
has been shown recently that appropriately functionalized35 and highly-purified
SWCNTs can be nontoxic and well-tolerated in vivo.36 In addition, CNTs are
considered to be relatively bioinert, even though one study has reported that they
can be degraded in vivo by neutrophils.37 Several reports have demonstrated that
CNTs readily cross cell membranes due to their intrinsic lipophilic character and
high aspect ratio (needle-like structure) and are able to shuttle biological molecules
including drug molecules, proteins, plasmid DNA, and siRNA into cells.27,28,38,39
Relying on their optical properties including strong near-infrared (NIR) optical
absorption, resonance Raman scattering, and photoluminescence, SWCNTs have
been exploited as optical imaging or contrast agents for tracking, detecting, and
10
imaging purposes.22,40–43 Moreover,
optical or radiofrequency
wavelength
irradiation of SWCNTs can also selectively be used for photothermal and
photoacoustic therapy.24,44,45 Finally, CNT materials, whether derivatized or
underivatized, have been observed to be eliminated from animals via the
kidneys.36,46 Taken together accumulating evidence suggests that, CNTs have the
potential to play a significant role in the diagnosis and therapy of cancer.
2.4. CNTs as nanocapsules for drug delivery
Drug delivery is one of the most promising biomedical applications of CNTs,
and over the years, several CNT-based systems have been developed with the
intention of obtaining better results for drug delivery. In these systems, drug
molecules have been mainly attached to the surface or sidewalls of CNTs either by
specific adsorption or by covalent bonding.20,21,29,30,47–49 In addition to surface
attachment, it has been shown that such molecules can be encapsulated within the
interior cavity of CNTs.50–52 Encapsulation of drug molecules within the interior of
CNTs may to be more beneficial than attachment to the external surface for several
reasons. First, the cytotoxicity of drug molecules can be sequestered within CNTs
interior by providing an insulating environment for the drug that prevents
degradation and leakage during circulation before the drug reaches its target sites.
Secondly, attaching a drug molecule to the external walls of CNTs typically involves
covalent or non-covalent conjugations which may alter the pharmacological activity
of the drug. Moreover, the interior of CNTs has a favorable binding energy for the
adsorption of lipophilic molecules, making it possible to encapsulate lipophilic drug
11
molecules by simple adhesive forces. Finally, encapsulation of drug molecules
within the CNT interior space also preserves the external surface of CNTs for further
chemical modification for desired cancer cell targeting with peptides and/or
antibodies.
Although the ideal length of CNTs for biomedical application is uncertain, but
US-tubes (~1.4 nm × 20 - 80 nm), which are produced from full-length SWCNTs via
a fluorination and pyrolysis procedure53 (Figure 2.2), may be best suited due to
their short and relatively uniform-lengths (ca. 95% ≤ 50 nm) which might help them
avoid the reticuloendothelial system (RES), while enhancing their cellular uptake
properties and eventual elimination profiles. US-tubes, with sidewall defects from
the chemical cutting process, have proven a convenient platform as nanocapsules
for the loading and containment of ions, molecules, and drugs whose cytotoxicity is
sequestered within the US-tubes.54–58 Additionally, it has been demonstrated that
the exterior surface of US-tubes can be modified with chemical moieties for
enhanced solubilization, with peptides for biological targeting purposes and with
monoclonal antibodies (MAbs) for the specific targeting of cancer cells.59,60
Moreover, a medical imaging agents derived from US-tubes (gadonanotubes) has
been shown to translocate into cells without exhibiting significant toxicity.61 Finally,
one recent study36 has shown that highly-purified SWCNTs and US-tubes are welltolerated in vivo by Swiss mice, even at very high doses (~0.5 g kg-1 b.w.).
12
Figure 2.2 − Preperation of US-tubes from SWCNTs.
Chapter 3
Cisplatin@US-tube carbon nanocapsules for
enhanced chemotherapeutic delivery1
3.1. Introduction
Chemotherapy is one of the main treatments for both localized and
metastasized cancer, and it is typically-used in conjunction with surgery and/or
radiotherapy. For chemotherapy, it is desirable to deliver a sufficient quantity of
drug to malignant cells, while minimizing undesirable side effects. However, the
efficacy of many drugs is hindered due to complications with administration such as
limited solubility, rapid elimination, inefficient distribution, inability to cross
1
Major portions of this chapter have been published in the following research article: Guven, A.;
Rusakova, I. A.; Lewis, M. T.; Wilson, L. J. Cisplatin@US-tube Carbon Nanocapsules for Enhanced
Chemotherapeutic Delivery. Biomaterials 2012, 33, 1455–1461.
13
14
cellular barriers, and the inability to differentiate between normal cells and cancer
cells. CDDP is one of the most potent and widely-used anticancer drugs for the
treatment of a variety of solid tumors, including testicular, ovarian, bladder, cervical,
head and neck, oesophageal, small-cell lung and breast cancers.4,5 The biological
activity of CDDP is derived from its interactions with DNA since CDDP interferes
with the normal transcription and replication processes of the cell which results in
cell death.5,11 Despite its success, the clinical use of CDDP and its derivatives are
limited due to severe side effects including nephrotoxicity, neurotoxicity,
ototoxicity, nausea, and vomiting.8
Thus, the development of efficient drug delivery systems that selectively
increase the concentration of chemotherapeutics, such as CDDP, in diseased cells
while limiting their concentration in normal cells continues to be of great interest.
Although a variety of drug delivery systems, including microspheres,62,63
silica nanoparticles,64,65 dendrimers,66 polymeric micelles,67–69 liposomes,70–72 and
carbon nanohorns,73,74 can be used as drug delivery agents, CNTs offer a number of
special properties which suggest that they too might be engineered into desirable
drug delivery platforms to compliment other more typically-used materials. To date,
a number of CNT based drug delivery systems have been explored. In these systems,
drug molecules mainly have been attached onto the surface or sidewalls of the
nanotubes either by specific adsorption or by covalent bonding.21,29 In addition to
surface attachment, it has been shown that small drug molecules can also be
embedded75 or encapsulated50 within SWNT materials. In this chapter, we describe
the preparation, characterization, and in vitro testing of a new US-tube-based drug
15
delivery platform for the treatment of cancer, which allows for the possible design
of a cancer-specific enzyme-activatable US-tube-based drug delivery platform that
releases its drug cargo within cancer cells only.
3.2. Materials and Methods
3.2.1. Preparation of US-tubes
US-tubes were prepared from full-length SWCNTs (purchased from Carbon
Solutions Inc., CA, USA), which were produced by the electric-arc discharge method,
as previously reported.53 To remove amorphous carbon and metal catalyst
impurities, US-tubes were first bath-sonicated in concentrated HCI for 1 h, then
collected by filtration on a glass filter and finally washed several times with distilled
water. These purified US-tubes were then chemically reduced by a metallic Na°
/THF reduction procedure to create individual US-tubes.76
3.2.2. Encapsulation of CDDP within US-tubes
Chemically debundled US-tubes (20 mg) were dispersed in deionized water
(20 mL) via bath-sonication for 1 h. CDDP (20 mg) was then added to the US-tube
suspension and vigorously stirred for 24 h. After stirring, the solution was left
undisturbed overnight whereupon CDDP-loaded US-tubes (CDDP@US-tubes)
flocculated from solution. After removing the supernatant solution, CDDP@US-tubes
were collected on a glass filter. To remove the excess CDDP from the outer surface of
the US-tubes, the CDDP@US-tubes were washed forty times with deionized water
16
(total of 60 mL) until all exterior CDDP was removed, as judged from Pt analysis on
the filtrate aliquots by ICP-OES (see below). The collected CDDP@US-tubes were
then dried in an oven at 80 ˚C.
3.2.3. Characterization of CDDP@US-tubes
To confirm the encapsulation of CDDP within the US-tubes, HR-TEM studies
were performed using a JEOL 2000 FX microscope equipped with an energydispersive spectrometer (EDS) and operated at 200 kV. Conventional and HR-TEM
imaging methods were used. Precautions were taken to minimize structural changes
in the sample caused by electron-beam heating effects. One such precaution was the
use of a cold sample stage which kept the sample temperature in the range of -70 ˚C
to -50 ˚C. The HR-TEM samples were prepared by placing a small amount of
CDDP@US-tube sample on a copper grid coated with amorphous carbon holey film.
No solvents were used during the procedure.
To confirm that CDDP was removed from the exterior surface of US-tubes by
washing, washings were collected and analyzed by ICP-OES. Elemental analyses of
CDDP@US-tube samples were carried out by multiple analytical techniques: XPS,
EDS, and ICP-OES. For XPS analysis, solid samples were placed on indium foil
(Sigma-Aldrich) and then examined using a PHI Quantera Spectrometer with photo
and Auger emissions produced via a monochromatic Al Ka X-ray source with a pass
energy of 140 eV for survey and 30 eV for high-resolution scans. EDS measurements
were carried out using an OXFORD EDS system installed in a JEOL-2100F TEM
operating at 200 kV. Samples for EDS measurements were prepared via bath-
17
sonication in methanol for a few min, and a drop of dispersed solution was then
placed on a TEM copper grid. ICP-OES analyses were made using a Perkin Elmer
Optima 4300 DV instrument with a CDD detector. The quantity of CDDP internalized
within the US-tubes was quantified by measuring the Pt concentration of each
sample. Samples for ICP-OES measurements were prepared by digesting the
CDDP@US-tubes with 26% HCIO3, as previously described.61 The platinum signal
was observed at 265.95 nm. Five scans were performed for each sample, and the
concentration was determined from the average of scans. Yttrium was used as the
internal standard.
3.2.4. CDDP release studies from CDDP@US-tubes
CDDP release studies from US-tubes were performed by enclosing the
CDDP@US-tubes inside a dialysis-membrane (20,000 MW) cylinder, which was
immersed in PBS at 37 ˚C for one week. During immersion, the solid CDDP@US-tube
sample in the dialysis-membrane cylinder was sampled periodically by filtration
and collection on a glass frit filter, followed by washing with distilled water to
remove PBS. The Pt concentrations of solid CDDP@US-tube samples were then
determined by ICP-OES. CDDP release with time from CDDP@US-tube samples was
also determined after dispersing the CDDP@US-tube samples in a 0.17% (w/v)
Pluronic®-F108 solution via probe sonication for 2 min to produce Pluoronic®F108- wrapped CDDP@US-tubes (W-CDDP@US-tubes). Pluronic®-F108 is a
neutrally charged, non-cytotoxic surfactant that is commonly used to administer
18
carbon nanotube materials for in vitro and in vivo testing.61 Release of CDDP from
W-CDDP@US-tubes and CDDP@US-tubes was then compared.
3.2.5. Cell viability studies
The cytotoxic activity of CDDP released from W-CDDP@US-tubes was
evaluated against two different breast cancer cell lines (MCF-7 and MDA-MB-231)
by using MTT cell proliferation assay (Invitrogen). This colorimetric assay quantifies
cell proliferation and viability by measuring the reduction of the tetrazolium
component (MTT) into an insoluble formazan product by the mitochondria of viable
cells. The cells were cultured in Minimum Essential Medium (MEM) supplemented
with 10% FBS, 1% penicillin/streptomycin, and 0.15% insulin (10 mg mL -1) at 37 ˚C
under a humidifying atmosphere containing 5% CO2. The cells were then seeded
onto 96-well plates at a density of 5 × 103 cells well-1 in 150 µL MEM. After 24 h of
incubation to allow cell adhesion, the medium in each well was replaced with 150
µL of new medium containing the serial dilution of either CDDP,W-CDDP@US-tubes,
or W-US-tubes at various concentrations (5 - 50 µM), and cells were then incubated
at 37 ˚C in 5% CO2 for 24 h, and 48 h. An equivalent US-tube concentration of
CDDP@US-tubes and US-tubes was used for each of the concentration point by
calculating the US-tubes concentration of CDDP@US-tubes from Pt wt % values
obtained by ICP-OES analysis. After incubation for each time period, the medium in
each well containing CDDP, W-CDDP@US-tubes, or W-US-tubes was aspirated and
the cells washed with phosphate-buffered saline (PBS). For cell viability
measurements, 100 µL of fresh medium (free of phenol red) and 10 µL of MTT
19
reagent (5 mg mL-1 in PBS) was added to each well and incubated for 4 h to allow
the formation of formazan crystals. After removing all but 25 µL of medium from the
wells, 50 µL of dimethyl sulfoxide (DMSO) was added to each well (to solubilize the
formazan) and mixed thoroughly and incubated at 37 ˚C for an additional 10 min.
The absorbance values were measured using a microplate reader (Bio-Rad Model
680) at 570 nm to determine relative viabilities.
3.2.6. Cell internalization rate studies
To determine the comparative cell internalization rate of free CDDP and
WCDDP@US-tubes into MCF-7 cells, the cells were plated on a cell culture dishes at
5 × 105 cell/dish and allowed to reach up to 70 - 80% confluence. After removing
the medium in each dishes, the attached cells were first washed with PBS and then
incubated with free CDDP and W-CDDP@US-tube samples in MEM at 10 mm CDDP
concentration for various times (3, 6, 12, 24, and 48 h). After each incubation time,
the cells were washed with PBS, collected from the dishes by lifting up with trypsinEDTA, counted with a cell counter (Cellometer Auto T4), and prepared for ICP-OES
analysis to quantify Pt in each sample.
3.2.7. Transmission electron microscopy
MCF-7 cells were routinely cultured as described earlier and grown on a cell
culture dish, and incubated with W-CDDP@US-tubes (10 μM CDDP) at 37 ˚C. After
24 h incubation, the cell monolayers were briefly rinsed with PBS, and fixed in situ
with a primarily fix solution (2% PF + 2.5% glutaraldehyde + 0.1M cacodylate +
20
2mM CaCl2 in sodium cacodylate buffer, pH7.4) for 1 h at room temperature (RT) on
a tissue rotator, washed with buffer (3x5min in 0.1M sodium cacodylate buffer +
2mM CaCl2, pH 7.4), post-fixed with 1% OsO4 in 0.1M sodium cacodylate buffer for 1
hour, rinsed with H2O (3x5min), saturated (stained) with aq. uranyl acetate for 45
min in the dark (foil wrapped), washed with H2O (3x5min), dehydrated through a
series of graded alcohol washes in the following order: 2x10min in 30%, 50%, 70%,
and 90%, 3x15min in 95%, and 3x20min in 100% ethanol, and changed 3 times in
Epon (1 h each). After removing the last change of Epon, the cell monolayers were
embedded in a fresh change of Epon plastic and polymerized at 70 ˚C over night. For
comparison, in a separated dish, MCF-7 cells without incubating with W-CDDP@UStubes were also grown and prepared for TEM as a control group. Several 1 μm
sections were cut and stained with 1% methylene blue and 1% basic fuchsin. Ultrathin sections (80 nm) were cut from the sample block using an RMC MTXL ultra
microtome and mounted on 100-mesh copper grids. The grids were stained with
2% alcoholic uranyl acetate and Reynold’s lead citrate. The samples were prepared
by the Integrated Microscopy Core at Baylor College of Medicine, and examined with
a JEOL 1230 TEM at 120kV equipped with a digital imaging system at Rice
University.
3.2.8. Statistical evaluation
Unless otherwise noted, all data are expressed as mean ± standard
deviation(SD) of at least three independent experiments. Wherever appropriate, the
statistical significance of data were analyzed using the Microsoft Excel statistical
21
package. A one-tail homoscedastic unpaired t-test was used to determine statistical
significance; a p-value of less than 0.05 (p < 0.05) was considered statistically
significant.
3.3. Results and Discussion
3.3.1. Encapsulation of CDDP within US-tubes
The encapsulation of CDDP within US-tubes has been achieved as detailed in
the Materials and Methods Section and as outlined in Figure 3.1. After an extensive
washing procedure (>20×) with deionized water, 10 mL aliquots of each washing
were collected and analyzed for Pt content by inductively-coupled plasma optical
emission spectrometry (ICP-OES) to determine CDDP release over time. As shown in
Figure 3.2, the majority of CDDP was removed by the first few washings with water.
Since no additional removal of CDDP was observed after 20 washings, it was
concluded that all exterior CDDP was removed by 20 washings.
22
Figure 3.1 − Preparation and purification of CDDP@US-tubes.
23
P t c o n c e n t r a t io n ( m g / L )
2 .0
1 .5
1 .0
0 .5
0 .0
0
10
20
30
40
N u m b e r o f w a s h in g s
Figure 3.2 − Pt determination by ICP-OES analysis of each collected aliquot of
H2O wash solution for a typical CDDP@US-tube sample. The data are
expressed as mean ± SD of three independent experiment. Error bars may be
smaller than symbols.
After the extensive washing procedure, the CDDP@US-tubes were collected
by filtration and dried in an oven at 80 ˚C. To determine the quantity of CDDP within
the US-tubes, the Pt concentration was determined via ICP-OES. Because of the
limited solubility of CDDP in water, CDDP@US-tube preparation was also attempted
in a few different solvent mixtures in which CDDP is more soluble, and the results
are summarized in Table 3.1. Although CDDP has greater solubility in ethanol
(EtOH) and dimethylformamide (DMF), greater loading was achieved when water
24
alone was used. For this reason, further preparations were performed in water to
enhance CDDP@US-tube yields.
Material
Loading Solvent
wt% Pt
(after one
washing with
H2O)
wt% Pt
(after 40
washings with
H2O)
CDDP@US-tubes
H2O
8.70 ± 0.43
6.37 ± 0.57
CDDP@US-tubes
H2O:EtOH (1:1)
6.66 ± 0.31
4.42 ± 0.35
CDDP@US-tubes
H2O:DMF (1:1)
8.51 ± 0.34
3.38 ± 0.30
CDDP@SWCNTs
H2O
6.27 ± 0.89
0.48 ± 0.11
Table 3.1 − Pt content by ICP-OES of CDDP@US-tubes under different loading
solvent and washing conditions. EtOH is ethyl alcohol and DMF is
dimethylformamide. Table presents the wt% Pt for an average of three trials
under each condition and the standard deviation.
A CDDP@US-tube sample has been characterized by high-resolution
transmission electron microscopy (HR-TEM). As seen in Figure 3.3a, CDDP clusters
are observed as dark spots encapsulated along the interior axis of the US-tubes, with
both Pt and Cl content confirmed by electron-dispersion spectroscopy (EDS). For
comparison, empty US-tube images do not reveal such dark spots (Figure 3.3b).
Since the dimension of an individual CDDP molecule is about 0.4 nm × 0.6 nm × 0.1
nm,77 CDDP molecules have the ability to easily fit within US-tubes (ca. 1.4 nm in
diameter) and cluster at the sidewall defect sites. Based on the CDDP aggregate sizes
25
(black spots) which ranged in length from 0.75 to 1.0 nm, up to four or five CDDP
molecules exist in each aggregate. Earlier theoretical calculations have also
concluded that CDDP can easily fit within the confines of SWCNTs.78
C
c)
a)
Cl
Cu
Cu
b)
Pt
Cl
0
2
Cu Pt
Ni
8
10
Energy (keV)
Figure 3.3 − HR-TEM images of a) bundled CDDP@US-tubes and b) bundled
empty US-tubes c) EDS spectrum of CDDP@US-tubes showing carbon and
nickel peaks (metal catalyst) from US-tubes, copper peaks (from the TEM Cugrid), and platinum and chloride peaks (from CDDP).
EDS analysis was also performed on a solid CDDP@US-tube sample (Figure 3.3c).
EDS detected carbon, nickel (metal catalyst) from US-tubes, copper (from the TEM
grid), and platinum and chloride (from CDDP). EDS also determined the same Pt:Cl
(1:1) mole ratio as that of the XPS data.
To determine whether the CDDP Pt center remains in its original oxidation
state (Pt2+) after encapsulation within US-tubes, the X-ray photoelectron
26
spectroscopy (XPS) spectra of the Pt 4f peaks of CDDP@US-tubes, metallic platinum
(Pt0), and sodium hexachloroplatinate hexahydrate, (Na2PtCl6.6H2O; Pt4+), were
obtained. Figure 3.4a shows the survey XPS spectrum of CDDP@US-tubes,
confirming the presence of platinum, chloride, and nitrogen from CDDP and carbon
and oxygen from US-tubes. Figure 3.4b shows that the Pt 4f peaks of the CDDP@UStube sample were positioned between the Pt 4f peaks of metallic platinum and
Na2PtCl6.6H2O, confirming that the CDDP@US-tube sample contains Pt2+ and not
reduce Pt0 or oxidize Pt4+. For comparison, we also obtained the Pt 4f spectrum of
free CDDP (Strem Chemicals) which displayed peaks at 72.30 eV and 75.65 eV. On
the other hand, the CDDP@US-tube sample exhibited Pt 4f peaks at 72.90 eV and
76.25 eV, corresponding to a +0.60 eV shift from that of free CDDP sample. This
difference apparently arises from the environment of the encapsulating US-tubes.
XPS also confirmed a 2:1 N:Cl ratio and a 2:1 N:Pt ratio, suggesting that the
extensive washing process with water to prepare the CDDP@US-tube sample nearly
halved the amount of chloride ion present in the original CDDP sample, likely
replacing them with water molecules.79 Since the loss of chloride ions from CDDP is
desirable before CDDP binds to DNA and other cellular targets,13 the loss of chloride
ions in the CDDP@US-tube sample should not affect the efficacy of the Pt2+ species
released from the US-tubes.
27
0
m e t a l lic p l a t in u m ( P t )
a)
b)
4
4 1 0
4
- C 1s
6 1 0
C D D P (P t
2+
2+
)
)
N a 2 P t C l 6 .6 H 2 O ( P t
4+
)
68
70
72
74
76
78
80
B in d i n g E n e r g y ( e V )
82
- O KLL
- O 1s
- N 1s
- Pt 4d
5
- Pt 4d
- Cl 2s
4
- Cl 2p
2 1 0
- Pt 4f
7
c /s
3
8 1 0
4
C D D P @ U S -tu b e s (P t
0
0
200
400
600
800
1000
B in d i n g E n e r g y ( e V )
Figure 3.4 − XPS spectrum of a) CDDP@US-tubes (survey), b) Pt 4f peaks for
various samples: i) metallic platinum (Pt0), ii) CDDP@US-tubes (Pt2+), iii)CDDP
(Pt2+) and iv) Na2PtCI6.6H2O (Pt4+).
3.3.2. Release studies of CDDP from CDDP@US-tubes
Release of CDDP from a drug delivery system is an important parameter that
should occur in a controlled fashion. To test the release of CDDP from CDDP@UStubes, we prepared a sample in which the CDDP@US-tube sample was dispersed via
probe sonication for 2 min in an aqueous 1.7 mg mL-1 Pluronic®-F108 surfactant
solution. The pluronic-wrapped CDDP@US-tube sample (W-CDDP@US-tubes) was
expected to slow CDDP release since the sidewall defects and ends of US-tubes
should be, at least, somewhat blocked by surfactant. Dialysis was performed on both
CDDP@US-tubes and W-CDDP@US-tubes in phosphate-buffered saline (PBS) at 37
˚C. The CDDP release rate for these two samples was investigated by determining
the Pt content of samples over time by ICP-OES analysis. Figure 3.5 displays the
28
comparative release profiles of CDDP from CDDP@US-tubes and W-CDDP@UStubes. From the obtained data, we observed that the W-CDDP@US-tube system
leaked only 6.1% CDDP in 24 h and only 16.9% over one week. However,
unwrapped CDDP@US-tubes showed a much greater rate of release, with almost
50% of CDDP released in the first 3 h and about 80% after one week. From this data,
we infer that the release of CDDP from W-CDDP@US-tubes is retarded because the
pluronic wrapping around the US-tubes helps prevent drug release.
C D D P @ U S -tu b e s
100
R e le a s e d C D D P ( % )
80
60
W -C D D P @ U S -tu b e s
40
20
0
0
50
100
150
200
250
T im e ( h )
Figure 3.5 − Comparative time release profiles of CDDP released from
CDDP@US-tubes and W-CDDP@US-tubes (Pluronic-F108-wrapped) in PBS at
37 °C. The data are expressed as mean ± SD of three independent experiments.
29
3.3.3. In vitro cytotoxicity studies
A series of in vitro studies have been conducted to determine the cytotoxicity
profiles
of
W-CDDP@US-tubes
using
the
well-established
MTT
(3-(4,5
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay.
The performance of W-CDDP@US-tubes was evaluated against two different breast
cancer cell lines (MCF-7 and MDA-MB-231), which are known to be resistant to
CDDP treatment,80 at 24 h and 48 h incubation times, and their efficacies compared
to that of free CDDP. Figure 3.6 displays the comparative cytotoxicities of CDDP, WUS-tubes, and W-CDDP@US-tubes in a dose- and time-dependent manner. The
cytotoxicity of CDDP and W-CDDP@US-tubes both increased with increasing CDDP
concentration. While the US-tubes alone have a relatively small effect on cell
viability for concentrations ≤20 mM, the W-CDDP@US-tubes clearly exhibited
greater cytotoxicity for both cell lines and even greater cytotoxicity when compared
to free CDDP at all CDDP concentrations. Although, the W-CDDP@UStubes displayed
greater efficacy for each incubation time than free CDDP at an equivalent dose, the
efficacy of the W-CDDP@US-tube sample was greater at 24 h than 48 h. To
investigate this result, we decided to determine the Pt concentration per cell for the
MCF-7 cell line treated with CDDP and W-CDDP@US-tubes as a function of time.
30
CDDP
W -C D D P @ U S -tu b e s
120
120
100
100
80
*
60
*
*
40
20
*
C e l l v i a b il i t y ( % )
C e l l v i a b il i t y ( % )
M C F - 7 c e ll s
W -U S - t u b e s
80
60
40
*
24h
#
0
0
10
20
30
40
50
0
C D D P c o n c e n tr a tio n ( M )
120
120
100
100
80
*
60
*
40
*
*
20
10
20
30
40
50
C D D P c o n c e n tr a tio n ( M )
C e l l v i a b il i t y ( % )
C e l l v i a b il i t y ( % )
#
48h
0
M D A - M B - 2 3 1 c e lls
*
20
80
60
#
40
20
24h
48h
0
0
0
10
20
30
40
50
C D D P c o n c e n tr a tio n ( M )
0
10
20
30
40
50
C D D P c o n c e n tr a tio n ( M )
Figure 3.6 − Time and concentration dependency of cell viability studies for
MCF-7 and MDA-MB-231 cells incubated with CDDP, W-CDDP@US-tubes, and
W-US-tubes. The quantity of US-tubes at each CDDP concentration is
equivalent for the CDDP@US-tube and US-tube samples, with the
concentration being calculated from their wt % values obtained by ICP-OES
for Ni and Y which is contained within US-tube materials as the metal catalysts
for carbon nanotube growth. The data are expressed as mean ± SD for three
independent experiments. (*) and (#) indicate a statistically significant
difference between W-CDDP@US-tube data compared to free CDDP data (*p <
0.01, #p<0.05).
31
These results are given in Figure 3.7 which demonstrate that the cells treated with
W-CDDP@US-tubes have significantly more Pt than cells treated with free CDDP
over the first 24 h time period, however by 48 h, the Pt concentration in per cell is
about the same for both W-CDDP@US-tubes and free CDDP. Thus, the US-tube
structure apparently assists the delivery of free CDDP from W-CDDP@US-tubes into
the cells over the first 24 h. In addition, the release rate of CDDP from W-CDDP@UStubes in vitro is likely greater than in only PBS (ca. 6% after 24 h) because
previously observed aggregation of US-tubes within mesenchymal stem cells has
indicated the removal of the Pluronic coating from US-tubes upon passage through
the cell membrane.61 All of the above results taken together demostrate that WCDDP@US-tubes are more cytotoxic than free CDDP at the 24 h time point (p <
0.05).
32
CDDP@US-tube
ns
CDDP
8E+10
Number of Pt ions per cell
ns
*
6E+10
*
4E+10
*
2E+10
0
3
6
12
24
48
Time (h)
Figure 3.7− Comparative internalization rate of CDDP and W-CDDP@US-tubes
into MCF-7 cells as a function of incubation time at 10 µM CDDP concentration.
The graph presents data for the average number of Pt2+ ions per cell for an
average of three trials. Error bars are standard errors for the three different
experiments. (ns=not significant, *p-value<0.05)
We have also confirmed the uptake of CDDP@US-tubes into the cytoplasm of
MCF-7 cells (Figure 3.8), although the uptake mechanism pathway or pathways have
not been fully investigated yet. The cellular uptake of W-CDDP@US-tubes, which
appears as irregular electron-dense aggregates within the cytoplasm as opposed to
free MCF-7 cells (Figure 3.8), has been confirmed by TEM analysis; however, both
vesicularized and free CDDP@US-tube aggregates have been observed to exist in the
cells. To determine the dominant intracellular uptake mechanism of CDDP@US-
33
tubes within MCF-7 cells will require more extensive studies, which are currently
underway and will be reported subsequently.
(a)
(c)
(e)
(b)
(d)
(f)
Figure 3.8 − (a, b) TEM images of a single MCF-7 cell from different angles. (c,
d, e, f)TEM images of MCF-7 cells incubated with W-CDDP@US-tubes at 10 µM
CDDP for 24 h. White arrows point to (a, b) ‘vesicularized’, and (c, d) ‘free’
CDDP@US-tube aggregates in the cytoplasm. Scale bar = 2 µm.
Since both cell lines used in this study are resistant to CDDP, and it is wellknown that reduced drug uptake is a major cause of CDDP resistance in cancer cell
lines,11,13 the present data suggests that CDDP@US-tube materials help to overcome
CDDP resistance by increasing drug accumulation in resistant cells.
34
3.4. Conclusions
In this study, CDDP encapsulation within US-tubes has been confirmed by
HR-TEM. Elemental analysis (XPS, and EDS) of CDDP@US-tubes has revealed that
about half of the chloride ions of the encapsulated CDDP are replaced with water
molecules during the aqueous washing procedure to prepare CDDP@US-tubes. It
has also been shown that CDDP@US-tubes release CDDP upon dialysis in PBS at 37
°C much more slowly when CDDP@US-tubes are wrapped with Pluronic-F108
surfactant. Finally, the CDDP@US-tube platform exhibited greater cytotoxicities
against MCF-7 and MDA-MB-231 cells at 24 h, when compared to free CDDP. These
results suggest that by wrapping CDDP@US-tubes with cancer-specific enzymeactivatable peptide sequences, that cancer-cell-specific chemotherapeutics might be
developed using the carbon nanocapsule platform of this study.
Chapter 4
Remotely-Triggered Cisplatin Release From
Carbon Nanocapsules by Radiofrequency
Fields2
4.1. Introduction
Maximizing therapeutic efficacy and minimizing adverse effects remain the
fundamental goals of drug delivery in cancer therapy. A vast majority of cancer
chemotherapeutics, while potent agents, cannot be employed to full clinical benefit
because of toxicity concerns. Over the past decade, drug-loaded nanocarriers have
been widely fabricated and studied to enhance tumor specific delivery. Most
2
This chapter has been published in the following research article: M. Raoof, B. T. Cisneros, A.
Guven, S. Phounsavath, S. J. Corr, L. J. Wilson, S. A. Curley. “Remotely triggered cisplatin release from
carbon nanocapsules by radiofrequency fields” Biomaterials 2013, 34, 1862-1869.
35
36
strategies that involve either active or passive targeting have been successful in
demonstrating tumor-specific accumulation of these nanocarriers to achieve anticancer toxicity in pre-clinical models. However, recent studies demonstrate that
intra-tumoral distribution of nanocarriers is patchy rather than homogenous.81–84
Nanocarriers extravasate from the vascular space and tend to accumulate in the
peri-vascular space through fenestrations in tumor vasculature (also called
enhanced permeation and retention effect).85 There are a multitude of factors that
limit subsequent permeation of nanocarriers from the peri-vascular space to tumor
cells, including the size of the nanocarriers, interstitial fluid pressure within the
tumor, variations in pH in the microenvironment, composition of the extracellular
matrix, and lymphatic clearance of nanocarriers.84,86–89 The spatial separation
between drug-loaded nanocarriers and tumor cells often renders this strategy futile.
To take advantage of intra-tumoral drug depots that are formed by these
nanocarriers, environmentally- or remotely-triggered drug release mechanisms are
being developed. The premise is based on the notion that free drug, because of its
smaller molecular size, can rapidly diffuse along its concentration gradient to cells
distant from the nanocarrier depots. Environmentally sensitive nanocarriers exploit
pH,90 temperature,91–93 or the presence of tumor-specific enzymes (such as matrix
metalloproteinases94) to trigger drug release. This strategy is inherently limited by
the
nonhomogeneous
microenvironment.95
pH
and
enzyme
Temperature-sensitive
concentrations
nanocarriers
in
(such
the
tumor
as
certain
liposomes) depend on a strategy that raises bulk tumor temperature above a critical
transition temperature to accomplish a phase change.96 However, raising bulk
37
tumor temperature selectively and safely remains a significant challenge. Because of
these limitations, nanocarriers that can be remotely-activated or disrupted by
focused ultrasound, light, or alternating magnetic fields to trigger drug release are
under investigation. In each case, the nanocarrier is comprised of a nano-susceptor
that absorbs energy and dissipates it locally to actuate release. Alternating magnetic
fields offer full-body-penetration but acoustic and photo-triggered drug release are
less ideal because of the non-uniformity and limited depth of penetration. These
studies highlight the need for innovative strategies to enhance efficacy of
nanocarriers to increase drug or biologic agent release after tumoral delivery.
In developing remotely-triggered nanocarriers, carbon nanotubes are of
particular interest to us. SWCNTs have been derivatized, functionalized, and loaded
or conjugated with chemotherapeutics.97 More importantly, SWCNTs exhibit unique
properties in radiofrequency (RF) electric fields such as enhancement of local Efields, absorption and dissipation of RF energy as heat, and alignment parallel to the
incident E-field.24,98,99 Because RF fields at low megahertz frequency offer the
advantage of full-body-penetration, nanotube-RF coupling can potentially be
exploited to triggered release. However, full-length SWCNTs have a very high aspect
ratio and, therefore, may not be ideal for drug delivery applications. To address this
problem, US-tubes have been fabricated using a fluorination and pyrolysis process.53
The defects created in the US-tube sidewalls by this procedure allow for easy
loading of cytotoxic chemotherapeutics, e.g. cisplatin (CDDP), contrast agents (Gd3+,
I2),
or
radioisotope
agents
(211AtCl).54,58,100
Moreover, in vivo studies
have
demonstrated that US-tubes are non-toxic and well tolerated by mice even at very
38
high doses (∼0.5 mg/kg) making them a suitable candidate for nanocarrier
development.36
We hypothesized that we could produce US-tubes loaded with CDDP and
wrapped with a Pluronic coating to prevent CDDP release from the US-tubes. We
further hypothesized that heating of the surface of US-tubes by a non-invasive RF
field would lead to loss of Pluronic coating and CDDP release (Figure 4.1). Our
studies were designed to confirm and characterize Pluronic-wrapped CDDP@UStubes (W-CDDP@US-tubes), to measure CDDP release from W-CDDP@US-tubes
following RF field exposure, and to demonstrate RF-induced CDDP release at levels
relevant to produce cancer cell cytotoxicity.
39
Figure 4.1 − Schematic describing synthesis, purification, and RF-triggered
disruption of Pluronic®-F108 and subsequent release of CDDP from WCDDP@US-tubes.
4.2. Methods
4.2.1. Sample Preperation
Pluoronic-F108-wrapped
CDDP-US-tubes
(W-CDDP-US-tubes)
were
prepared as described previously.100 In brief, full-length SWCNTs (Carbon Solution
Inc., Riverside, CA), produced by an electric-arc discharge method, were cut into UStubes by fluorination followed by pyrolysis at 1000 °C under an inert atmosphere.
40
US-tubes were first purified in concentrated HCl by bath-sonication to remove
amorphous carbon and metal catalyst impurities (nickel and yttrium), then
chemically reduced by a metallic Na°/THF reduction procedure to produce
individual US-tubes.76 The debundled US-tubes were dispersed in deionized water
via bath-sonication for 60 min, and then CDDP was added to the US-tube suspension
and vigorously stirred for 24 h. The reaction vessel was then left undisturbed
overnight whereupon CDDP@US-tubes flocculated from solution. The CDDP@UStubes were collected by filtration on a glass filter, washed with excess deionized
water to remove all exterior CDDP from the outer surface of the US-tubes (as judged
from Pt analysis on the filtrate aliquots by ICP-OES), and dried at 80 °C. WCDDP@US-tubes were prepared by suspending dry CDDP-US-tube samples in a
0.17% (w/v) Pluronic-F108 solution via probe sonication for 2 min, followed by
centrifugation at 3200 rpm for 10 min (3×) to remove unsuspended CDDP@UStubes. Extensive characterization of this CDDP@US-tubes material has been
reported previously.100
4.2.2. Inductively-coupled plasma optical emission spectrometry (ICP-OES)
ICP-OES analyses were carried out by a Perkin Elmer Optima 4300 DV
instrument with a CCD detector. The quantity of CDDP in each sample was
determined by measuring the Pt concentration, which was detected at 265.95 nm.
For all ICP-OES measurements, the collected samples were transferred into glass
vials, then digested with 26% HClO3. The samples were then diluted with 2% trace
41
metal grade HNO3. The Pt concentrations were determined from an average of five
scans for each sample. Yttrium (371.029 nm) was used as the internal standard.
4.2.3. RF generator setup
We employed the Kanzius External RF (13.56 MHz) Generator System
(ThermMed, LLC, Erie, PA) as described previously.101 The generator utilizes a
capacitively-coupled transmitter and receiver head to generate a uniform RF field
within an air-gap of 10 cm. The generator input power was arbitrarily set at 950 W
for all experiments. To demonstrate heating of US-tubes or CDDP@US-tubes, a
suspension was created using 0.17 % (w/v) Pluronic® F-108 in deionized water or
in PBS. Varying concentrations were loaded in a 1.3 mL quartz cuvette. The cuvette
was placed in a Teflon holder and positioned at 5/16 inch from the transmitter
head. The incident E-field intensity at this position was estimated to be 700 kV/m.
The temperature of the cuvette was recorded using an infrared thermal camera
(FLIR Systems, Boston, MA). Heating rates were calculated between 5 and 120 s of
RF exposure where the Time vs. Temperature plots were noted to be linear. In order
to expose 12-well cell culture plates to the RF field for the CDDP release studies and
cell studies, the 12-well plate was positioned at an arbitrary point in the RF field on
a Teflon holder. At this point the ionic heating of 1 mL PBS in a well of a 12-well
plate was noted to be (∼1 °C/min). This was used as an internal control before,
during, and after RF field exposures. The 12-well plates loaded with samples were
equilibrated to 37 °C in a cell culture incubator prior to each exposure. The plates
were then removed from the incubator and placed on the Teflon holder at an
42
ambient temperature of (20–22 °C). The plates were allowed to cool to 30 °C before
starting the RF exposure experiments. This step was necessary to distinguish bulk
from local heating effects.
4.2.4. CDDP release from W-CDDP@US-tubes
CDDP release studies from US-tubes were performed as previously described
but with some modification.100 Briefly, lyophilized CDDP@US-tubes were suspended
in PBS with 0.17% (w/v) Pluronic F108 at a US-tube concentration of approximately
700 mg/L. In order to expose the CDDP@US-tubes to various experimental
conditions, 1 mL of sample was placed in each well of a 12-well plate. The plate was
then exposed to the RF field or to constant temperature water bath hyperthermia at
appropriate experimental parameters as detailed in the results section. The samples
were then placed inside a dialysis-membrane (20,000 MW) cylinder, which was
immersed in 800 mL PBS at room temperature (22–24 °C) for 168 h. During
immersion, the W-CDDP-US-tube sample in the dialysis-membrane cylinder was
sampled periodically. The PBS solution in the container was under constant
circulation and was replaced at least 4 h before each sampling to maintain a
favorable concentration gradient.
4.2.5. Fluorescence measurements
For fluorescence studies, samples were exposed to varying parameters in a
12-well cell culture plate and after exposure, were transferred to a 96-well culture
plate at 0.2 mL per well. Fluorescence was measured using a Fluostar Omega (BMG
43
Labtech, Ortenberg, Germany) apparatus with the appropriate excitation and
emission filter sets for allophycocyanin. To demonstrate temperature-dependent
Pluronic disruption from the W-CDDP-US-tube surface, fluorescence was measured
using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA).
4.2.6. Cell studies
Both human liver cancer cell lines (Hep3B and HepG2) were purchased from
American Type Culture Collection (Manassas, VA) and maintained as per the
instructions of the supplier. Media, i.e., MEM (for HepG2 or Hep3B) was
supplemented with 10% (v/v) fetal bovine serum. Additional supplementation was
performed with sodium pyruvate, non-essential amino acids, Penicillin G and
Streptomycin. Cells were cultured in T-75 or T-150 tissue culture flasks (Corning
Inc., Corning, NY). For each cell line, the short tandem repeat fingerprint was
confirmed by the Cell Line Characterization Core Service (M. D. Anderson Cancer
Center, Houston, TX) within one year of all experiments. All media and supplements
were purchased from Gibco (Life Technologies, Grand Island, NY). The cells were
passaged approximately every three to five days before reaching confluency. Media
was replaced every three days.
Release-dependent cytotoxicity was assessed in a 0.4-micron trans-well
system (Corning Inc., Corning, NY). The W-CDDP@US-tubes were loaded in the top
well and sub-confluent monolayers were grown in the bottom well. Aggregates of
W-CDDP@US-tubes failed to pass through the filter as confirmed by UV–Vis (not
shown). Released CDDP could, however, diffuse through the filter and enter the
44
bottom compartment containing adherent cells with resultant CDDP-induced
cytotoxicity. Viability was assessed 120 h after drug exposure using a standard MTT
assay.
4.2.7. Statistical analysis
Unless otherwise stated, data points represent average from 3 to 6
independent experiments and error bars represent standard error of the mean. For
inferential statistics, a p-value < 0.05 was considered significant. Two group
comparisons were made using two-tailed t-test for an unpaired sample. Multiple
group comparisons were made using one-way analysis of variance (ANOVA) with
Bonferroni post-hoc test.
4.3. Results
4.3.1. RF absorption by US-tubes
We prepared US-tubes loaded with and without CDDP as detailed in the
methods. Heating of full-length SWCNTs has been demonstrated and theoretically
predicted under the influence of RF irradiation.40 It was desirable to determine if
US-tubes dissipate heat when exposed to low-frequency radiowaves. The Kanzius
RF generator was used for this purpose. US-tubes loaded with or without CDDP
were subjected to gentle filter centrifugation through a 10 kDa centrifugation filter
and resuspended in a solution of 0.17% (w/v) Pluronic-F108. This was repeated
several times until the filtrate heating rate was about the same as a solution of
45
0.17% Pluronic® F-108. This was necessary to eliminate ionic heating effects in the
RF field that have been previously reported.101
The heating rates for the two suspensions and their background are shown in
Figure 4.2. Here, it can be noted that both US-tube and CDDP-US-tube suspensions
heat in the RF field significantly above the background ionic heating. We also
observed that the heating rates were concentration-dependent. Comparing the
relative heating of US-tubes to CDDP@US-tubes, we found that drug loading
somewhat attenuated the heating of US-tubes under RF field exposure.
Figure 4.2 − Heating rates of W-CDDP@US-tubes and W-US-tubes suspended in
0.17% pluronic F108 in comparison with filtrate alone as a function of
concentration.
46
4.3.2. Localized heating of US-tubes in a physiological environment
In order to understand the heating of CDDP@US-tubes in a more
physiologically-relevant environment, CDDP@US-tubes in 0.17% (w/v) Pluronic®
F-108 were suspended in phosphate buffered saline (PBS, pH 7.4). Significant ionic
heating under RF exposure was observed when the W-CDDP@US-tubes were
suspended in PBS, however, there were no statistically significant differences in the
heating rates of filtrate and suspension. We conclude that RF exposure of
CDDP@US-tubes in PBS does not result in significant enhancement of the bulk
temperature. Furthermore, these findings suggest that the ionic heating of PBS
either overshadows or abrogates the heating contribution due to CDDP@US-tubes
alone.
To address this hypothesis, an experiment was designed to probe localized
heating of CDDP@US-tubes in PBS. It has previously been observed that uncoated
SWCNTs have non-specific affinity for proteins and it is also known that Pluronicwrapping attenuates the binding of proteins to the surface of SWCNTs.102 Using this
information, a relatively thermostable fluorescent protein (allophycocyanin (APC))
was utilized to probe the disruption of Pluronic on CDDP@US-tubes surfaces. The
principle of this assay is demonstrated in Figure 4.3A. W-CDDP@US-tubes prevent
APC binding and a fluorescent signal can be detected. Local heating of US-tubes or
bulk ionic heating should disrupt Pluronic-wrapping above the melting point of
Pluronic F-108 (∼57.5 °C) allowing APC to bind to US-tube surfaces and thus
produce quenching of APC fluorescence. Similar fluorescence quenching behavior
47
has been described previously for other carbon-based nanomaterials such as carbon
nanotubes103 fullerenes104 and nanographene sheets.105
Figure 4.3 − Panel A. Schematic demonstrating the principle of the
temperature-dependent Pluronic disruption assay. Panel B and C.
Temperature-dependent fluorescence quenching with a transition
temperature close to the melting point of Pluronic F108 is indicative of
Pluronic disruption from the w-CDDP-US-tube surface. Panel D. RF durationdependent quenching of fluorescence demonstrates localized heating of WCDDP@US-tubes in PBS. Water bath temperature controls (HT) fail to
demonstrate quenching of APC fluorescence suggesting lack of Pluronic
disruption due to bulk temperature. The RF-dependent quenching is therefore
not a result of non-thermal denaturation of APC.
48
In order to test this hypothesis further, APC was added to a suspension of WCDDP@US-tubes in PBS and a temperature-dependent decrease in fluorescence was
observed. This was normalized to fluorescence measurements from a control
solution of APC in 0.17% (w/v) Pluronic-F108 to exclude effects from
photobleaching, temperature-dependent fluorescence quenching, or thermal
denaturation of APC. The normalized curve demonstrated quenching of APC due to
the presence of Pluronic-wrapped W-CDDP@US-tubes (Figure 4.3B). The first
derivative of this curve revealed the peak transition at 59.7 °C (Figure 4.3C), which
is consistent with the transition temperature of Pluronic-F108 (∼57.5 °C).
Therefore, we attribute the quenching of APC fluorescence to the temperaturedependent disruption of Pluronic-F108 on CDDP@US-tube surface due to the rise in
bulk temperature.
Next, this system was used to study localized heating of W-CDDP@US-tubes
in the RF field while suspended in PBS. The results, shown in Figure 4.3D,
demonstrated that RF field exposure resulted in a dose-dependent quenching of
fluorescence of APC, suggesting Pluronic disruption. We monitored the bulk
temperature during this exposure, which reached a maximum of 42.5 °C after 5 min
of RF field exposure. This experiment was then repeated by exposing the CDDP-UStube suspension to 42.5 °C in a water bath for 5 min instead of exposing it to RF.
Fluorescence measurements did not show significant quenching of APC in the water
bath experiment. This implies that the Pluronic disruption and APC fluorescence
quenching is not due to a rise in bulk temperature. It is possible that fluorescence
quenching of APC could be due to non-thermal denaturation of APC in the RF field
49
but it was found that exposing APC in 0.17% (w/v) Pluronic-F108 solution to the RF
field for 5 min does not result in significant fluorescence quenching, thus, excluding
this possibility. We conclude that these data demonstrate localized heating at the
surface of W-CDDP@US-tubes in a physiologic salt solution as well as disruption of
Pluronic on the W-CDDP@US-tube surface.
4.3.3. RF-triggered release of CDDP from US-tubes
Previously it was demonstrated that CDDP can be loaded into US-tubes
through sidewall defects and wrapping the CDDP@US-tubes with Pluronic can
significantly retard subsequent release of CDDP.100 Thus, based on these previous as
well as present studies, it was reasoned that Pluronic disruption caused by localized
heating of US-tubes should enhance release of CDDP from W-CDDP@US-tubes in an
RF field-triggered fashion. In order to test this hypothesis, W-CDDP@US-tubes were
suspended in PBS and subjected to varying durations of RF exposure. Samples not
exposed to RF were used as a negative control. In order to differentiate release of
CDDP due to bulk temperature and that due to localized heating of W-CDDP@UStubes, water bath temperature controls were used. These samples were incubated
in a water bath for a duration that matched the RF experiments. The incubation
temperature for the water bath experiments was chosen as the final average
temperature reached after 2.5 or 5 min of RF exposure. After the appropriate
exposure, samples were immediately loaded in dialysis cassettes and CDDP release
at room temperature was determined by ICP measurements.
50
Data shown in Figure 4.4 demonstrated that RF field exposure approximately
doubles the release of CDDP from W-CDDP@US-tubes after 2.5 or 5 min of RF
exposure at each time point measured (relative to no-RF controls). Relative areaunder-curve (AUC) depicts cumulative release relative to the no-RF control.
Regardless of the duration (2.5 or 5 min), RF exposure enhanced cumulative release
of CDDP by 1.9 fold after 168 h. Samples exposed to RF reached an average bulk
temperature of 38.7 °C after 2.5 min and 49.5 °C after 5 min of RF exposure. We then
exposed a W-CDDP@US-tubes suspension to these temperatures and durations in a
water bath and measured the release at room temperature similar to RF exposed
samples. For the sample exposed at 49.5 °C for 5 min, we found significant
enhancement of release that was similar to but less than the RF exposed samples
(cumulative release, 1.7 fold), suggesting bulk temperatures lower than Pluronic's
transition temperature of 57.5 C can allow release of CDDP. However, the
temperature control sample exposed at 38.7 C for 2.5 min demonstrated far less
enhancement in cumulative release (1.3-fold). These results demonstrate and
distinguish between CDDP release from W-CDDP@US-tubes caused by bulk and
local (at the surface of the CDDP@US-tubes) temperature rise, when exposed to the
RF field.
51
Figure 4.4 − CDDP release from W-CDDP@US-tubes with or without RF. Panel
A. Release of CDDP from W-CDDP@US-tubes with or without RF exposure for
2.5 or 5 min is shown. In addition, bulk temperature controls are
demonstrated to evaluate the contribution of bulk ionic heating to CDDP
release from W-CDDP@US-tubes. Panel B. Relative areas-under-curve depicts
cumulative CDDP release over 168 h relative to untreated control.
4.3.4. RF-triggered release and cytotoxicity
To evaluate the significance of these findings, liver cancer cells were exposed
to W-CDDP@US-tubes in a trans-well system such that the CDDP@US-tubes
nanocarriers were spatially separated from the cellular compartment. Only released
CDDP could diffuse through the trans-well membrane (pore size <400 nm). This
system is designed to model the limitations of in vivo drug delivery using
nanocarriers where the drug-loaded carriers can be spatially separated from their
52
intended site of delivery in cancerous tissue because of multi-scale barriers to
transport. The experimental scheme is represented in Figure 4.5.
Figure 4.5 − Release-dependent cytotoxicity of W-CDDP@US-tubes. Hep3B
and HepG2 human liver cancer cells were plated in the bottom well of the
trans-well plate and treated with PBS, W-US-tubes or W-CDDP@US-tubes
followed with or without RF exposure for 3 min once, twice, or three times at
24 h intervals. The cell viability was assessed 120 h after treating with WCDDP@US-tubes.
Approximately 5000 human liver cancer cells (Hep3B or HepG2) were plated
on the bottom well of a 12-well trans-well system. After 24 h, the cells were found to
be adherent at which point a second compartment was placed on top and the cells
were treated with PBS, W-US-tubes (13.7 mg/L) or W-CDDP@US-tubes (CDDP
2.5 μM, US-tube 13.7 mg/L). This concentration was the approximate IC50 for WCDDP@US-tubes for both cells lines at 120 h. The trans-well system was or was not
53
subjected to a 3-min RF field exposure 1, 2, or 3 times at 24 h intervals. Viability was
assessed at 120 h when the PBS treated control samples reached confluence. For
both cell lines, it was noted that even the US-tubes alone are slightly cytotoxic at this
concentration. It was also noted that this slight cytotoxicity was not enhanced by
multiple RF exposures. The data in Figure 4.5 demonstrated that Hep3B and HepG2
cells possess different sensitivities to RF field exposure. As shown, Hep3B cells are
not affected by multiple 3-min RF field exposures while HepG2 cells demonstrate
slight toxicity to RF. Interestingly, this toxicity is not enhanced after repeated RF
exposures. The W-CDDP@US-tube treatment reduced the viability of both cell lines
to 50% due to the spontaneous release of CDDP. This cytotoxicity was significantly
enhanced by a single 3-min RF exposure (p < 0.01). For Hep3B cells, repeated 3-min
RF exposure demonstrated slightly higher cytotoxicity but this was not statistically
significant. For the HepG2 cells, multiple RF exposures did not further enhance the
observed cytotoxicity. Overall, these data clearly demonstrated cytotoxicity
attributable to RF-triggered CDDP release from CDDP@US-tubes.
4.4. Discussion
Nanoparticle-mediated drug delivery is widely studied as it may help to
overcome drug resistance and enhance tumor-selective toxicity. However, several
challenges remain before this potential can be fully realized. While studies have
demonstrated successful accumulation of nanocarriers within tumors, it can be
argued that the therapeutic efficacy of these nanomaterials is limited by their
sequestration in the peri-vascular space. The slow passive release of drugs from
54
nanocarriers is therefore often not sufficient to allow therapeutic levels to be
achieved within the tumor. Indeed, this argument is supported by recent studies
that demonstrate patchy distribution of nanocarriers in the vascularized areas of
the tumor, while certain avascular areas are completely devoid of the
chemotherapeutic agent.82–84,87 The barriers to transport are incompletely
understood and are likely to be multifaceted, involving both host and nanoparticle
properties. While host parameters (interstitial fluid pressure, extracellular matrix
composition, pH, and cellular stroma components) are difficult to modulate,
nanoparticle design can be readily altered.88,89 It is well known that smaller
nanoparticles permeate through the tumor interstitium better than larger
nanoparticles.106 However, small nanoparticles are less likely to take advantage of
the EPR effect and could be readily cleared by the renal excretion. Hence,
nanoparticle properties that govern optimal tumor accumulation are different from
those that allow optimal tumor-wide permeation. An alternative approach provides
a practical solution to this problem and utilizes remotely-triggered release of drugs
after intra-tumoral accumulation of nanocarriers.
In this study, we have described a carbon nanocapsule-based system that can
readily release its drug cargo in response to a non-invasive RF-trigger. Carbon
nanotube interaction with an RF field has been previously studied.24 It was noted
that under RF field exposure, full-length nanotubes dissipate energy as heat.
Furthermore, this energy is sufficient to raise the bulk temperature of the
surrounding environment allowing the use of SWCNTs as remotely-activated
thermal agents. The heating of US-tubes by an RF field has not been previously
55
described. As for SWCNTs, the remote heating of W-CDDP@US-tubes and W-UStubes in deionized water by an RF field has been demonstrated here. However, bulk
heating of W-US-tubes or W-CDDP@US-tubes has only been observed in a non-ionic
environment. When suspended in a physiologically relevant ionic environment
(PBS), the bulk temperature rises due to ionic heating of PBS and the local heating of
US-tubes cannot be measured. Theoretical studies have predicted that only metallic
SWCNT can elevate bulk temperatures of ionic solvents through a “lightning-rod”
effect where the metallic SWCNTs polarize in the presence of an E-field and enhance
E-field strength by several orders of magnitude.99 This can lead to a dissipation
mechanism in the bulk ionic solvent that increases with increasing conductivity of
the solvent. Contrary to metallic SWCNTs, the power dissipation by semi-conducting
SWCNTs is thought to occur predominantly in the nanotube core and therefore is
unlikely to enhance bulk temperature in a conductive host such as PBS. Defects
created in the sidewalls of US-tubes during the fabrication process can alter the
electronic properties of SWCNTs from which they are derived. Based on the
experimental observations of this study, it is infer that the US-tubes behave as a
poor dielectric with predominant energy dissipation within the core of the US-tube.
This heating can be somewhat abrogated by the CDDP loading, the reason for which
is not clear at this time but could be due to basic changes in the dielectric properties
of the US-tube.
Furthermore, it has been demonstrated here that despite the lack of
contribution to temperature change in the bulk ionic media, W-CDDP@US-tubes can
dissipate energy at the molecular level that is sufficient to disrupt the Pluronic
56
coating in an RF dose-dependent manner, which subsequently allows CDDP release.
Several studies have demonstrated remotely-triggered drug release. For instance,
ultrasound-triggered release has been demonstrated from acoustically-responsive
gold nanocages, drug-loaded liposomes, or microbubbles.107–109 While significant
tumor regression was noted in these studies, small animal models do not
recapitulate the limits imposed by depth and variability of penetration of
ultrasound. Similarly, light has been extensively studied as a trigger to drug release
with reports dating back to the 1980s. Photo-activation utilizes photoisomerization,
photo crosslinking, photosensitization-induced oxidation, or photo-thermal
activation of gold nanoparticles (See Ref.110 for a review). Regardless of the
mechanism of light-triggered drug release, penetration of light even at THz
frequencies is limited to a few centimeters, and cannot be utilized for deep-seated
tumors, thus necessitating alternative deep-penetrating strategies. Recently, Peiris
et al. utilized an alternating magnetic fields (AMF) at radiowave frequencies
(10 kHz) to trigger drug release from iron oxide nanoparticles cross-linked to drugloaded liposome in a serial chain.81 Similar to W-CDDP@US-tubes, they noted that
drug release could be accomplished without raising bulk ionic temperature. They
attributed this behavior to nanoscale vibration of magnetic iron oxide particles in an
AMF. In contrast, W-CDDP@US-tubes are a less magnetic material but a material
that can be activated by the incident electric field of RF exposure as demonstrated
by the present work. Similar to an AMF, RF electric fields can easily penetrate to
deep-seated tumors and have well-documented safety. The present study has
demonstrated that RF exposure can significantly enhance drug release of CDDP from
57
carbon nanocapsules to enhance cytotoxicity. We have also found that RF-triggered
drug release from the carbon nanocapsules exhibits cytotoxicity in a drug releasedependent manner. Increasing the duration of RF exposure or frequency of RF
exposure does not further enhance drug release suggesting an irreversible change in
the Pluronic coating on the W-CDDP@US-tube surface. Unlike iron oxide crosslinked liposomes, US-tubes serve the dual purpose of an encapsulating agent and
that of a susceptor in an RF field, thereby eliminating the need for complex
preparation methods. While it has been demonstrated here that a Pluronic coating
significantly retards drug release from the nanocapsules, up to 30% of CDDP can be
passively released despite Pluronic coating. Future studies already underway are
evaluating alternative temperature-sensitive coatings to allow stealth nanoparticles
that only permit drug release in the presence of an RF field. In addition, preliminary
data in our laboratory has demonstrated that the US-tube platform can be
functionalized with antibodies, which will allow tumor specific targeting in
conjunction with the known EPR effect.
4.5. Conclusion
A carbon-nanocapsule system has been developed that can be remotely
triggered by RF irradiation to release CDDP. Mechanistic insight into the process has
revealed thermal disruption of a Pluronic coating by US-tube heating in the RF field
without appreciable rise in bulk temperature at an RF dose that is minimally toxic to
cancer cells. Furthermore, a release-dependent cytotoxicity in human liver cancer
58
cell lines in a trans-well system has been observed that mimics spatial limitations in
delivery of chemotherapeutics by drug-loaded nanocarriers in vivo.
Chapter 5
In vivo Biodistribution and Anticancer
Activity of Cisplatin Delivered by Ultra-short
Carbon Nanotube Capsules3
5.1. Introduction
In our previous studies (Chapter 3),100 we have shown that the
CDDP@US-tubes exhibited greater cytotocities against MCF-7 and MDA-MB-231
breast cancer cell lines in vitro, when compared to free CDDP. It was also
demonstrated that the US-tube platform apparently assists the delivery of
encapsulated CDDP which suggested that CDDP@US-tube materials help to
overcome CDDP resistance by increasing drug accumulation in otherwise resistant
3
A. Guven, G. J. Villares, L. J. Wilson and M. T. Lewis. Manuscript in preparation.
59
60
cell. In this chapter, we report the in vivo biodistribution behaviors and therapeutic
efficacy of the CDDP@US-tubes material after the systematic administration on
three different human breast cancer xenograft models in mice.
5.2. Materials and Methods
5.2.1. Sample preparation:
US-tubes, CDDP@US-tubes and W-CDDP@US-tubes were prepared as
previously reported.100 Briefly, full-length SWCNTs (Carbon Solution Inc.), produced
by the electric-arc discharge method, were cut into US-tubes by fluorination
followed by pyrolysis at 1000 °C under an inert atmosphere.53 US-tubes were first
purified in concentrated HCl for 1h by bath-sonication to remove amorphous carbon
and metal catalyst impurities (nickel and yttrium), then chemically reduced by a
Na0/THF reduction procedure to produce individual US-tubes.76 Next, the US-tubes
were refluxed for 5 min in 6N HNO3 and repeatedly washed with deionized water.
The debundled US-tubes were dispersed in deionized water via bath-sonication for
60 min, and then CDDP was added to the US-tube suspension and vigorously stirred
for 24 h, which was then left undisturbed overnight whereupon CDDP-loaded UStubes (CDDP@US-tubes) flocculated from solution. The CDDP@US-tubes were
collected by filtration on a glass filter, washed excessively with deionized water to
remove all exterior CDDP from the outer surface of the US-tubes, as judged by
platinum analysis on the filtrate aliquots by inductive-coupled plasma optical
emission spectroscopy (ICP-OES) and then dried at 80 °C. Extensive characterization
61
of this CDDP@US-tube material has been also reported previously.100 W-US-tube
and W-CDDP@US-tube samples were prepared by suspending either dry US-tubes
or CDDP@US-tube samples in a 0.17% (w/v) Pluronic® F-108 solution via probe
sonication for 2 min, followed by centrifugation at 3200 rpm for 10 min (3x) to
remove unsuspended CDDP@US-tubes.
5.2.2. Establishment of xenografts and treatment
All animal experiments were performed under a protocol approved by
Baylor College of Medicine Institutional Animal Care and Use Committee in
accordance with the National Institutes of Health Guide for the Care and Use of
Experimental Animals. MCF-7 and MDA-MB-231 human breast xenografts were
generated by inoculation of 1×106 of either MCF-7 or MDA-MB-231 cells suspended
in 20 µL Matrigel-PBS into the epithelium-free “cleared” fat pads of SCID/Beige
mice. Human, patient-derived breast cancer xenografts (PDX) (BCM-4272
transplant) were established as described previously.111 For each xenograft
experiment, a total of forty human breast-tumor-bearing mice were used. The tumor
volumes were monitored weekly by caliper measurement. When the average tumor
volume reached at least 100 mm3, mice were randomly divided into treatment or
vehicle groups for each experiment. For treatment, 100 µL of different formulations
of CDDP, CDDP@US-tubes, W-US-tubes or W-CDDP@US-tubes in saline was
administered via i.p. once a week. The administration dose was normalized to be 2.5
mg CDDP per kg (2.5 mg/kg). Tumor size was measured by a caliper two times a
week and calculated as the volume = [(tumor length) × (tumor width) 2] / 2. At the
62
end of the experiment, the animals were sacrificed and tissues collected to obtain a
quantitative measurement of the biodistribution of CDDP and US-tube samples in
each tissue by quantifying platinum (Pt) and yttrium (Y) concentration in each
sample via inductively-coupled plasma mass spectrometry (ICP-MS). Pieces of tissue
(heart, liver, lung, spleen, kidney, and tumor) were taken from animals of each
group, formalin fixed, paraffin embedded, frozen, and processed to 4-µm slides by a
tissue-processing core laboratory at BCM. One slide from each tissue sample was
stained with hematoxylin and eosin.
5.2.3. Biodistribution and blood circulation studies
For biodistribution studies, a total of 45 MDA-MB-231 tumor-bearing mice
(tumor size, ~200 mm3) were randomly divided into three groups (CDDP,
CDDP@US-tubes, and W-CDDP@US-tubes) of 15 mice each. Three mice from each
treatment group were sacrificed at 4, 24, 48, 72, and 168 h after receiving 100 µL of
either CDDP, CDDP@US-tube or W-CDDP@US-tube samples in saline at a dose of 2.5
mg CDDP per kg mouse via an i.p injection. The blood samples were collected by
cardiac puncture for the blood circulation studies. After harvest, the organs/tissues
were immediately frozen in liquid nitrogen, lyophilized to dryness and weighted.
Blood and the dried tissue samples were digested by first heating with concentrated
HNO3 and then with 26% HClO3. The concentration of Pt and Y were then
determined by ICP-MS for blood circulation and biodistribution analysis.
63
5.2.4. Inductively-coupled plasma-optical emission mass spectrometry
(ICP-MS)
ICP-MS analyses were carried out using a Perkin Elmer Elan Optima 9000 DV
instrument with a CDD detector. The quantity of CDDP in each sample was
determined by measuring the platinum concentration, which was detected at 265.95
nm. For all ICP-MS measurements, the collected samples were transferred into glass
vials and digested first using 10 ml of concentrated HNO3 at elevated temperature
(~ 230 °C), and then with 2 ml 26% HClO3. Following digestion, the samples were
diluted with 2% v/v trace metal grade HNO3. The concentrations were determined
from an average of five scans for each sample. Germanium (371.029 nm) was used
as the internal standard.
5.2.5. Statistical analysis
All the data are expressed as mean values ± standard error (SE). Graphs were
constructed and all statistical analyses were done using GraphPad© Prism 6.0
(GraphPad Software, Inc.). Statistical comparison between two experimental groups
was done using a t test (unpaired, two-sided), comparison of multiple groups was
performed with two-way ANOVA using Tukey's multiple comparison test. A value of
less than 0.05 (p < 0.05) was used for statistical significance.
64
5.3. Results
5.3.1. Tumor suppressing effect of CDDP@US-tubes on MCF-7 xenograft in
SCID/Beige mice
In previous in vitro studies,100 we determined the efficacy of CDDP@US-tubes
against two different breast cancer cell lines (MCF-7 and MDA-MB-231), which are
known to be CDDP resistant and showed that US-tubes helped increase the
accumulation of the drug in cells compared to free CDDP. To examine the ability of
US-tubes to deliver anticancer drugs and to determine the efficacy of CDDP@UStubes on tumor growth suppression in mice in vivo, a series of treatment studies
were conducted using three different breast cancer xenograft models (MCF-7, MDAMB-231, and BCM-4272).
To determine the in vivo efficacy of the CDDP@US-tubes, we first tested MCF7 xenografts established in SCID/Beige mice. A total of forty MCF-7 tumor bearing
mice (weighing 20 ± 2 g) with established xenografts (as detailed in the Materials
and Methods Section) were randomly divided into five groups of eight mice each
and intraperitoneal (i.p.) injections were started when the average tumor volume
reached 150 mm3. Animals in Group 1 (G1) received saline solution (0.9 % w/v
NaCl) as a control. Group 2 (G2) was treated with empty Pluronic-wrapped US-tubes
(W-US-tubes) as a vehicle control. Group 3 (G3) was treated with CDDP dissolved
and diluted in saline solution. Group 4 (G4) was treated with Pluronic-wrapped
CDDP@US-tubes (W-CDDP@US-tubes). Finally, Group 5 (G5) was treated with
CDDP@US-tubes dispersed in saline solution via bath-sonication. The final
65
concentration of CDDP in G3, G4, and G5 was normalized to be 2.5 mg/kg mouse
body weight. The quantity of US-tubes was the same for G2, G4, G5, with the
concentration being calculated from their weight% values obtained by ICP-OES for
Ni and Y which remains within US-tubes as remnant metal catalysts from the growth
of the original full-lengths CNTs from which the US-tubes are derived. Treatment
was performed once a week until the end of the experiment.
66
C o n tro l
W -C D D P @ U S -tu b e s
20
16
R e la t iv e t u m o r v o lu m e
4
3
2
*
0
0
10
D a y s a fte r 1
* *
*#
20
st
T u m o r d o u b lin g tim e s
C
1 .0
P r o p o r t io n e v e n t - f r e e m ic e
24
5
C D D P @ U S -tu b e s
B o d y w e ig h t (g )
A
1
CD D P
W -U S -tu b e s
0 .5
0
0 .0
30
0
10
tre a tm e n t
20
30
T im e (d a y s )
*p<0.05 CDDP vs. CDDP@US-tubes
8
p < 0 .0 0 5
6
ns
4
p < 0 .0 5
2
es
b
es
P
D
C
w
-C
D
D
D
P
w
@
@
U
U
S-
S-
tu
tu
b
D
D
C
-U
S-
Sa
tu
li
b
n
P
es
0
e
B
F o ld i n c r e a s e in t u m o r v o l u m e
#p<0.05 CDDP vs. W-CDDP@US-tubes
Figure 5.1 − Suppression of tumor growth by intraperitoneal injection of
CDDP@US-tubes to an MCF-7 breast cancer xenograft. (A), Tumor growth of
MCF-7 tumor-bearing mice after receiving different treatments as indicated.
The mean body weight changes is also plotted in A. Fold increase in tumor
volume (B) and Kaplan-Meier survival curves (C) depicting the tumor
doubling times for different treatment groups.
67
Figure 5.1 displays the comparative in vivo therapeutics efficacy of the given
treatment groups. As shown in Figure 5.1A, a time-related increase in tumor volume
was observed in all treatment groups except for the CDDP@US-tube treated group.
Compared to the control injection, W-US-tube injected mice showed similar tumor
growth rate, although it was not as effective as CDDP alone or the W-CDDP@UStubes. As showed in Figure 5.1A, there was also no significant difference between
the mean body weight changes in all treatment groups over the course of the
experiment. The mean fold increase in tumor volume (from day 0 to day 28) of the
CDDP@US-tube treated mice was significantly lower compared to CDDP alone or WCDDP@US-tubes (Figure 5.1B). Five out of eight tumors from mice treated with
CDDP@US-tubes failed to grow at all, while all control groups got larger.
Determination of the tumor growth doubling time (Figure 5.1C) revealed that
CDDP@US-tubes significantly improved mouse survival.
A
n g P t / m g d r y tis s u e
68
50

40
10
30
5
20
n g Y / m g d r y tis s u e
C o n tro l
w -U S -tu b e s
CDDP
w -C D D P @ U S -tu b e s
C D D P @ U S -tu b e s
*
0
*
Tum or
10
#
0
Tum or
B

*
H eart
Lung
S p le e n
15
 
0 .8
10
K id n e y
S to m a ch
C o n tro l
w -U S -tu b e s
CDDP
w -C D D P @ U S -tu b e s
C D D P @ U S -tu b e s
0 .6
0 .4
0 .2
5
L iv e r

0 .0
 
Tum or
0
Tum or
* p < 0 .0 5

H eart
Lung
C D D P v s C D D P @ U S -tu b e s,
S p le e n
#
L iv e r s
S to m a ch
p < 0 .0 5 C D D P v s w - C D D P @ U S - t u b e s ,
p < 0 .0 5 w - C D D P @ U S - t u b e s v s C D D P @ U S - t u b e s ,

K id n e y

p < 0 .0 5 w - U S - t u b e s v s C D D P @ U S - t u b e s ,
p < 0 .0 5 w - U S - t u b e s v s w - C D D P @ U S - t u b e s
Figure 5.2 − Platinum and yttrium concentration in tissue from mice treated
over a period of ~ 45 d with weekly saline, w-US-tubes, CDDP, CDDP@UStubes, and W-CDDP@US-tubes administration via i.p. injection. (A)
Concentration of platinum (from cisplatin) and (B) yttrium (from US-tubes) in
tissue (mean ± s.e., n = 8). Statistically significant different (p < 0.05) are
marked with symbols as indicated above (unpaired, two-sided t-test).
At the end of the experiment, animals from each treatment group were
sacrificed and tissues were collected to obtain a quantitative measurement of the
accumulation of CDDP and US-tubes samples in each tissue by quantifying Pt (for
CDDP) and Y (for US-tubes) in each tissue sample via ICP-MS analysis. ICP-MS
analysis revealed that the CDDP@US-tube treated mice have significantly more Pt in
69
their tumor, spleen and stomach compare to other treatment groups (Figure 5.2A).
By analyzing the Y concentration in tissue, it has also been determined that the level
of Y detected in spleen, liver, kidneys, and stomach was found to be greatest, while
the amount found in other organs was much lower (Figure 5.2B). The analysis
showed that CDDP@US-tube treated mice had significantly more Y in their spleen
and stomach compare to mice treated with empty W-US-tube, and W-CDDP@UStubes.
5.3.2. Tumor suppressing effect of CDDP@US-tubes on MDA-MB-231
xenograft in SCID/Beige mice
In the second experiment, the in vivo anticancer efficacy of CDDP@US-tube
materials was tested in mice bearing MDA-MB-231 human breast cancer xenograft
in a manner similar to that described above for MCF-7 xenograft. The tumors were
established as described in Materials & Method section and i.p. injections were
started when the average tumor volume reached ~100 mm3. Changes in relative
tumor volume and body weight are shown in Figure 5.3A. As plotted in Figure 5.3A,
the mean body weights of mice in all treatment groups showed similar an increase
and there was no significant difference between the treatment groups over course
of the experiment. As seen in Figure 5.3A, the control and the empty W-US-tube
treated group showed a fast growth pattern, resulting in an approximately 12-fold
increase in their tumor volume (from day 0 to day 28). No significant difference in
the relative tumor growth ratio (V/V0) of these two treatment group confirmed that
the empty W-US-tubes themselves do not affect the tumor growth in mice. On the
70
other hand, the relative tumor growth ratios (V/V0) of CDDP, W-CDDP@US-tubes,
and CDDP@US-tubes were similar (Figure 5.3A) and there was no statistically
significant difference between the treatment groups. However, the mean fold
increase in tumor volume (from day 0 to day 28) of CDDP@US-tube (7.45 ± 0.77)
treated mice was significantly lower than mice in control treatment group (13.03 ±
2.02, p = 0.03) (Figure 5.3B), while the fold increase in mice treated with free CDDP
(9.09 ± 1.73), and W-CDDP@US-tube (9.57 ± 1.88) was not significant different (p =
0.16, and p = 0.24, respectively). The determination of tumor growth tripling time
showed that the median survival of the CDDP@US-tube, and W-CDDP@US-tube
treatment groups, (10 d and 14 d, respectively) was longer than that of mice treated
with free CDDP, 7 d (Figure 5.3C).
71
w -C D D P @ U S -tu b e s
16
15
12
9
6
3
0
T u m o r t r ib lin g t im e s
1 .0
0 .5
0
10
0 .0
20
0
tre a tm e n t
10
15
20
25
p < 0 .0 5
B
ns
ns
25
20
15
10
5
es
b
es
tu
b
S-
tu
U
SP
D
C
w
-C
D
D
D
P
w
@
@
U
-U
S-
Sa
tu
li
b
n
P
es
0
e
F o ld i n c r e a s e in t u m o r v o l u m e
5
T im e (d a y s )
D
D a y s a fte r 1
st
D
0
C
R e l a t iv e t u m o r r a t io ( V / V o )
20
C
P r o p o r tio n E v e n t - F r e e
24
C D D P @ U S -tu b e s
B o d y w e ig h t (g )
A
CDD P
w -U S -tu b e s
C o n tr o l
Figure 5.3 − Suppression of tumor growth by intraperitoneal injection of
CDDP@US-tubes to an MDA-MB-231 breast cancer xenograft. (A), Tumor
growth of MDA-MB-231 tumor-bearing mice after receiving different
treatments as indicated. (* indicates p < 0.05, unpaired, two-sided t-test, n = 8;
error bars = s.e.) The mean body weight changes is also plotted in A. Fold
increase in tumor volume (B) and Kaplan-Meier survival curves (C) depicting
the tumor tribling times for different treatment groups.
72
Although the efficacy of free CDDP, W-CDDP@US-tube, and CDDP@US-tube
on the MDA-MB-231 xenograft was found to be similar (Figure 5.3A), the ICP-MS
analysis of tissue collected from the animals at the end of the experiment revealed
that the mice treated with CDDP@US-tubes (4.28 ± 1.17, p = 0.02) and WCDDP@US-tubes (2.16 ± 0.16, p = 0.01) had significantly more Pt in their tumor than
B
30
8
#
6
20
*
#
*
#
4
2
*
S a lin e
W -U S -tu b e s
CDDP
W -C D D P @ U S -tu b e s
C D D P @ U S -tu b e s
0
10
Tum or
*#
0
Tum or
n g Y / m g d r y tis s u e
A
n g P t / m g d r y t is s u e
mice treated with free CDDP (1.17 ± 0.18) (Figure 5.4A).
H eart
Lung
S p le e n
20
 
0 .6
15
K id n e y
0 .2
 
0 .0
5
S to m a ch
S a lin e
W -U S -tu b e s
CDDP
W -C D D P @ U S -tu b e s
C D D P @ U S -tu b e s
0 .4
10
L iv e r
 
Tum or
0
Tum or
* p < 0 .0 5

H eart
Lung
S p le e n
C D D P v s C D D P @ U S -tu b e s,
#
K id n e y
L iv e r
S to m a ch
p < 0 .0 5 C D D P v s w - C D D P @ U S - t u b e s ,
p < 0 .0 5 w - C D D P @ U S - t u b e s v s C D D P @ U S - t u b e s ,

p < 0 .0 5 w - U S - t u b e s v s C D D P @ U S - t u b e s
Figure 5.4 − Platinum and yttrium concentration in tissue from mice treated
over a period of ~ 45 d with weekly saline, w-US-tubes, CDDP, CDDP@UStubes, and W-CDDP@US-tubes administration via i.p. injection. (A)
Concentration of platinum (from cisplatin) and (B) yttrium (from US-tubes) in
tissue (mean ± s.e., n = 8). Statistically significant different (p<0.05) are
marked with symbols as indicated above (unpaired, two-sided t-test).
73
However, the tumor Pt levels as stated above were considerably lower in
tumors than those of CDDP, W-CDDP@US-tubes, and CDDP@US-tubes observed
with MCF-7 tumor-bearing mice (2.77 ± 0.35, 3.09 ± 0.57, 9.74 ± 0.83, respectively).
This was apparently not sufficient to display tumor suppressing efficacy. Consistent
with MCF-7 xenograft study, the level of Y in mice treated with W-US-tubes, WCDDP@US-tubes, and CDDP@US-tubes was found highest in spleen followed by
stomach, liver, and kidney, confirming the higher retention of these materials in
these organs.
5.3.3. Tumor suppressing effect of CDDP@US-tubes on the BCM-4272 PDX
xenograft in SCID/Beige mice
To extend our studies beyond cell line xenografts, we evaluated the efficacy
of the CDDP@US-tube material with a human breast cancer patient-derived
xenograft (PDX) model, where human tumor tissue is surgically resected and
transplanted directly into immune-deficient mice. Because traditional cell lines have
been cultured for many years, and have accumulated many genetic/epigenetic
alterations, there is some argument as to whether their response to treatment
accurately reflects patient responses. It has been shown that PDX models display a
high degree of similarity in response as the tumor of origin in the patient when
challenged with a comparable agent.111–113 Because of their ability to predict clinical
tumor response, PDXs are increasingly being considered to be more relevant in vivo
preclinical models, and thus, are expected to increase the success of identifying new
active antitumor agents for clinical trials.112 BCM-4272 xenografts were established
74
as described previously.111 As shown in Figure 5.5A, the tumors in the control and
the empty W-US-tube groups showed a slow but stable growth. Compared with the
control group, the CDDP treatment group slowed down tumor growth, but it was not
as effective as the W-CDDP@US-tube or CDDP@US-tube treatment groups with both
of these treatment groups showing considerable tumor suppression (regression)
(p<0.005) compared to CDDP treatment. There were no significant body weight
changes between treatment groups until the end of the experiment.
75
C o n tr o l
w -U S -tu b e s
w -C D D P @ U S -tu b e s
22
20
6
1 .0
5
4
3
2
1
* *
* *# *#
*#
0
T u m o r d o u b l in g t im e s
P r o p o r tio n E v e n t - F r e e
R e l a t iv e t u m o r r a t io ( V / V o )
24
C
B o d y w e ig h t (g )
A
0 .5
0 .0
0
0
10
D a y s a fte r 1
20
st
CDD P
C D D P @ U S -tu b e s
0
30
10
20
30
T im e (d a y s )
tre a tm e n t
p < 0 .0 5
12
p < 0 .0 0 5
ns
8
4
s
e
s
P
D
C
w
-C
D
D
D
P
w
@
@
U
U
S-
S-
tu
tu
b
b
e
P
D
D
-U
S-
C
tu
li
b
n
e
e
s
0
Sa
B
F o ld i n c r e a s e in t u m o r v o l u m e
*p < 0.05 CDDP vs. CDDP@US-tubes
#p < 0.05 CDDP vs. W-CDDP@US-tubes
Figure 5.5 − Suppression of tumor growth by intraperitoneal injection of
CDDP@US-tubes to an Patient-derived, 4272 transplant, xenograft. (A), Tumor
growth of Patient-derived tumor-bearing mice after receiving different
treatments as indicated. (unpaired, two-sided t-test, n=9; error bars= s.e.) The
mean body weight changes is also plotted in A. Fold increase in tumor volume
(B) and Kaplan-Meier survival curves (C) depicting the tumor doubling times
for different treatment groups.
76
The mean fold increase in tumor volume (from day 0 to day 28) of control (4.88 ±
0.32) and W-US-tubes treated group (5.32 ± 0.49) were similar, however the
CDDP@US-tube (2.57 ± 0.25) treated group displayed a significantly lower fold
increase than those of free CDDP treated mice (4.02 ± 0.48, p = 0.01) which was also
not significantly different than the control group (4.88 ± 0.32, p = 0.15). The
increase in tumor volume for the W-CDDP@US-tubes (3.21 ± 0.26) was found to be
significantly lower than the control group (p = 0.001), while it was not statistically
different from the free CDDP treatment group (Figure 5.5B). As a result, an
increased survival of animals was observed in mice treated with CDDP@US-tubes or
W-CDDP@US-tubes compared to mice treated with free CDDP (Figure 5.5C).
ICP-MS analysis of the tissues showed that CDDP@US-tube and WCDDP@US-tube treated mice had significantly more Pt in their tumor, spleen and
kidney than the mice treated with free CDDP, while the free CDDP treated mice
displayed higher Pt accumulation in heart compare to other treatment groups
(Figure 5.6A). Consistent with results from the above MCF-7 experiment, for the
MDA-MB-231 xenograft studies, the Y concentration was found highest in the spleen
followed by kidney, liver and stomach, indicating higher retention of US-tube
materials in these organs (Figure 5.6B). Compared with W-CDDP@US-tube and WUS-tube treated mice, the level of Y in the spleen was found significantly higher in
mice treated with CDDP@US-tubes.
B
30
8
#
6
20
*
#
*
#
4
*
2
S a lin e
W -U S -tu b e s
CDDP
W -C D D P @ U S -tu b e s
C D D P @ U S -tu b e s
0
10
Tum or
*#
0
Tum or
n g Y / m g d r y tis s u e
A
n g P t / m g d r y t is s u e
77
H eart
Lung
S p le e n
20
 
0 .6
15
K id n e y
0 .2
 
0 .0
5
S to m a ch
S a lin e
W -U S -tu b e s
CDDP
W -C D D P @ U S -tu b e s
C D D P @ U S -tu b e s
0 .4
10
L iv e r
 
Tum or
0
Tum or
* p < 0 .0 5

H eart
Lung
S p le e n
C D D P v s C D D P @ U S -tu b e s,
#
K id n e y
L iv e r
S to m a ch
p < 0 .0 5 C D D P v s w - C D D P @ U S - t u b e s ,
p < 0 .0 5 w - C D D P @ U S - t u b e s v s C D D P @ U S - t u b e s ,

p < 0 .0 5 w - U S - t u b e s v s C D D P @ U S - t u b e s
Figure 5.6 − Platinum and yttrium concentration in tissue from mice treated
over a period of ~ 45 d with weekly saline, W-US-tubes, CDDP, CDDP@UStubes, and W-CDDP@US-tubes administration via i.p. injection. (A)
Concentration of platinum (from cisplatin) and (B) yttrium (from US-tubes) in
tissue (mean ± s.e., n = 8). Statistically significant different (p < 0.05) are
marked with symbols as indicated above (unpaired, two-sided t-test).
5.3.4. Blood circulation and the biodistribution studies
To understand the treatment efficacy of CDDP@US-tube materials, it was
important to investigate the biodistribution of the CDDP@US-tubes in a timedependent manner. The biodistribution studies has been conducted after a single i.p.
administration of CDDP, W-CDDP@US-tubes, and CDDP@US-tubes (CDDP
concentration 2.5 mg/kg) for MDA-MB-231 tumor-bearing mice by determining the
78
Pt (from CDDP) and the Y (from US-tubes) concentrations in plasma and in main
organs/tissues (tumor, liver, spleen, kidney, heart, lungs, and stomach) via ICP-MS
analysis at different time points (4, 24, 48, 72, and 168 h). The mice from each
treatment groups were sacrificed and harvested for blood circulation and
biodistribution analysis (see Materials and Methods Section).
P t c o n c e n t r a t io n
A
in b l o o d ( n g / m L )
CD D P
w -C D D P @ U S -tu b e s
C D D P @ U S -tu b e s
600
*
400
*
#
*
200
#
*
0
4h
24h
48h
72h
168h
4h
24h
48h
72h
168h
Y c o n c e n t r a t io n
in b l o o d ( n g / m L )
B
60
40
20
0
Figure 5.7 − Blood circulation for CDDP, W-CDDP@US-tubes, CDDP@US-tubes
in MDA-MB-231 tumor-bearing mice as determined by measuring Pt (from
CDDP) and Y (from US-tubes) concentration in blood via ICP-MS at different
time points post injection (mean ± s.e., n = 3). CDDP@US-tubes showed
prolonged blood circulation compared to free CDDP.
Figure 5.7A illustrates the comparative blood circulation data of free CDDP,
W-CDDP@US-tubes and CDDP@US-tubes for MDA-MB-231 tumor-bearing mice as a
79
function of time. ICP-MS analysis of the blood samples revealed that CDDP@UStubes and W-CDDP@US-tubes exhibited a much longer blood circulation time than
CDDP which was quickly cleared from the blood. At 4 h, the Pt concentration in
blood for mice treated with CDDP, W-CDDP@US-tubes, and CDDP@US-tubes was
271.55 ± 21.51 ng/mL, 250.40 ± 57.03 ng/mL, and 456.00 ± 71.40 ng/mL,
respectively. In comparison, CDDP-treated mice showed a significantly lower Pt
concentration in the plasma at 24 h (102.78 ± 9.92) which gradually declined until
the end of the experiment, while the mice treated with W-CDDP@US-tube and
CDDP@US-tube materials maintained relatively higher Pt concentrations (221.11 ±
33.26 and 327.03 ± 80.01, respectively) at 24 h, and continued to be relatively
higher until the end of the study. The concentration of Pt in blood at 168 h p.i., the
last point of evaluation, in mice treated with W-CDDP@US-tubes and CDDP@UStubes was 2.3-fold (48.27 ± 7.41) and 3.8-fold (78.62 ± 12.83), respectively, higher
than in mice treated with free CDDP (20.88 ± 5.14) (p < 0.05).
ICP-MS analysis showed that the Pt concentration of CDDP-treated mice in
each tissue gradually declined over time. The Pt levels detected in liver, spleen, and
kidney was highest, while the amount measured for other organs was much lower.
On the contrary, mice treated with W-CDDP@US-tubes and CDDP@US-tubes
displayed a significantly different biodistribution than mice treated with free CDDP
(Figure 5.8), with much higher Pt concentrations in tumor, spleen, and kidney, while
being similar in lung, stomach, heart, and liver (Figure 5.8A).
80
As shown in Figure 5.8, the concentration of Pt in tumors of all treatment
groups was similar at 4 h, and then started to increase, and became significantly
higher at 48 h and peaked at 72 h in CDDP@US-tube treated mice (3.09 ± 0.48
ng/mg dry tissue), which was more than 5-fold higher than in mice treated with free
CDDP (0.56 ± 0.08 ng/mg dry tissue) and approximately 3-fold higher than WCDDP@US-tube treated mice (1.08 ± 0.12). There was also a significant difference
between the CDDP and W-CDDP@US-tube treatment groups at 72 h (p = 0.024). At
168 h, the Pt concentration levels in tumors of all treatment groups decreased;
however, the accumulation of Pt for the CDDP@US-tubes (1.96 ± 0.44 ng/mg dry
tissue) was still significantly higher than that in the CDDP treated groups (0.34 ± 0.1
ng/mg dry tissue, p = 0.022). The Pt concentrations detected in kidney, liver, and
spleen after free CDDP administration was found highest, while the amount found in
other organs was much lower. The Pt concentration in kidney from mice treated
with free CDDP (9.27 ± 1.26), and W-CDDP@US-tubes (7.64 ± 0.18) were highest
and found significantly higher than mice treated with CDDP@US-tubes (4.87 ± 0.05)
at 4 h, and then gradually declined until the end of the study. Contrary to this,
CDDP@US-tube treated mice displayed a relatively consistent Pt concentration in
kidney up to 48 h, followed by a decreased and significantly higher Pt concentration
at 72 h (4.14 ± 0.3) compare with both the free CDDP (1.38 ± 0.49, p = 0.005), and
W-CDDP@US-tube (2.16 ± 0.82, p = 0.02) treatment groups. Although the Pt levels
detected in the liver were lower than those measured in the kidneys in the first 24 h,
in the long-term (at 168 h), the Pt levels were greater in the liver than in the
kidneys. The liver Pt levels in all treatment groups displayed a similar trend at all
81
evaluated time points except 4 h, with greater Pt concentration for CDDP@US-tubes
(6.05 ± 0.32) compared to free CDDP (4.87 ± 0.11, p = 002). Figure 5.8 shows that
the CDDP@US-tubes and W-CDDP@US-tubes afforded considerably higher Pt
accumulation in the spleen compare to free CDDP at 48 h and 72 h, while the mice
treated with free CDDP exhibited higher Pt accumulation only at 4 h, and then
gradually decreased over time. As shown in Figure 5.8B, ICP-MS analysis confirmed
the high retention of CDDP@US-tube and W-CDDP@US-tube materials in the spleen,
where the Y concentration was higher than for all other organs.
82
CDDP
n g P t / m g d r y tis s u e
A
w -C D D P @ U S -tu b e s
4
*
*
*
*
3
*

2
#
#
*
1
0
4h
24h
48h
72h 168h
4h
24h
n g P t / m g d r y tis s u e
Tum or
4h
24h
48h
72h 168h
Lung
#
10
5
0
24h
48h
72h 168h
4h
24h
48h
72h 168h
4h
24h
48h
72h 168h
K id n e y
S p le e n
CDD P
n g Y / m g d r y tis s u e
72h 168h
15
L iv e r
w -C D D P @ U S -tu b e s
C D D P @ U S -tu b e s
0 .6
0 .4
0 .2
0 .0
4h
24h 48h 72h 168h
4h
24h 48h 72h 168h
4h
24h 48h 72h 168h
H eart
Tum or
n g Y / m g d r y tis s u e
48h
H eart
20
4h
B
C D D P @ U S -tu b e s
5
Lung
4
3
2
1
0
4h
24h 48h 72h 168h
L iv e r
* p < 0 .0 5
4h
24h 48h 72h 168h
4h
24h 48h 72h 168h
S p le e n
C D D P v s C D D P @ U S -tu b e s,

#
K id n e y
p < 0 .0 5 C D D P v s w - C D D P @ U S - t u b e s ,
p < 0 .0 5 w - C D D P @ U S - t u b e s v s C D D P @ U S - t u b e s
83
Figure 5.8 − Time-dependent in vivo biodistribution of intraperitoneally
injected CDDP, W-CDDP@US-tubes, and CDDP@US-tubes in MDA-MB-231
tumor bearing mice. (A) Concentration of platinum (from CDDP), (B)
Concentration of yttrium (from US-tubes) in tissue (mean ± s.e., n = 3).
Statistically significant different (p < 0.05) are marked with symbols as
indicated above (unpaired, two-sided t-test).
5.4. Discussion
The in vivo biodistribution and pharmacokinetic studies of CNTs have been
extensively studied by a number of research groups using different CNT materials,
different surface functionalizations, and tracking techniques, after different
administration technique (i.p. vs. i.v.) resulting in varying and sometimes
controversial results.22 However, the present study is the first to explore the in vivo
delivery of a chemotherapeutic agent (CDDP) encapsulated within a CNT material.
Although CDDP is one of the most potent anticancer drugs, its potential use
in cancer therapy is hampered due to its severe side effects caused by the lack of
selectivity and resistance issues which is mainly attributed to reduced drug
accumulation in tumor cells. Because of its reactive nature, CDDP has limited
bioavailability. Upon administration, CDDP rapidly binds to albumin and other
plasma proteins leading to irreversible deactivation of nearly 90% of the CDDP dose
before it accumulates in the tumor by the EPR effect. Because this form of CDDP is
inactive, it has no biological effects.114 Thus, an ideal drug delivery systems is
expected to not only increase the concentration of CDDP in tumor cells, but also
protect it from plasma deactivation.
84
Recently, we developed a new CNT-based drug delivery system that is
comprised of CDDP encapsulated within the US-tubes (CDDP@US-tubes) that
showed enhanced in vitro antitumoral efficacy against two different breast cancer
cell lines, MCF-7 and MDA-MB-231, which are known to be CDDP resistant,
compared to free CDDP.100 In the present study, we have investigated the
biodistribution and in vivo efficacy of the CDDP@US-tube material in mice with
three different breast cancer xenograft models. Previous in vivo toxicity assessment
studies have demonstrated that high doses of US-tubes (up to 1000 mg/kg b.w.)
after i.p. administration are well tolerated by Swiss mice and showed that a large
portion of small aggregates and well-individualized US-tubes reached the systemic
circulation and subsequently accumulated in a variety of tissues and cells. 36 From
this study, it was reasoned that i.p. administration of CDDP@US-tube materials
would also avoid the biodistribution complications associated with i.v. injection
such as possible mechanical blockage of the vasculature system. In addition, i.p.
administration of free CDDP has been also studied extensively, and the dose of free
CDDP that can be given safely by i.p. is substantially higher compared to i.v.
administration. Moreover, the concentration of active cisplatin in the plasma is not
decreased after i.p. administration comprised to i.v. injection. To determine
pharmacokinetics and tissue specific distribution of CDDP vs. US-tubes, we
quantified the Pt (from CDDP) and Y (within the US-tubes as remnant metal
catalyst) concentrations in samples via ICP-MS analysis which allows to
measurement of exact Pt and Y concentration in tissue samples because of its high
sensitivity (1 ppb) and specificity due to the absence of background Pt and Y in
85
samples. To our knowledge, this is the first study that measures the remnant metal
catalyst, such as Y in our study, to trace and quantitatively determine the blood
clearance, and biodistribution behaviors of a CNTs material in vivo. To date, most of
the in vivo biodistribution and pharmacokinetic studies of CNT materials reported in
the literature have been carried out using radiolabels or spectroscopic tags for
indirect detection of CNTs,
26,46,115
which may gradually dissociate, decay, and/or
lose activity over time.116 In other studies, rather insensitive CNT Raman scattering
intensities have been sometimes employed as a tracer.41
The results of the present study demonstrate that CDDP@US-tubes have
greater tumor suppression efficacy than free CDDP in both MCF-7 and BCM-4272
PDX breast tumor models, while it was found to be similar in a MDA-MB-231
xenograft. In all three xenograft studies, the CDDP@US-tubes have afforded higher
CDDP uptake in tumors compared to free CDDP. The similar tumor suppression
effects of CDDP@US-tubes and CDDP on the MDA-MB-231 xenograft is possibly due
to greater aggressive tumor growth of the MDA-MB-231 xenograft compared to
MCF-7 and BCM-4272 PDX xenografts, which resulted in considerable lower Pt
concentration in the tumor per mg of dry tissue. In fact, it has been observed that
the mean fold increase (from day 0 to day 28) in tumor volume of the control
(saline) treated group for the MDA-MB-231 xenograft (11.415 ± 1.906) was notably
higher compared to the MCF-7 (3.486 ± 0.494) and BCM-4272 PDX (4.88 ± 0.319)
xenografts.
86
The reason for the higher CDDP uptake in tumor for CDDP@US-tubes is likely
due to their prolonged blood circulation time compare to free CDDP which
facilitates substantial tumor targeting by the EPR effect. Consistent with previous
reports, we observe short blood circulation time for free CDDP.117,118 The adsorption
of free CDDP occurred very rapidly by various organs especially kidney, liver and
spleen. However, the mice treated with W-CDDP@US-tubes and CDDP@US-tubes
displayed a significantly different biodistribution behavior than mice treated with
free CDDP, with much higher Pt accumulation in tumor, spleen, and kidney, while
being similar in lung, stomach, heart, and liver. By also analyzing the Y
concentration in tissue, we have determined the higher retention of US-tubes in
spleen, liver, kidneys, and stomach, while the amount found in other organs was
much lower after i.p. administration.
87
Chapter 6
Conclusion and Future Perspective
Over the past decade, a number of nanoscale materials have been explored that
exhibit many unique intrinsic physical and chemical properties and have much to offer in
the field of medicine. Among the diverse classes of nanomaterials, CNTs, or more
specifically US-tubes, hold great promise in the area of nanomedicine for the
development of new nanoplatforms. In only a few short years, the US-tubes have been
shown to be a popular, new platform for the delivery of various medical agents for both
imaging and therapeutic purposes.
In this work, for the first time, we have shown that US-tubes can be also utilized
as a drug delivery platform to deliver the chemotherapeutic drug, CDDP. The therapeutic
efficacy of a CDDP@US-tube material has been evaluated both in vitro and in vivo and
found to exhibits superior efficacy when compared to free CDDP. The studies have
revealed the ability of the US-tube platform to assist the delivery of encapsulated CDDP
by increasing the accumulation of drug in resistance cells which suggests how the
88
CDDP@US-tube materials helps to overcome CDDP resistance. Although it has been
demonstrated that passive tumor accumulation of CDDP@US-tubes in vivo occurs by the
EPR effect. Tumor specific targeting of CDDP can be passively also achieved by
functionalizing the US-tube platform with antibodies to further boost accumulation in
tumor cells.
We have also demonstrated that CDDP release from W-CDDP@US-tubes can
be stimulated by radiofrequency (RF) fields to produce heat that disrupts the
Pluronic coating and triggers CDDP releases in an RF-dependent manner. RFinduced release-dependent cytotoxicity of W-CDDP@US-tubes has been evaluated in
vitro against two different liver cancer cell lines, Hep3B and HepG2, and found to
exhibit superior cytotoxicity to W-CDDP@US-tubes not exposed to RF. While it has
been demonstrated that a Pluronic coating significantly retards drug release from
the CDDP@US-tubes, up to 30% of CDDP is passively released despite the Pluronic
coating. Future studies will evaluate alternative temperature-sensitive coatings to
allow stimulated drug release only in the presence of an RF field.
From the above encouraging results, we believe that it might be possible to
control CDDP release from CDDP@US-tubes by wrapping them with covalentlyattached activatable cancer-specific peptide sequences instead of surfactant
molecules. Recently, a catalytic method to functionalized US-tubes with various
amino acids and peptides was reported which improved solubility, biocompatibility,
and biological targeting capabilities.60 In future work, using this functionalization
protocol, a peptide chain, containing an enzyme-specific substrate sequence for a
89
cancer cell antigen, such as the well-known prostate specific antigen (PSA) and/or
matrix metalloproteinase-2 (MMP-2) can be synthesized and covalently-attached to
CDDP@US-tubes. This peptide chain should cover the sidewall defect sites and ends
of the US-tubes as a sheath and help prevent premature drug release until the
peptide is cleaved upon PSA and/or MMP-2 activation either within or in the
extracellular environment of cancer cells, as shown schematically in Figure 6.1.
Inserting different cancer specific peptide sequences into the peptide chain will also
allow for the treatment of a wide variety of cancers. Using this strategy, peptidewrapped CDDP@US-tube could become a smart drug delivery platform that would
activate CDDP drug release within cancer cells.
PSA
MMP-2
Figure 6.1 − Hydrolysis of the peptide sequence on a CDDP@US-tube by PSA
and/or MMP-2 .
90
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