DOXORUBICIN CONJUGATED TO D-α-TOCOPHERYL
POLYETHYLENE GLYCOL 1000 SUCCINATE (TPGS):
IN VITRO CYTOTOXICITY, IN VIVO
PHARMACOKINETICS AND BIODISTRIBUTION
CAO NA
(B.ENG., XI’AN JIAOTONG UNIVERSITY)
A THESIS SUBMITTED FOR THE DEGREE OF MASTER
OF ENGINEERING
DEPARTMENT OF CHEMICAL AND BIOMOLECULAR
ENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE
2007
ACKNOWLEDGEMENTS
First of all, I would like to take this opportunity to express my deepest gratitude and
appreciation to the following people:
My supervisor, Associate Professor Feng Si-Shen, for his invaluable advice, guidance,
unconditional support and encouragement during the period of my research in Chemical &
Biomolecular Engineering. I have learnt how to carry out research work, how to overcome
the difficulty in research.
My senior, Zhang Zhiping, for his unconditional support and invaluable advice in the
study. His sharing on research experience as well as his help in training is greatly
appreciated.
My Laboratory colleagues, Dr. Dong Yuancai, Miss Lee Siehuey, Miss Chen Shilin, Dr.
Mei Lin, Mr. Pan Jie, Ms Sun Bingfeng and others, for their kind support.
Lab officers, Ms. Tan Mei Yee Dinah, Mr. Beoy Kok Hong, Ms. Chai Keng, Mr. Chia Pai
Ann, Ms. Sandy Koh, Dr. Yuan Zeliang and others I may neglect to mention here, for
their kind assistance.
The financial support provided by National University of Singapore in the form of GST
stipend is greatly acknowledged.
i
TABLE OF CONTENT
ACKNOWLEDGEMENTS ................................................................................................. i
TABLE OF CONTENT...................................................................................................... ii
SUMMARY...................................................................................................................... vii
NOMENCLATURE .......................................................................................................... ix
LIST OF TABLES............................................................................................................. xi
LIST OF FIGURES .......................................................................................................... xii
LIST OF SCHEMES........................................................................................................ xiv
1 INTRODUCTION ....................................................................................................... 1
1.1 General Background ...................................................................................... 1
1.2 Objective and Thesis Organization................................................................ 4
2 LITERATURE SURVEY............................................................................................ 6
2.1 Cancer and Cancer Chemotherapy................................................................. 6
2.1.1 Cancer ..................................................................................................... 6
2.1.2 Cancer Treatment.................................................................................... 7
2.1.3 Cancer Chemotherapy............................................................................. 8
2.1.4 Problems in Chemotherapy..................................................................... 9
2.1.5 Chemotherapeutic Engineering............................................................. 10
2.2 Polymeric Drug Carrier................................................................................ 11
2.2.1 Polymers as Drug Carrier...................................................................... 11
2.2.1.1 Biodegradable Polymers................................................................ 11
2.2.1.2 Polyethylene Glycol ...................................................................... 15
ii
2.2.2 Polymeric Drug Formulations............................................................... 16
2.2.2.1 Paste............................................................................................... 16
2.2.2.2 Micelles ......................................................................................... 17
2.2.2.3 Liposomes ..................................................................................... 19
2.2.2.4 Microspheres ................................................................................. 21
2.2.2.5 Nanoparticles................................................................................. 22
2.3 Prodrug......................................................................................................... 23
2.3.1 Design and Synthesis of Polymeric Prodrugs....................................... 24
2.3.1.1 N-hydroxysuccinimide (NHS) Ester Coupling Method................ 25
2.3.1.2 Carbodiimide Coupling Method.................................................... 26
2.3.1.3 Dextran-prodrug ............................................................................ 28
2.3.1.4 N-(2-hydroxypropyl)methacrylamide (HPMA)-prodrug .............. 30
2.3.1.5 Dendrimer Conjugates................................................................... 31
2.3.2 PEGylated Drug Conjugation ............................................................... 34
2.3.2.1 PEGylation of Small Organic Molecules ...................................... 34
2.3.2.2 PEGylation of Polypeptide (Peptides and Proteins)...................... 36
2.3.2.3 Targeting PEGylation.................................................................... 37
2.4 Vitamin E TPGS .......................................................................................... 38
2.4.1 Chemistry of Vitamin E TPGS ............................................................. 38
2.4.2 Solubilizer for Water-insoluble Compounds ........................................ 40
2.4.3 Absorption Enhancer ............................................................................ 41
2.4.4 Bioavailability Enhancer....................................................................... 41
2.4.5 Anticancer Enhancer ............................................................................. 44
2.4.6 Drug Delivery Applications.................................................................. 45
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2.5 Doxorubicin ................................................................................................. 46
2.5.1 History................................................................................................... 46
2.5.2 Mechanism of Action............................................................................ 47
2.5.3 Side Effects and Limitations................................................................. 49
2.5.4 Formulations ......................................................................................... 50
3 SYNTHESIS AND CHARACTERIZATION OF THE TPGS-DOX CONJUGATE52
3.1 Introduction.................................................................................................. 52
3.2 Experiment Section...................................................................................... 52
3.2.1 Materials ............................................................................................... 52
3.2.2 Synthesis of TPGS-DOX ...................................................................... 53
3.2.2.1 TPGS Succinoylation .................................................................... 53
3.2.2.2 TPGS-DOX Conjugation .............................................................. 54
3.2.3 Characterization of TPGS-DOX Conjugate.......................................... 55
3.2.3.1 FT-IR ............................................................................................. 55
3.2.3.2 1H NMR......................................................................................... 56
3.2.3.3 GPC ............................................................................................... 56
3.2.3.4 Drug Conjugate Efficiency............................................................ 56
3.2.3.5 Stability of the Conjugate.............................................................. 57
3.3 Results and Discussion ................................................................................ 57
3.3.1 FT-IR Spectra........................................................................................ 57
3.3.2 1H NMR Spectra ................................................................................... 58
3.3.3 GPC Results .......................................................................................... 59
3.3.4 Drug Loading Capacity......................................................................... 61
3.3.5 In Vitro Stability ................................................................................... 62
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3.4 Conclusions.................................................................................................. 62
4 IN VITRO STUDIES ON DURG RELEASE KINETICS, CELLULAR UPTAKE
AND CELL CYTOTOXICITY OF THE TPGS-DOX CONJUGATE............................ 63
4.1 Introduction.................................................................................................. 63
4.2 Materials and Methods................................................................................. 63
4.2.1 Materials ............................................................................................... 63
4.2.2 In Vitro Drug Release ........................................................................... 64
4.2.3 Cell Culture........................................................................................... 64
4.2.4 In Vitro Cell Uptake Efficiency ............................................................ 64
4.2.5 Confocal Laser Scanning Microscopy .................................................. 65
4.2.6 In Vitro Cytotoxicity ............................................................................. 65
4.2.7 Statistics ................................................................................................ 66
4.3 Results and Discussion ................................................................................ 66
4.3.1 In Vitro Drug Release ........................................................................... 66
4.3.2 In Vitro Cellular Uptake........................................................................ 68
4.3.3 Confocal Laser Scanning Microscopy .................................................. 72
4.3.4 In Vitro Cytotoxicity ............................................................................. 74
4.4 Conclusions.................................................................................................. 79
5 IN VIVO INVESTIGATION ON PHARMACOKINETICS AND
BIODISTRIBUTION OF THE TPGS-DOX CONJUGATE ........................................... 81
5.1 Introduction.................................................................................................. 81
5.2 Materials and Methods................................................................................. 81
5.2.1 Animal................................................................................................... 81
5.2.2 In Vivo Pharmacokinetics ..................................................................... 82
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5.2.2.1 Drug Administration...................................................................... 82
5.2.2.2 Blood Collection and Sample Analysis......................................... 82
5.2.2.3 Pharmacokinetic Parameters ......................................................... 83
5.2.3 In Vivo Biodistribution.......................................................................... 84
5.2.3.1 Drug Administration...................................................................... 84
5.2.3.2 Tissues Collection and Samples Analysis ..................................... 84
5.2.3.3 Statistics......................................................................................... 85
5.3 Results and Discussion ................................................................................ 85
5.3.1 Pharmacokinetics .................................................................................. 85
5.3.2 Biodistribution ...................................................................................... 88
5.4 Conclusions.................................................................................................. 91
6 CONCLUSIONS AND RECOMMENDATIONS .................................................... 93
6.1 Conclusions.................................................................................................. 93
6.2 Recommendations........................................................................................ 95
7 REFERENCE............................................................................................................. 96
vi
SUMMARY
Polymer-drug conjugation is one of the major strategies for drug modifications, which
manipulate therapeutic agents at molecular level to increase their solubility, permeability
and stability, and thus biological activity. Polymer-drug conjugation can significantly
change biodistribution of the therapeutic agent, thus improving its pharmacokinetics (PK)
and pharmacodynamics (PD), increasing their therapeutic effects and reducing their side
effects, as well as provide a means to circumvent the multi-drug resistance (MDR). D-α-
tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or simply, TPGS), a
water-soluble derivative of natural Vitamin E, is a PEGylated vitamin E, which is formed
by esterification of vitamin E succinate with PEG 1000. Its amphiphilic structure,
comprising lipophilic alkyl tail and hydrophilic polar head, is bulky and has large surface
areas, which enables it to be an effective emulsifier and solubilizer. TPGS has been
intensively used in our research either as an effective macromolecular emulsifier or as a
component for a novel biodegradable copolymer PLA-TPGS for nanoparticle formulation
of therapeutic agents, which resulted in high drug encapsulation efficiency, high cellular
uptake and high in vitro cytotoxicity and in vivo therapeutic effects. Some reports
demonstrated that TPGS can increase the oral bioavailability and enhance cytotoxicity of
drugs. TPGS-drug conjugation should thus be an ideal solution for the drugs that have
problems in their adsorption, distribution, metabolism and excretion (ADME).
The aim of this study was to develop a novel TPGS-DOX conjugate to enhance the
therapeutic potential and reduce the systemic side effects of the drug, doxorubicin. In this
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research a novel prodrug, TPGS-doxorubicin conjugate, was successfully developed. The
hydroxyl terminal group of TPGS was activated by succinic anhydride (SA) and interacted
with the primary amine group of doxorubicin. The polymer-drug conjugation was
confirmed by 1H NMR, FT-IR and GPC to characterize the molecular structure and
molecular weight. The efficiency was determined to be 8% and stability of the conjugate
was also favorable for required storage period. The drug release from the conjugate was
pH dependent without significant initial burst. The cellular uptake, intracellular
distribution, and cytotoxicity of the polymer-drug conjugation were accessed with MCF-7
breast cancer cells and C6 glioma cells as in vitro model. The conjugate showed higher
cellular uptake and broader distribution within the cells. Judged by IC50, the conjugate was
found 31.8, 69.6, 84.1% more effective with MCF-7 cells and 43.9, 87.7, 42.2% more
effective with C6 cells than the pristine drug in vitro after 24, 48, 72 h culture,
respectively. In vivo pharmacokinetics confirmed the advantages of the prodrug. The area-
under-the-curve (AUC) was found to be 6,810 h·ng/mL for the prodrug but 289 h·ng/mL
for the doxorubicin, which implied 23.6 times more effective, and the half-life of the drug
is 9.65±0.94 h for the TPGS-DOX conjugation but 2.53±0.26 h for the original DOX,
which implied 3.81 times longer (p<0.01), at the 5 mg/kg DOX dose i.v. injection. Effects
of the conjugation on biodistribution showed the impaired systemic toxicity, especially for
heart, stomach and intestine.
The TPGS-DOX prodrug showed great potential to become a novel dosage form of
doxorubicin and may also be applied to other anticancer drugs.
viii
NOMENCLATURE
ACN Acetonitrile
AUC The area under the curve
BBB Blood brain barrier
BD Biodistribution
CL Clearance
CLSM Confocal laser scanning microscopy
CMC Critical micelle concentration
CyA Cyclosporine A
DCC N,N'-dicyclohexylcarbodiimide
DCM Dichloromethane
DMAP Dimethylaminopyridine
DMEM Dulbecco’s modified eagle medium
DMSO Dimethyl sulfoxide
DOX Doxorubicin
FBS Fetal bovine serum
FT-IR Fourier transform infrared spectroscopy
GI Gastrointestinal
GPC Gel permeation chromatography
HPLC High performance liquid chromatography
HPMA N-(2-hydroxypropyl)methacrylamine
IC50 The drug concentration at which 50% of the cell growth is inhibited
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MDR Multi-drug resistance
MRT Mean residence time
MTD Maximum tolerated dose
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
NHS N-hydroxysuccinimide
NMR Nuclear magnetic resonance spectroscopy
PBS Phosphate-buffered saline
PEG Poly(ethylene glycol)
PGA Poly(L-glutamic acid)
P-gp P-glycoprotein
PK Pharmacokinetics
SA Succinic anhydride
t1/2 Half-life time
TEA Triethylanmine
teffect Therapeutic effective period
THF Tetrahydrofuran
TPGS Vitamin E TPGS, d-α-tocopheryl polyethylene glycol 1000
succinate
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LIST OF TABLES
Table 4-1 IC50 values (in equivalent µM DOX) of MCF-7 and C6 cancer cells cultured with the TPGS-DOX conjugate vs the pristine DOX in 24, 48, 72 h ............................... 77 Table 5-1 Pharmacokinetic parameters of the TPGS-DOX conjugate vs the pristine DOX i.v. injected in the SD rats at the equivalent 5 mg/kg dose ............................................... 86 Table 5-2 AUC values (µg·h/g) of biodistribution in various organs after intravenous injection of the free DOX and the TPGS-DOX conjugate to rats at 5 mg/kg equivalent dose ................................................................................................................................... 91
xi
LIST OF FIGURES
Fig 2-1 Schematic of a micelle ......................................................................................... 18
Fig 2-2 Liposomes for drug delivery ................................................................................ 20
Fig 2-3 Schematic presentation of (a) polymeric conjugate with targeting moiety and (b) hyperbranched polymeric conjugate with targeting and imaging components................. 25
Fig 3-1 FT-IR spectra of DOX (red line), TPGS (purple line) and TPGS-DOX (blue line)........................................................................................................................................... 58
Fig 3-2 1H NMR spectra of (a) TPGS, (b) TPGS-SA, (c) DOX, (d) TPGS-DOX with the insert for a magnification of the region between 6 and 10 ppm ....................................... 60
Fig 3-3 Gel permeation chromatogram (GPC) of the TPGS-DOX conjugate and the unconjugated TPGS .......................................................................................................... 61
Fig 4-1 Release of DOX from TPGS-DOX conjugate incubated in phosphate buffer at 37 oC (mean ± SD and n=3)................................................................................................... 68
Fig 4-2 (a) MCF-7 and (b) C6 cell uptake efficiency cultured with the pristine DOX or the TPGS-DOX for 0.5, 1, 4, 6 h respectively at equivalent drug concentration 1 µg/mL (mean ± SD and n=6)........................................................................................................ 70
Fig 4-3 Cell uptake of the TPGS-DOX and DOX in (a) MCF-7 and (b) C6 cells after 4 h incubation (mean ± SD and n=6) ...................................................................................... 71
Fig 4-4 Confocal laser scanning microscopy (CLSM) of MCF-7 cells after 4 h incubation with (a) the pristine drug DOX and (b) with the TPGS-DOX conjugate, and that of C6 cells with (c) the DOX and (d) the TPGS-DOX conjugate at the equivalent 1 µg/mL DOX concentration..................................................................................................................... 74
Fig 4-5 Cellular viability of (a) MCF-7 breast cancer cells and (b) C6 glioma cells after 24, 48, 72 h culture with the DOX-TPGS conjugate respectively in comparison with that of the pristine DOX at various equivalent DOX concentrations (mean ± SD and n=6) ....... 76
xii
Fig 4-6 Cellular viability of MCF-7 breast cancer cells and C6 glioma cells after 24, 48, 72 h culture with TPGS respectively at various equivalent concentrations in the conjugate (mean ± SD and n=6)........................................................................................................ 78
Fig 5-1 Pharmacokinetic profile of the pristine DOX and the DOX-TPGS conjugate after intravenous injection in rats at a single equivalent dose of 5 mg/kg (mean ± SD and n=4)........................................................................................................................................... 85
Fig 5-2 The DOX levels (µg/g) in heart, lung, spleen, liver, stomach, intestine and kidney after i.v. administration at 5 mg/kg equivalent drug of (a) the free DOX, (b) the TPGS-DOX conjugate (mean ± SD and n=3).............................................................................. 90
xiii
LIST OF SCHEMES
Scheme 2-1 PEO families ................................................................................................. 13
Scheme 2-2 Chemical structure of polyanhydride............................................................ 14
Scheme 2-3 Chemical structure of some polyesters ........................................................ 15
Scheme 2-4 Chemical structure of PEG ........................................................................... 16
Scheme 2-5 NHS esters compounds react with nucleophiles to release NHS leaving group........................................................................................................................................... 26
Scheme 2-6 Carbodiimide amide coupling scheme.......................................................... 27
Scheme 2-7 Chemical structure of DCC, DIC and EDC, respectively............................. 27
Scheme 2-8 Chemical structure of dextran....................................................................... 28
Scheme 2-9 Chemical structure of dextran-methylprednisolone hemisuccinate.............. 29
Scheme 2-10 Synthesis of FITC-labeled CM dextran ...................................................... 30
Scheme 2-11 Examples of synthesis of HPMA-drug antibody conjugates ...................... 32
Scheme 2-12 Typical architecture of a third generation dendrimer ................................. 33
Scheme 2-13 Chemical Structure of (a) monomethoxy-PEG and (b) di-hydroxyl PEG.. 34
Scheme 2-14 Synthetic schemes of (a) PEG-Ara-C4 and (b) PEG-Ara-C8 conjugates .... 36
Scheme 2-15 Architecture of multi-block PEG-Dox with targeting antibody conjugated38
Scheme 2-16 Chemical structure of TPGS: red = hydrophobic vitamin E, green = hydrophilic PEG chain, black = succinate linker.............................................................. 39
Scheme 2-17 Chemical structure of doxorubicin.............................................................. 47
xiv
Scheme 3-1 Scheme of TPGS succinoylation .................................................................. 54
Scheme 3-2 Scheme of TPGS-DOX conjugation............................................................. 55
xv
1 INTRODUCTION
1.1 General Background
Prodrug is a pharmacological substance in an inactive (or significantly less active) form
that is formulated through transient modification of a given drug. In other words, a
prodrug is an inactive precursor of a drug. The involved temporary chemical modification
in prodrug can be metabolized in the body in vivo and leave the inherent pharmacological
properties of the parent drug intact (Saltzman 2001). A conjugation of a drug to a polymer
was called “polymeric prodrug”, which has led a new era of polymer drug delivery system
(Pasut and Veronese 2007). Polymeric prodrug has quite a few merits over the precursor
drug, such as increased solubility, enhanced bioavailability, improved pharmacokinetics,
ability of targeting and protected activity of protein drug (Khandare and Minko 2006). In
particular, Polymer-drug conjugation can significantly change biodistribution of the
therapeutic agent, thus improving its pharmacokinetics (PK) and pharmacodynamics (PD),
increasing their therapeutic effects and reducing their side effects, as well as provide a
means to circumvent the multi-drug resistance (MDR), which is caused by overexpression
of MDR transporter proteins such as p-glycoproteins (p-gp) in the cell membrane that
mediate unidirectional energy-dependent drug efflux and thus reduce intracellular drug
levels. The MDR transporter proteins are rich in the liver, kidney, and colon cells. Tumors
also acquire drug resistance through induction of MDR transport proteins during treatment
(Harris and Hochhauser 1992; Borst and Schinkel 1996; Schinkel 1997; Gottesman, Fojo
et al. 2002; Liscovitch and Lavie 2002; Müller, Keck et al. 2003). The medical solution
1
for MDR is to apply inhibitors of MDR transporters such as cyclosporine A, which may
also suppress the body immune system and thus cause medical complications. Moreover,
the inhibitors themselves may have problems in formulation and delivery. The engineering
solution is thus preferred, which does not use any p-glycoprotein inhibitor. Instead, it
applies high technologies such as nanotechnology and polymer-drug conjugation, to
engineering the drugs to avoid being recognized by the p-glycoproteins (Feng and Chien
2003; Feng 2004; Feng 2006).
Various architectures of polymers have been utilized as carrier to deliver drugs, some of
which have stepped into clinical development and some have shown promise (Kopeček,
Kopečková et al. 2001; Duncan 2003). In synthetic polymers, N-(2-
hydroxypropyl)methacrylamine (HPMA) copolymers (Kopeček, Kopečková et al. 2000;
Chytil, Etrych et al. 2006), Poly(ethylene glycol) (PEG) (Greenwald, Choe et al. 2003;
Harris and Chess 2003), and poly(L-glutamic acid) (PGA) (Li, Price et al. 1999) have
been predominantly utilized as the carriers of anticancer drugs such as doxorubicin,
campothecin and platinates. In particular, PEG is water soluble, biocompatible and
nontoxic, facilitating its application for conjugation with paclitaxel (Feng, Yuan et al.
2002), camptothecin (Conover, Greenwald et al. 1998), methotrexate (Riebeseel,
Biedermann et al. 2002) and doxorubicin (Veronese, Schiavon et al. 2005) to improve
their water solubility, plasma clearance and biodistribution.
D-α-Tocopheryl Polyethylene glycol 1000 succinate (vitamin E TPGS or simply, TPGS),
a water-soluble derivative of natural vitamin E, is formed by esterification of vitamin E
succinate with PEG 1000. Its amphiphilic structure enables it to be an effective emulsifier
2
and solubilizer (Fischer, Harkin et al. 2002). In our lab, TPGS has been successfully
applied as a novel emulsifier in preparation of PLGA nanoparticles and as a component of
a novel biodegradable polymer, PLA-TPGS for nano-carrier of anticancer agents, which
exhibited high emulsification efficiency, high drug encapsulation efficiency and improved
cellular uptake, cytotoxicity and therapeutic effects (Feng 2004; Feng 2006). TPGS was
also demonstrated possessing the ability to enhance the oral bioavailability of
cyclosporine A, vancomycin hydrochloride and talinolo in animals (Prasad, Puthli et al.
2003; Bogman, Zysset et al. 2005). Besides, co-administration of TPGS could enhance the
cytotoxicity of doxorubicin, vinblastine and paclitaxel by the inhibition on p-glycoprotein
mediated MDR (Dintaman and Silverman 1999).
Doxorubicin, an anthracyclinic antibiotic, is a DNA-interacting chemotherapeutic drug
(http://en.wikipedia.org/wiki/Doxorubicin; Takakura and Hashida 1995), which is
effective in treating breast cancer (Bonfante, Ferrari et al. 1986) as well as ovarian (ten
Bokkel Huinink, van der Burg et al. 1988), prostate (Raghavan, Koczwara et al. 1997),
cervix (Hoffman, Roberts et al. 1988) and lung cancers (Schuette 2001). However, clinical
use of doxorubicin is limited because of the short half-life (Al-Shabanah, El-Kashef et al.
2000) and acute systemic toxicity (Blum and Carter 1974). Additionally, the intracellular
level of doxorubicin can be reduced by the MDR effects (Krishna and Mayer 2000). This
triggered us to take the advantages of TPGS and involve it as a carrier by conjugation with
doxorubicin to enhance the drug’s therapeutic potential in vitro and in vivo as well as to
reduce systemic toxicity.
3
1.2 Objective and Thesis Organization
This thesis tells a story of TPGS-DOX conjugation for chemotherapy. The novel prodrug,
TPGS-DOX conjugate, was investigated on drug loading efficiency, conjugate stability,
drug release property, intracellular uptake, in vitro cytotoxicity and in vivo
pharmacokinetics and biodistribution. This project provides a novel dose form of
doxorubicin with improved therapeutic effects, whose methodology can also be utilized in
other anti-cancer drugs.
The thesis consists of six chapters. First, an introduction with general background and
thesis organization are included in chapter 1. Second, chapter 2 gives a comprehensive
literature review on polymeric-prodrug, highlighting the TPGS advantages in
chemotherapy. Then chapter 3 is dedicated to the synthesis and characterization of TPGS-
DOX conjugate. TPGS was first activated by succinic anhydride through ring opening
reaction. Then it was covalently attached to the primary amine group in doxorubicin. The
resultant product was characterized by FT-IR and NMR for the chemical structure, GPC
for the molecular weight and the distribution. The doxorubicin content conjugated to
TPGS was determined by fluorescence detection using microplate reader. The stability of
the prodrug was investigated in PBS at 4°C. Chapter 4 shows the in vitro results including
drug release from the conjugate, the intracellular uptake efficiency of the conjugate using
MCF-7 and C6 cancer cells as in vitro model with comparison of free DOX, and
cytotoxicity at various drug concentrations. At last, in chapter 5, pharmacokinetics and
biodistribution via intravenous administration of the TPGS-DOX and free DOX are
4
included, followed by final conclusions and some recommendations for future work in
chapter 6.
5
2 LITERATURE SURVEY
2.1 Cancer and Cancer Chemotherapy
2.1.1 Cancer
Cancer, caused by disordered division of cells, has been seriously threatening human
health. It is always combined with malignant behavior of these cells, which tend to spread
by direct invasion into adjacent tissue or metastasis to distant sites
(http://en.wikipedia.org/wiki/Cancer). Although rapider diagnosis of cancer has been
achieved than before, many forms are still incurable. According to BBC news, cancer has
become NO. 1 killer with 135,000 death per year compared to 110,000 from heart disease
(http://news.bbc.co.uk/1/hi/health/1015657.stm).
So far, the specific mechanisms of formation and spreading of cancer have not been well
explained. The external effects and internal factors are the most important two
contributors to the cause of cancer. Radiation, overexposure to certain chemical
substances, infectious agents, diet and lifestyle can initiate and promote carcinogenesis. In
particular, the tobacco products can even cause about 80% of all cancers, especially in
high risk of larynx, oesophagus, pancreas, bladder, kidney, cervix and lung cancers.
Regarding the internal factors, many theories have been demonstrated. The most important
seven contributors conclude failure of apoptosis, overactivation of oncogenes, inactivation
of tumor suppressor genes, cell cycle activation of quiescent cells, acquisition of
metastatic behavior by malignant cells, disordered responses to cellular growth factors,
6
and immune system surveillance failure (Hanahan and Weinberg 2000). These factors
may act together or sequentially, no matter the external or internal ones.
2.1.2 Cancer Treatment
Cancer cells may break away and grow into a distant point of a normal tissue, which is
called metastasize. Therefore, tumors are classified to two types, benign and malignant.
Benign tumors do not spread, which are localized in one part of a body without lethal
threaten. In contrast, malignant tumors can spread from the original location to other parts
via the blood or/and the lymphatics, which usually happens in late stage of cancer.
Different cancer cells have specific propensity in metastasis. Prostate cancer intends to
spread into bones, while colon cancer prefers liver (Fausto).
Prevention is more active measure than cure to defense against cancer, which is classified
into primary and secondary type for a patient without and with a history of disease,
respectively. It was reported that low meat intake and certain coffee consumption are
associated with the reduced risk of cancer (Ward, Sinha et al. 1997; Sinha, Peters et al.
2005; Larsson and Wolk 2007). Moreover, some vitamins (Lieberman, Prindiville et al.
2003), β-carotene (http://www.cancer.gov/cancertopics/factsheet/Prevention/betacarotene)
and other chemoprevention agents, such as tamoxiten (Vogel, Costantino et al. 2006) and
finasteride (Baron, Sandler et al. 2006), have been demonstrated to be protective against
cancer. When prevention fails, cure is necessary.
Cancer can be treated through several methods, the effective ones among which are
surgery, radiotherapy, chemotherapy, hormone therapy and immunotherapy, although they
7
possess their own advantages and disadvantages. According to the stage of the cancer and
the location and grade of the tumor, as well as the condition of the patients, different
therapy or a combination will be employed. Removing the cancer completely without
damage to the normal tissue is the ideal solution of the treatment. It can be achieved
sometimes by surgery, but this is not always feasible, especially when the cancer can
metastasize to other sites in body. Besides, patients have to suffer from physical pain and
even the danger of infections. What is worse, it may speed up the growth rate of the
remaining cancer cells and even cause death by metastatic cancer. The larger excised
tumor causes the greater possibility of death. Radiation therapy utilizes ironing radiation
to kill cancer cells and shrink tumors, which can be almost employed into every type of
solid tumors. It can damage not only the cancer cells but also normal cells. However, the
normal cells can recover itself after the treatment. As radiotherapy is a localized strategy
like surgery, it is not effective to the patients suffering metastasis. Hormonal therapy
employs or blocks certain hormones to inhibit the cancer cell growing, which may cause
quite a few side effects such as nausea, swelling of limbs and weight gain. Immunotherapy
is a therapeutic strategy, which induces the patient’s own immune system to fight against
cancer. Cancer chemotherapy, using chemotherapeutical agents to killing or suppressing
cancer cells, first succeeded in 1950s and was widely employed in 1960s. However, most
chemotherapeutic agents cause serious side effects, which limits its application (Feng and
Chien 2003). Combination of chemotherapy with other strategies is a prominent treatment
recently in cancer cure.
2.1.3 Cancer Chemotherapy
8
Chemotherapy sometimes is defined as the use of chemical substances to treat disease
(http://en.wikipedia.org/wiki/Chemotherapy), which, however, is often toxic and even
life-threatening. Chemotherapy of cancer utilizes chemotherapeutic agents to control or
kill cancer cells, usually, via damaging to DNA or RNA of cancer cells
(http://www.chemocare.com/whatis/cancer_cells_and_chemotherapy.asp). So far,
hundreds of anti-cancer drugs have been found for clinical application, some of which are
natural extracts while others are synthetic or semi-synthetic agents. The majority of the
drugs can be classified into: alkylating agents, antimetabolites, anthracyclines, plant
alkaloids, topoisomerase inhibitors, monoclonal antibodies, etc. Some drugs perform
better while working together with other anti-cancer drugs, which is called combination
chemotherapy. Nanotechnology has been designed and investigated to achieve cancer
chemotherapy at home (Feng 2004).
2.1.4 Problems in Chemotherapy
Chemotherapy is an effective and complicated procedure for treating cancer. However,
anti-cancer drugs not only damage cancer cells, but also affect normal cells, which usually
leads to serious side effects including hair loss, nausea and vomiting, diarrhea or
constipation, anemia, malnutrition, memory loss, depression of the immune system,
hemorrhage, secondary neoplasms, cardiotoxicity, hepatotoxicity, hephrotoxocity,
ototoxicity and even death (http://en.wikipedia.org/wiki/Chemotherapy). The specific side
effects are up to what and how much of the chemotherapeutic agent is administrated and
how the body may response. One or more of the above effects may be observed after the
treatment.
9
Most of anticancer drugs are hydrophobic agents, thus adjuvants are required to make
them available in clinical use. For example, paclitaxel, a diterpenoid extracted from the
bark of Pacific yew tree, exhibits a wide anti-cancer spectrum. However, it is too
hydrophobic with a water solubility ≤0.5 mg/l to be directly administrated into body
(Mathew, Mejillano et al. 1992). Therefore, Cremophor EL was used as an adjuvant in
dose form but it causes serious side effects, such as hypersensitivity reactions,
nephrotoxicity, neurotoxicity and cardiotoxicity (Weiss, Donehower et al. 1990; Li, Price
et al. 1999).
Moreover, although chemotherapy increasingly succeeds, especially in initial stage, it
usually becomes less effective in the long-term therapy, which is associated with drug
resistance. The drug resistance can be due to three mechanisms: pharmacokinetic
resistance, kinetic resistance and genetic resistance. In a combinational chemotherapy,
multidrug resistance (MDR) may develop, which is found to be relevant with an
overexpression of P-glycoprotein (P-gp). P-gp is well known for removal of toxic
substance in normal function. It effluxes the drugs out of cells by an energy mediated
unidirectional process. These MDR transporter proteins are rich in the liver, kidney, and
colon cells and also involved in the various physiological drug barriers such as the
gastrointestinal (GI) drug barrier for oral chemotherapy, the blood-brain barrier (BBB) for
brain tumor treatment and the intratumoral barrier for efficient drug delivery in the tumor
(Harris and Hochhauser 1992; Schinkel 1997; Krishan 2000; Liscovitch and Lavie 2002;
Müller, Keck et al. 2003).
2.1.5 Chemotherapeutic Engineering
10
Chemotherapeutic engineering refers to the application and development of chemical
engineering principles and methodologies for chemotherapy so that better efficacy can be
achieved with fewer side effects. In order to solve the above problems, effective drug
delivery system seems as important as the discovery of new drugs, which makes the
development of chemotherapeutic engineering emergent. In resent decades, the
development of diversity drug delivery system with improved efficacy but reduced side
effects became a hot spot in chemotherapy. Numerous drug delivery strategies have been
intensively investigated, among which microspheres (Couvreur and Puisieux 1993),
nanoparticles (Couvreur, Roblot-Treupel et al. 1990; Brigger, Dubernet et al. 2002),
liposomes (Daoud, Hume et al. 1989; Pinto-Alphandary, Andremont et al. 2000), micelles
(Jones and Leroux 1999), cyclodextrins (Utsuki, Brem et al. 1996), pastes (Li, Price et al.
1999) and prodrug (Senter, Svensson et al. 1995; Soyez, Schacht et al. 1996; Springer and
Niculescu-Duvaz 1996; Khandare and Minko 2006) have attracted much attention.
2.2 Polymeric Drug Carrier
2.2.1 Polymers as Drug Carrier
In 1974, the discovery of the ability of macromolecules to localize to lysosomes started a
new era for macromolecules used as drug carriers (De Duve, De Barsy et al. 1974). Since
1975, when the rationale of polymeric targeted drug delivery was formulated on the basis
of previous investigations (Ringsdorf 1975), polymers, either natural or synthetic origin,
have been widely used in drug delivery system.
2.2.1.1 Biodegradable Polymers
11
The natural polymers, such as collagen, fibrin, alginate, silk, hyaluronic acid and chitosan,
are abundant and have good biodegradability and biocompatibility, which counteract their
drawbacks such as immunogenicity, instability and lack of chemical group for
modification (Duncan and Kopeček 1984). Meanwhile, perhaps synthetic polymers are the
most widely used materials as growth factor delivery carries, especially for biodegradable
synthetic polymers, which provide excellent chemical and mechanical properties that
natural polymers often fail to possess. The frequently used biodegradable polymers
include poly(ortho esters) (POE), polyanhydride and polyesters.
POE was developed in early 1970s, since when, four families of POE have been indicated
as shown in Scheme 2-1 (Tomlinson, Heller et al. 2003). The polymer can degrade to a
diol and a lactone, which will further produce γ-hydroxybutyric acid. The high
hydrophobic property of the polymer results in the limitation of the amount that water can
penetrate the polymer. As the erosion rate of the polymers is extremely slow, they can be
as a compression molded disk in an aqueous environment. More importantly, POE II and
POE IV have been found to have excellent potential as a delivery system in ocular
application (Tomlinson, Heller et al. 2003).
12
Scheme 2-1 PEO families
Polyanhydrides are a kind of biodegradable polymers in which anhydride bonds connect
monomer units of the chain. The chemical structure of a polyanhydride molecule with n
repeating units is shown in scheme 2-2. Water-labile anhydride bonds in polyanhydrides
give two carboxylic acid groups, which results in easy metabolization and
biocompatibility. Polyanhydrides consist of three main classes, which are aliphatic,
unsaturated and aromatic polyanhydrides (Hazra, Golenser et al. 2002). Polyanhydrides
can degrade uniformly into non-toxic metabolites and thus become useful for controlled
drug delivery (Hazra, Golenser et al. 2002). Gliade®, made of a polyanhydride wafer
containing a chemotherapeutic agent, can deliver carmustine to the malignant glioma
tumor, telling a successful story of polyanhydrides (Mishra and Jain 2000). However, as
polyanhydrides are quite sensitive to water, some hydrophobic monomers are employed to
resist the water penetration and increase the stability of polyanhydrides in water (Saltzman
2001).
13
Scheme 2-2 Chemical structure of polyanhydride
Polyesters, which contain the ester functional group in the main chain, have been
approved by FDA and are widely used as biodegradable polymers. The homo or
copolymers based on glycolic and lactic acid are significantly common, which have been
studied for more than 50 years (Lowe 1954; Schneider 1955). Polyglycolide (PGA) is the
simplest linear, aliphatic polyester. It exhibits somewhat unique solubility, in that the high
molecular weight form is insoluble in almost all the organic solvents while the low
molecular weight form is more soluble. On the other hand, PGA is soluble in fluorinated
solvents, which can be utilized for melt spinning and film preparation (Schmitt 1973). The
initial application of PGA was limited because of its hydrolytic instability. Recently, PGA
and its copolymers with lactic acid, ε-caprolacone or trimethylene carbonate are in wide
use as materials for absorbable sutures and being investigated in biomedical area (Gilding
and Reed 1979; Middleton and Tipton 1998). Polylactide is thermoplastic, aliphatic
polyester synthesized from lactide by ring opening polymerization. Due to the chiral
nature of lactide, there are two optical stereoisomers, poly(L-lactide) (PLLA), poly(D-
lactide) (PDLA) and poly(D,L-lactide) (PDLLA). As PDLLA degrades faster than others
do, it attracts more attention as a drug delivery system (Conti, Pavanetto et al. 1992).
Poly(lactic-co-glycolic acid) (PLGA), also a FDA approved copolymer, can be
14
synthesized by random ring-opening co-polymerization of glycolic acid and lactic acid,
producing different forms depending on the ratio of two monomers. PLGA can be
degraded by hydrolysis in water. Besides, PLGA can be dissolved in a wide range of
common solvents not as the poor solubility of PLA and PGA in organic phase. So far,
PLA and PLGA are in a dramatically wide application among a variety of biomedical
devices because of their satisfactory biodegradability, biocompatibility and restorability
(Holland, Tighe et al. 1986; Van Rensburg, Jooné et al. 1998; Riebeseel, Biedermann et al.
2002). In addition, poly (ε-caprolactone) (PCL) is another biodegradable polyester, which
degrades by hydrolysis quite slow in physiological conditions. Thus it is more attractive
for the preparation of implantable devices and used in controlled release and targeted drug
delivery (Sinha, Bansal et al. 2004; Zhang, Lee et al. 2007).
Scheme 2-3 Chemical structure of some polyesters
2.2.1.2 Polyethylene Glycol
Poly (ethylene glycol) (PEG, Scheme 2-4) is a unique polyether diol, which has been
approved by FDA for human intravenous, oral and dermal application. It can be
manufactured by interaction of ethylene oxide with water, ethylene glycol or ethylene
glycol oligomers. The ratio of reagents determines the chain length, producing a variety of
molecular weights of polymers (http://chemindustry.ru/Polyethylene_Glycol.php).
Initiation of ethylene oxide polymerization using anhydrous alkanols results in a
15
monoalkyl capped PEG such as methoxy PEG, which is amphiphilic and can dissolve in
both organic solvents and water. In addition to linear PEG chains, there are also some
branched PEGs in which two or more PEG chains may be joined through linkers such as
lysine (Monfardini, Schiavon et al. 1995) and triazine (Abuchowski, van Es et al. 1977).
Similarly, amphiphilic copolymers have been developed such as PLA-PEG (Ramaswamy,
Zhang et al. 1997; Nguyen, Allemann et al. 2003; Youk, Lee et al. 2005), PCL-PEG
(Gyun Shin, Yeon Kim et al. 1998; Kim and Lee 2001; Luo, Tam et al. 2002), PVL-PEG
(Tomlinson, Heller et al. 2003), PGA-PEG (Smith, Schimpf et al. 1990; Kim, Shin et al.
1999), etc., which contains both hydrophilic portion and hydrophobic part. Although PEG
is known to be non-degradable, the non-toxic property (Pang 1993), excellent solubility in
aqueous solution (Powell 1980), low level of protein or cellular absorption, extremely low
immunogenicity and antigenicity (Dreborg and Akerblom 1990), and satisfactory
pharmacokinetic and biodistribution behavior (Yamaoka, Tabata et al. 1994) enable it to
be an ideal material in pharmaceutical applications (Hooftman, Herman et al. 1996).
Scheme 2-4 Chemical structure of PEG
2.2.2 Polymeric Drug Formulations
2.2.2.1 Paste
Local administration or direct tumor injection of chemotherapeutic agents has been
expected by a method that can maximize local drug level in tumor environment but
16
minimize systemic exposure to normal organs. Chemotherapeutic polymer-paste is one of
the strategies to reduce local recurrence of disease at tumor site in the surgery. To date,
paclitaxel-loaded polymeric surgical paste has been designed and well developed. PCL,
which has low melting range (50-60 °C) and biodegradation lifetime of 6-9 months in vivo
(Pitt, Gratzl et al. 1981), was employed as the base component. Paclitaxel was added into
melt PCL and then poured into a prepared mould. The polymer with paclitaxel would
solidify after cooling down to obtain the paste. In surgical application, paste was melt and
delivered via injection directly to the tumor resection as a liquid which formed a solid
conform at surgical wound under body temperature (Winternitz, Jackson et al. 1996).
Adding mPEG was reported to reduce the onset of the melting temperature by 5-10 °C
(Winternitz, Jackson et al. 1996), while gelatin, albumin or methylcellulose can speed up
the release of paclitaxel from the matrix (Dordunoo, Oktaba et al. 1997). Therapeutic drug
level can be maintained in the region of implanted site but reduced at non-targeting distant
site. Optimally, all paste formulations showed no great effect on body weight of the
treated animals, revealing the well tolerated drug dose (Zhang, Jackson et al. 1996).
However, the brittle and inflexible properties and difficult manipulation limit the further
development of the paste as the optimal formulations of chemotherapeutic agents.
2.2.2.2 Micelles
A micelle is an aggregate of surfactant molecules dispersed in a liquid colloid, which
contains normal phase micelles (oil-phase-water) and inverse phase micelles (water-in-oil).
When the concentration is higher than the Critical Micelle Concentration (CMC), which is
the concentration of a monomeric amphiphile the micelle appears (Charman 1992), almost
17
all of the polymers can formulate to be micelles as a core-shell structure (Fig 2-1) with a
small particle size (less than 100 nm). Polymeric micelles are manufactured from
copolymers comprising both hydrophilic and hydrophobic polymers as the core and
hydrophobic drug can be mainly entrapped into core. For the hydrophobic part, a
biodegradable polymer such as PLA, PCL, PGA and PLGA is used and for the
hydrophilic part, PEG is the most used polymer to yield high in vitro stability (Yokoyama,
Sugiyama et al. 1993). The hydrophobic drug can be loaded in the amphiphiles by dialysis,
salting out, emulsion and solvent evaporation method.
Fig 2-1 Schematic of a micelle
As drug carriers, micelles can provide quite a few obvious advantages. They can solubilize
poorly soluble drugs and increase drugs’ bioavailability. The size of micelles permits them
to accumulate in body regions through leaky vasculature. The unique structure makes
them hardly reactive to blood or tissue components. By attachment via a certain ligand to
the outer shell, they can result in targeted efficacy. Last but not least, micelles can be
prepared in large quantities easily and reproducibly (Torchilin 2002). So far, some
chemotherapeutic agents have been formulated in micelles. Paclitaxel-loaded micelles of
PEtOx-PCL copolymers was fabricated by dialysis method, which had a size of only 20
18
nm with a drug loading efficiency of 0.5-7.6%. The micelles showed comparable
therapeutic efficacy to the commercial paclitaxel in vitro (Cheon Lee, Kim et al. 2003). By
conjugating folate to PCL-mPEG copolymer, Park et al (Park, Lee et al. 2006) formulated
the targeted micelles. Doxorubicin-loaded micelles of PEO-P(Asp) (Yokoyama, Kwon et
al. 1992), PEO-PBLA (Kwon, Naito et al. 1995), PEO-PDLLA (Pişkin, Kaitian et al.
1995), PAA-PMMA (Inoue, Chen et al. 1998), etc. have been investigated. Although
micelles can give some improvement in chemotherapy, the instable problems for storage
became a restriction in further application (Tomlinson, Heller et al. 2003).
2.2.2.3 Liposomes
Liposomes is a phospholipids spherical vesicle, consisting of an aqueous core surrounded
by a lipid bilayer or multilayer (Fig 2-2) (http://en.wikipedia.org/wiki/Liposome), usually
range in size from 0.05 to 5.0 µm. Phospholipids possess a polar head and two
hydrophobic tails, which is significant to the formulation of bilayer in aqueous solution.
Due to the unique structure, liposomes can protect and carry hydrophilic drug in aqueous
core or hydrophobic drug in the lipid layers. The preparation methods may varies
according to different size and characteristic liposomes, generally including thin-film
hydration, freeze-drying, detergent dialysis, calcium induced fusion, reverse-phase
evaporation, sonication and extrusion (Sharma and Sharma 1997).
19
Fig 2-2 Liposomes for drug delivery
Liposomes have attracted much attention in medical application, which is attributed to
their biocompatibility with cell membranes, capability to protect drug from degradation,
especially for protein drugs, and ability to add specific targeting ligands on surface (Lee
and Yuk). As liposomes are manufactured from lipids which are relatively non-toxic, non-
immunogenic, biocompatible and biodegradable, a broad range of water-insoluble drugs
such as cyclosporine and paclitaxel can be encapsulated. Sharma et al. (Sharma, Mayhew
et al. 1997) reported that paclitaxel liposomes could deliver drug effectively in body
system and improve therapeutic index in in vivo model. Traditional liposomes are taken up
by RES and cleared quickly from blood circulation (Poste, Bucana et al. 1982).
Cholesterol and PEG modified liposomes significantly impaired such limitations (Wheeler,
Wong et al. 1994). Moreover, PEGylated liposomes tagged with transferring exhibited 2-
to 3-fold higher targeting effect than the plain liposomes (Visser, Stevanovic et al. 2005)
and folate-targeted liposomes showed much higher affinity to tumor cells (Goren,
Horowitz et al. 2000).
20
Although liposomes have achieved some improvement in drug delivery system, issues like
stability, reproducibility, sterilization method, low drug loading efficiency, particle size
control and short circulation half-life in body are the remaining problems need to be
solved (Sharma and Sharma 1997).
2.2.2.4 Microspheres
Microspheres, especially biodegradable polymeric microspheres, have been studied
extensively. Solvent evaporation and spray drying are two commonly used methods for
microspheres preparation, as well as hot melt microencapsulation, solvent removal, phase
inversion microencapsulation (Vasir, Tambwekar et al. 2003). In solvent evaporation
method, spherical droplets can be formed by dispersing oil soluble monomers in aqueous
solution (oil in water, O/W) or water soluble monomers in an organic phase (water in oil,
W/O). Often, a double emulsion is employed, which means the first W/O emulsion in
which drug is loaded in aqueous phase is then dispersed in another aqueous medium to get
the final O/W emulsion. Microspheres are able to protect the drug molecules against
degradation, control their release after administration and facilitate their passage across
biological barriers. Some researchers have achieved a constant release of drug from the
polymeric microspheres via a W/O/W double emulsion solvent evaporation method after
the initial burst (Yang, Chung et al. 2000). Liggins et al (Liggins, D'Amours et al. 2000)
indicated that microparticles with less than 8 µm may be cleared from the peritoneum into
the lymph nodes. Recently, poly(ortho-ester) microspheres was made for delivering DNA
vaccines and tested in vivo (Wang, Ge et al. 2004). However, the relatively large size of
microspheres may limit its application in some cases such as intravenous delivery.
21
2.2.2.5 Nanoparticles
Polymer nanoparticles are microscopic particles with particles size less than 1 µm
diameter, which have became an attractive area for drug delivery application, especially
for biodegradable polymeric nanoparticles. Nanoparticles can be used to deliver
hydrophilic or hydrophobic drugs, proteins, vaccines, biological macromolecules, etc..
Nanoparticles can be fabricated by dispersion of polymers and polymerization of
monomers, which involves solvent extraction/evaporation method, salting-out method,
dialysis method, supercritical fluid spray technique and nanoprecipitation method (Feng
and Chien 2003). The most commonly used method is solvent extraction/evaporation,
which can encapsulate hydrophobic drugs by single emulsion and hydrophilic drugs via
double emulsion method.
Polymeric nanoparticles have been investigated as potential drug carriers because of their
unique advantages including providing a controlled release of drugs, targeting drugs to
tumors, showing available size for intravenous injection, reducing the uptake of drugs to
RES, improving biodistribution of drugs in body (Kim, Lee et al. 2003). It has been
indicated that the new-concept chemotherapy by nanoparticles of biodegradable polymers
can realize personalized chemotherapy with controlled dosage and duration, localized
chemotherapy by targeting, sustained and controlled chemotherapy with favorable release
profile of drugs, chemotherapy across biological barriers like GI and BBB, chemotherapy
at home via oral, nasal or ocular administration (Feng 2004). Innovatively fabricated PLA-
TPGS, PLGA-MMT and PLA-mPEG nanoparticles for paclitaxel formulation exhibited
great superiority over Taxol and even the PVA-emulsified PLGA nanoparticles (Li, Price
et al. 1999; Feng and Huang 2001; Feng, Yuan et al. 2002; Riebeseel, Biedermann et al.
22
2002; Mu and Feng 2003; Mu and Feng 2003; Yin Win and Feng 2005; Dong and Feng
2006; Win and Feng 2006; Zhang and Feng 2006). Besides, targeting (Song, Labhasetwar
et al. 1998; Nishioka and Yoshino 2001) and multifunctional (Kopelman, Lee Koo et al.
2005) nanoparticles also become attractive and have been investigated recently.
Nanoparticles, indeed, showed a promising approach, however, the initial burst and
incomplete release of the encapsulated molecules in microspheres and nanoparticles,
particularly for protein, may influence their application (Hong Kee Kim 1999; Kwon,
Baudys et al. 2001).
2.3 Prodrug
Polymer-drug conjugation is a major strategy for drug modifications, which manipulates
therapeutic agents in molecular level in order to increase their biological activity. Such a
strategy is based on a central assumption that the molecular structure of drugs can be
modified to make analogous agents, which are chemically distinct from the original
compound, but produce a similar or even better biological effect. Drug modifications are
frequently directed to alter the properties of the drug that influence its concentration
(solubility), its duration of action (stability), or its ability to move between compartments
in tissues (permeability). Polymer-drug conjugation can modify biodistribution of
therapeutic agents, thus improving their pharmacokinetics (PK) and pharmacodynamics
(PD), increasing their therapeutic effects and reducing their side effects, as well as provide
a means to circumvent the multi-drug resistance (MDR). Prodrug, as the pharmacological
substance in an inactive form, can be metabolized in the body in vivo into the active
compound once administered (Saltzman 2001). Polymer-anticancer drug conjugation has
been intensively investigated in the literature (Khandare and Minko 2006). Some
23
polymeric prodrugs have stepped into clinical development and several conjugates have
shown promise (Kopeček, Kopečková et al. 2001; Duncan 2003).
2.3.1 Design and Synthesis of Polymeric Prodrugs
Polymers have been a hot spot as carriers of drugs over the latest decades, especially
designed to be polymeric prodrugs. A number of polymeric conjugation methods have
been investigated since 1955, when peptamin-polyvinylpyrrolidone conjugates with
improved efficacy was reported (Jactzkewits 1955). In present, there are three major types
of polymeric prodrugs in use: (a) prodrugs which can be broken down in cells to release
active agents, (b) prodrugs in combination of more than one substance, (c) prodrugs with
targeting ability. Generally, an ideal polymeric prodrug was revealed to possess one or
more of the following components: (a) a polymeric backbone as a carrier, (b) active
therapeutic agents, (c) appropriate spacers, (d) an imaging substance and (e) a targeting
molecule (Fig 2-3) (Khandare and Minko 2006). The choice of an appropriate polymer
and a targeting agent is crucial for the success of a prodrug. The selection of a polymer
should meet the following criteria: (a) available chemical functional groups to permit
covalent linkage with drugs or targeting agents, (b) hydrophilic property to ensure water
solubility, (c) degradability to ensure excretion from the body, (d) biocompatibility to
avoid immunogenic response, (e) availability in reproduction and administration (Soyez,
Schacht et al. 1996). The selected polymers can be classified by the origin, chemical
structure, biodegradability and molecular weight. In addition, modification of a polymer is
significant, which depends upon the reactive chemical groups in polymer and the
functional group of the drug. Most of the biomolecules like ligands, peptides, proteins,
carbohydrates, lipids, polymers, nucleic acid and oligonucleotide possess functional
24
groups for conjugation or have potential to be activated for further conjugation. According
to different molecules, a suitable method, process and reagents are, indeed, vital to the
successful conjugation. The following are some strategies commonly used to obtain a
polymeric prodrug, mainly regarding the active group and polymer backbone.
Fig 2-3 Schematic presentation of (a) polymeric conjugate with targeting moiety and (b) hyperbranched polymeric conjugate with targeting and imaging components
2.3.1.1 N-hydroxysuccinimide (NHS) Ester Coupling Method
An N-hydroxysuccinimide (NHS) ester is perhaps the most common activation chemical
method for creating reactive acylating agents (Scheme 2-5). Carboxyl groups activated by
NHS esters show highly reactive to amine nucleophiles. NHS ester derivatives can be
prepared in nonaqueous condition employing water-insoluble carbodiimides or
25
condensing agents, or in aqueous media using the water-soluble carbodiimide EDC in situ
(Hermanson 1996). Due to the high reactivity at physiological pH, NHS is widely utilized
as an acylating agent and preferred for conjugation with amine terminal compounds (Cruz,
Iglesias et al. 2001; Luis J. Cruz 2001; Chockalingam, Gadgil et al. 2002; Kajiyama,
Kobayashi et al. 2004; Cabrita, Abrantes et al. 2005; Natarajan, Xiong et al. 2005).
However, both hydrolysis and amine reactivity would increase with the increase of pH.
Thus maintaining high concentrations of protein or other target molecules in the reaction
is quite necessary.
Scheme 2-5 NHS esters compounds react with nucleophiles to release NHS leaving group
2.3.1.2 Carbodiimide Coupling Method
A carbodiimide is a functional group comprising the formula N=C=N, which is used to
mediate the formation of an amide or phosphoramidate linkage between a carboxylate and
an amine or a phosphate and an amine, respectively (Scheme 2-6). The carbodiimides are
either water-soluble such as EDC or water-insoluble such as DCC and DIC. Water soluble
carbodiimides can be used for biomacromolecular conjugation in a variety of buffer
solutions. On the other hand, water-insoluble carbodiimides are preferred for conjugation
26
of polymers with drugs, imaging agents, targeting agents or other polymers in organic
solvents (Hermanson 1996).
Scheme 2-6 Carbodiimide amide coupling scheme
The most commonly used carbodiimides are N,N'-dicyclohexylcarbodiimide (DCC), N,N'-
diisopropylcarbodiimide (DIC) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC) (Scheme 2-7). DCC can bring high yielding amide coupling reaction
but lead dangerous allergic reaction contacted with skin and the insoluble byproduct N,
N’-dicyclohexylurea is difficult to be removed completely, which limits its application in
certain reactions. DIC, as a liquid, can be easily removed by extraction and does not lead
an allergic reaction, which has been developed as an alternative to DCC. With a favorable
solubility in water, EDC was widely used in peptide synthesis, protein crosslinking with
nucleic acids and immunoconjugation (Nakajima and Ikada 1995).
Scheme 2-7 Chemical structure of DCC, DIC and EDC, respectively
27
2.3.1.3 Dextran-prodrug
Dextran is a complex, branched, natural polysaccharide, which possesses a huge number
of primary and secondary carbohydrate hydroxyl groups (Scheme 2-8). Thus it can be
conjugated to drugs and proteins which have active chemical groups either by direct
conjugation or incorporation of a spacer. Dextran is manufactured from sucrose by certain
lactic-acid bacteria, the best-known as Leuconostoc Mesenteroides and Streptococcus
mutans. The homogeneous structure of dextran makes the synthesized conjugates easy to
characterize and the high water solubility can be restored (Larsen 1989).
Scheme 2-8 Chemical structure of dextran
Dextran has been extensively investigated as a polymeric carrier to deliver anticancer
drugs through conjugation. Mehtyprednisolone and dexamethasome were covalently
attached to dextran using succinate linker, which showed potential of the dextran prodrug
in colon-specific delivery of glucocorticoides (McLeod, Friend et al. 1993).
Methylprednisolone-dextran prodrug (Scheme 2-9) also employed succinic acid as a linker
and 1,1’-Carbonyldidmidaxole as coupling agent, which achieved controlled hydrolysis
(Mehvar, Dann et al. 2000). The multiple hydroxyl groups on dextran provide efficient
functional sites for conjugation. In camptothecin (CPT) analogue-carboxymethyl (CM)
28
dextran conjugate via Gly-Gly-Gly linker, fluorescein isothiocyanate (FITC) was also
conjugated to dextran, producing a prodrug with imaging moiety as shown in Scheme 2-
10 (Harada, Murata et al. 2001). A dextran-peptide-methotrexate conjugate with tumor
targeting capability was reported (Chau, Tan et al. 2004). Due to the conjugated linker,
Pro-Val-Gly-Leu-Ile-Gly peptide cleavable by matrix metalloproteinase II (MMP-2) and
IX (MMP-9) that are over expressed in tumors, the conjugate exhibited satisfactory
stability and high selectivity uptake.
Scheme 2-9 Chemical structure of dextran-methylprednisolone hemisuccinate
29
Scheme 2-10 Synthesis of FITC-labeled CM dextran
2.3.1.4 N-(2-hydroxypropyl)methacrylamide (HPMA)-prodrug
N-(2-hydroxypropyl)methacrylamide (HPMA) is a water-soluble, nonimmunogenic,
synthetic polymer, which has suitability as carriers for drug delivery (Kopecek 1990).
HPMA copolymers are not biodegradable but are highly biocompatible (Kopeček,
Kopečková et al. 2000). HPMA homopolymer was designed and manufactured in
Czechoslovakia as a plasma expander (Kopecek and Bazilova 1973). Many functional
groups such as charged groups, targeting moieties and drugs can be incorporated into the
polymer backbone. The spacers in this connection can be either nondegradable or
degradable. The dipeptide glycylglycin (GG) is always utilized as nondegradable spacer
(Drobník, Kopeček et al. 1976) while the acid sensitive cis-aconityl bond and the
30
lysosomal enzyme sensitive tetrapeptide GFLG are employed as degradable spacers
(Rejmanova, Kopecek et al. 1985).
HPMA copolymers and their conjugates with drugs have become one of the most
extensively investigated systems. An HPMA copolymer with doxorubicin conjugated with
the peptidyl linker GFLG exhibited superior cytotoxicity in vitro and successfully
overcame P-gp mediated drug resistance (Minko, Kopečková et al. 1999; Kopeček,
Kopečková et al. 2001), which also displayed significant effectiveness in reducing both
sensitive and resistant tumors in vivo as compared to free DOX (Kopeček, Kopečková et
al. 2000). Various targeting moieties, such as carbohydrates and antibodies, were attached
to HPMA copolymer conjugate, which achieved the biorecognizability at the plasma
membrane (Chytry, Letourneur et al. 1998; Chytil, Etrych et al. 2006). A targeted
galactosamine containing HPMA copolymer-GFLG-DOX was found to induce dramatic
tumor associated toxicity (Duncan 1992). These preclinical studies prepared the clinical
trials of the conjugate, which is available currently. A number of synthetic methods have
been developed (Scheme 2-11) for antibodies attached to HPMA copolymer conjugates
via amino groups and oxidized saccharide (Omelyanenko, Kopečková et al. 1996; Chytil,
Etrych et al. 2006).
2.3.1.5 Dendrimer Conjugates
Dendrimer, which was defined in the late 1970s and early 1980s, is a new class of
macromolecules with highly branched, three-dimensional features like the architecture of
a tree. A typical dendrimer (Scheme 2-12) consists of a multifunctional central core (C),
branched units (B) and surface groups (S). The branched units represent the repeating
31
monomer unit which is also called “generations” (Tomalia, Naylor et al. 1990).
Dendrimers are synthesized from monomers by a step-growth polymerization process,
including mainly two approaches: divergent (Tomalia 1996) and convergent (Hawker and
Frechet 1990). The gradual stepwise process of synthesis determines a well defined size
and structure. The surface characteristics facilitate conjugation of a broad range of
molecules with different functionality, no matter with drugs, imaging agents or targeting
moieties (Dufes, Uchegbu et al. 2005).
Scheme 2-11 Examples of synthesis of HPMA-drug antibody conjugates
The highly branched structure, the low polydispersity and modifiable end groups assure
that dendrimer can be used to develop novel drug conjugates. Polyamidoamine (PAMAM)
dendrimers were the first complete dendrimer family (G=0-7), which are prevalent
32
nanovehicles for delivery of bioactives. 5-fluorouracil (5FU)-PAMAM conjugates with
cyclic cores was synthesized using the “time-sequenced propagation” technique, which
exhibited slow release of the drug (Zhuo, Du et al. 1999). Conjugation of the famous
anticancer drug cisplatin with PAMAM dendrimers showed slower release, higher
accumulation in tumors and lower systemic toxicity than free cisplatin (Malik, Evagorou
et al. 1999). The use of dendrimers as vectors for tumor targeting was also investigated.
Fréchet et al. fabricated ester-terminated dendrimers containing folic acid, or methotrexate
(MTX) as drug delivery with tumor cell specificity (Kono, Liu et al. 1999). The conjugate
of folic acid to the dendrimer utilized coupling of γ-carboxyl group in folic acid to
hydrazide groups in dendrimers. Dendrimer-peptide conjugates were also reported for the
development of synthetic vaccines, which had multiple copies of the peptide so that small
concentrations could result in the required immune response (Tam 1988; McGeary,
Jablonkai et al. 2001).
Scheme 2-12 Typical architecture of a third generation dendrimer
33
2.3.2 PEGylated Drug Conjugation
PEG possesses an ideal array of properties such as very low toxicity, excellent solubility
in aqueous media, availability in various molecular weights, extremely immunogenicity
and antigenicity, ready excretion after administration although non-biodegradable,
favorable pharmacokinetics and biodistribution, which enable PEG conjugates to become
classical prodrugs with approval for human use (Goren, Horowitz et al. 2000). Most
commonly used PEGs for prodrug modification are monomethoxy PEG and di-hydroxyl
PEG (Scheme 2-13). PEG has limited conjugation capacity since only two terminal
functional groups can be conjugated to biocomponents. Recently, this limitation has been
overcome by coupling amino acid to double the number of active groups (Greenwald,
Choe et al. 2003). To date, a number of strategies have been developed in PEGylated
conjugates. Anticancer agents have particularly benefited from this technology as well as
applications to larger proteins.
Scheme 2-13 Chemical Structure of (a) monomethoxy-PEG and (b) di-hydroxyl PEG
2.3.2.1 PEGylation of Small Organic Molecules
34
Water-soluble PEG-drug conjugates are considered to be most promising in drug delivery
system, especially for delivering hydrophobic drugs with low molecular weight in cancer
therapy. The most straightforward strategy towards drug-PEG conjugates is relying on
involvement of the terminal primary hydroxyl groups of PEG to affect the conjugate
reaction. Direct conjugation of drugs to hydroxyl groups of PEG can be achieved by
forming ether, carbonate or urethane bonds. With the use of certain spacers like succinate,
PEG can be used in a larger range of drugs (Goren, Horowitz et al. 2000).
A prodrug strategy using PEG as solubilizing agent has been reported in the case of
paclitaxel (Greenwald, Gilbert et al. 1996), in which PEGs with different molecular
weight were employed and compared. Esters with PEG in the α-position were found quite
effective as linking groups in the design of the prodrug. In vivo test suggested that
molecular weight play a significant role in solubilizing effect. PEG polymers can be
modified by small amino acids or other aliphatic chains molecules. For example, The
antitumor agent 1-β-D arabinofuranosilcytosyne (Ara-C) was covalently attached to linear
or branched PEG through amino acid spacer for controlled release (Schiavon, Pasut et al.
2004). Furthermore, the hydroxyl group of PEG was functionalized with a bicarboxylic
amino acid to form a tetrafunctional derivative (Scheme 2-14), resulting in the conjugates
with four or eight Ara-C molecules in one PEG chain. Besides, PEG-doxorubicin
conjugate using amino acid as spacer was investigated (Veronese, Schiavon et al. 2005).
Although less cytotoxicity in vitro was observed, the prolonged plasma circulation time
and greater tumor targeting showed its promise compared to free drug.
35
Scheme 2-14 Synthetic schemes of (a) PEG-Ara-C4 and (b) PEG-Ara-C8 conjugates
2.3.2.2 PEGylation of Polypeptide (Peptides and Proteins)
The conjugation of PEG with the polypeptide (peptides and proteins) has been
accomplished mainly via reaction with available amino groups, while other reactive sites
such as histidine and cysteine can be utilized. So as to achieve successful coupling, PEG
36
must be activated first, which may be done using active ester, active carbonates,
maleimides or thiazolidine (Greenwald, Choe et al. 2003).
In the pioneering work, PEG-proteins were prepared by conjugation of bovine serum
albumin (BSA) with mPEG-dichlorotriazine, which exhibited significantly reduced
immunogenicity and antigenicity (Abuchowski, van Es et al. 1977). R-hirudin, an
effective antithrombotic peptide, can be modified with two strands of PEG through
urethane bonds to achieve a dramatically prolonged half-life time with enhanced
antithrombotic activity and no immunogenicity (Esslinger, Haas et al. 1997). PEGylation
of granulocyte-colony stimulating factor (G-CSF) contained a single strand of PEG-20000
covalently bound with the N-terminal amino group of the methionyl moiety of G-CSF
(Holmes, O'Shaughnessy et al. 2002). The resultant high molecular weight of the prodrug
significantly reduced renal clearance. Clinical investigation of several other PEGylated
protein drugs have also been reported, such as IL-2 (Yang, Topalian et al. 1995), growth
hormone antagonist (Trainer, Drake et al. 2000), and α-interferon (Wang, Youngster et al.
2000).
2.3.2.3 Targeting PEGylation
Targeting of the anticancer agents is crucial to the specific sites as it can maximize cancer
cell death effect and minimize normal cell survive effect. The prodrug that antibody-
targeted biodegradable PEG multi-block attached to N2,N5-diglutamyllysine tripeptide
with doxorubicin conjugated via acid-sensitive hydrazone bond has been designed and
synthesized (Ulbrich, Etrych et al. 2002). PEG was first activated with phosgene and N-
hydroxysuccinimide and then interacted with amine group of triethyl ester of tripeptide
37
N2,N5-diglutamyllysine to get a degradable multi-block polymer, which, later, was
converted to the polyhydrazide by hydrazinolysis of the ethyl ester with hydrazine hydrate.
A part of the hydrazide group in polymer was modified with succinimidyl 3-(2-
pyridyldisulfanyl) porpanoate to introduce a pyridyldisulfanyl group for conjugation with
an activated antibody. DOX was coupled to the remaining hydrazide groups by acid-labile
hydrazone bonds to the polymer (Scheme 2-15). This prodrug was found having very high
antitumor activity and significant decrease of non-specific accumulation in the heart, liver
and lungs. Several other targeting prodrugs, such as DU-257-PEG-KM231 mAb (Suzawa,
Nagamura et al. 2000) and luteinizing hormone-releasing-hormone-PEG-camptothecin
(Dharap, Qiu et al. 2003) conjugate, have also been investigated recently.
Scheme 2-15 Architecture of multi-block PEG-DOX with targeting antibody conjugated
2.4 Vitamin E TPGS
2.4.1 Chemistry of Vitamin E TPGS
D-α-tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS or TPGS) was
developed in the 1950s as a water-soluble derivative of lipid-soluble natural vitamin E,
which is prepared by esterification of d-α-tocopheryl succinate (TOS) with polyethylene
38
glycol 1000. TPGS appears a waxy solid at room temperature and stable in air. The
molecular weight is around 1542 Da. The thermal degradation and melt temperature are
199 °C and 38-41 °C, respectively (http://www.eastman.com). TPGS possesses a very low
critical micelle concentration (CMC) that is 0.02% and can form low viscosity solutions in
water until the concentration at 20 wt%, beyond which lipid crystalline phases may form
(Bogman, Zysset et al. 2005).
Scheme 2-16 Chemical structure of TPGS: red = hydrophobic vitamin E, green = hydrophilic PEG chain, black = succinate linker
TPGS is an amphiphile consisting of lipophilic alkyl tail (TOS) and hydrophilic polar
head portion (PEG) as shown in Scheme 2-16, which results in the bulky structure and
large surface area. The hydrophile-lipophile balance (HLB) of TPGS is about 13.2. The
unique chemical properties of TPGS have suggested its application as a solubilizer, an
emulsifier, an absorption enhancer, a plasticizer, a water-soluble source of vitamin E, and
a carrier of hydrophobic drugs. TPGS shows no human toxicity data to date. National
cancer institute reported that the safety oral dosage of TPGS can be up to 1 g/kg/day
39
(http://www.eastman.com). The acute oral LD50 of adults is larger than 7 g/kg for either
TPGS or its moieties, PEG 1000 and TOS (Krasavage and Terhaar 1977). The Japanese
government’s Ministry of Health, Labour and Welfare approved the application of TPGS
as a pharmaceutical excipient for oral drug formulations in 2005
(http://www.inpharmatechnologist.com).
2.4.2 Solubilizer for Water-insoluble Compounds
It is well known that quite a few drugs, especially anticancer drugs, are poorly water
soluble. There are many strategies improving solubility and TPGS is a powerful tool to
solubilize many hydrophobic agents. TPGS was used as solubilizing adjuvant to formulate
emulsion of an oily composition of several antitumor drugs (Kawata, Ohmura et al. 1986).
Another early step on the solubilizing effect of TPGS is the work by Ismailos et
al.(Ismailos, Reppas et al. 1994). The aqueous solubility of cyclosporine A (CyA) at three
different temperatures (5, 20 and 37 °C) showed 2- to 16-fold increase in the presence of
various concentrations of TPGS ranging from 0.01 to 0.05 mM. Later, Yu, L. et al (Li,
Price et al. 1999) reported that TPGS significantly improved solubility of the poorly water
soluble drug amprenavir, which is a potent HIV protease inhibitor. The solubility linearly
increased with increasing TPGS concentration through micelle solubilization since no
increase showed when the concentration of TPGS was below CMC. TPGS was also found
enhancing the solubility of another selective HIV-1 non-nucleoside reverse transcriptase
inhibitor, thiocarboxanilide UC-781 (Deferme, Van Gelder et al. 2002). Moreover, a
dramatic increase in estradiol solubility was found in the aqueous solvent as well as in
various concentration of ethanol with TPGS (Sheu, Chen et al. 2003). Recently, a new
40
successful story was documented involving anticancer drug paclitaxel (Varma and
Panchagnula 2005). Aqueous solubility of paclitaxel was significantly enhanced up to 38
times by TPGS, revealing TPGS is one of the best semi-solid oral dosage forms for
taxoids.
2.4.3 Absorption Enhancer
TPGS has caused much attention on its ability to enhance the absorption of several drugs
that, otherwise, have poor bioavailability. Sokol et al. (Sokol, Narkewicz et al. 1991),
Boudreaux et al. (Boudreaux, Hayes et al. 1993) and Chang et al. (Youk, Lee et al. 2005)
demonstrated that TPGS could enhance absorption of the lipophilic drug cyclosporine,
which is an immunosuppressive agent to prevent the rejection of transplanted organs. As a
consequence, the required daily dosage of cyclosporine was significantly reduced to
maintain therapeutic blood plasma concentration of the drug. TPGS also displayed well as
an absorption enhancer in amprenavir (Li, Price et al. 1999), which has been already used
in commercial amprenavir capsules, an agenerase that contains approximately 36 IU of
vitamin E in 50 mg capsule. Besides many examples of TPGS used for poorly water
soluble drugs, examples of TPGS used in poorly permeable drugs that are water soluble
were also reported. Vancomycin hydrochloride, a glycopeptide antibiotic, is water soluble
but poorly absorbed from the gastrointestinal tract. By introducing TPGS, the Cmax value
of vancomycin hydrochloride in plasma of rats increased from negligible to over 4.98
µg/mL (Prasad, Puthli et al. 2003).
2.4.4 Bioavailability Enhancer
41
As a water-soluble derivative of natural vitamin E, TPGS alone or TPGS/d-α-tocopheryl
acetate blend provides enhanced bioavailability in not only animals but also some
populations of human. Quite a few studies supported the use of TPGS to deliver vitamin E
in human exhibiting fat malabsorption (Traber, Kayden et al. 1986; Sokol, Heubi et al.
1987; Sokol, Butler-Simon et al. 1993). In the trial, Solol et al. dosed TPGS to 60 children
with chronic cholestasis, who were unresponsive even to 70-212 IU/kg/day of other oral
vitamin E forms. All children responded to TPGS at only 25 IU/kg/day oral dose of TPGS
and neurological function was improved or stabilized in most of patients. The vitamin E
trial was performed using an emulsified form of tocopheryl acetate, showing the
concentrations of tocopherols in plasma, erythrocytes and adipose tissues after
administration were high with TPGS. Thus TPGS provided better bioavailability than the
other forms. The proposed mechanism explained that the formed TPGS micelles could
cross from the intestinal lumen to enterocytes. TPGS may be hydrolyzed to release the
tocopherol which may be transferred into the enterocyte or the whole TPGS micelle could
be taken up by the enterocyte. Besides, TPGS can also improve the bioavailability of other
fat soluble vitamins, such as vitamin D (Argao, Heubi et al. 1992).
Not only can TPGS improve the bioavailability of vitamins, it also can enhance the
bioavailability of pharmacological compounds. Khoo found that by employing TPGS the
solid dispersion of halofantrine, an important anitmalarial agent, could result in 5-fold
improvement in absolute oral bioavailability (Khoo, Porter et al. 2000). TPGS enhanced
bioavailability of cyclosporine A in liver transplant patients and reduced daily dosage to
40-70% (Sokol, Butler-Simon et al. 1993). Paclitaxel, an antimicrotubule anticancer drug,
exhibits very poor absorption because of both poor solubility and permeability and thus it
42
is usually administered via intravenous injection. TPGS enhanced the bioavailability of
paclitaxel in the in vivo studies due to the increased solubility and permeability. It was
able to improve not only the bioavailability of oral paclitaxel but also that of other
biopharmaceutical classification system class II-IV drugs (Varma and Panchagnula 2005).
Regarding the mechanism of TPGS enhancing bioavailability, while Sokol originally
attributed the reason to micelles formation enhancing solubility, others later provided
evidence suggesting it could be due to P-glycoprotein (P-gp) inhibition resulting in
enhanced permeability (Dintaman and Silverman 1999; Wacher, Wong et al. 2002). P-gp
is primarily expressed on the luminal surface of epithelial cells from certain tissues such
as intestine, liver, kidney (Thiebaut, Tsuruo et al. 1987). TPGS can play a role as reversal
agent of P-gp mediated multidrug resistance (MDR) and inhibit P-gp substrate drugs
transporting. For example, TPGS significantly increased the bioavailability of P-gp
substrate talinolol in coadministration with talinolol form in vitro cell permeability on
Caco-2 cells and in vivo intraduodenal perfusion study (Bogman, Zysset et al. 2005). It
has also been reported that vitamin E was able to protect the human hepatocellular
carcinoma cells from free-radical induced lipid peroxidation and oxidative DNA damage
so as to inhibit the MDR phenotype (Fantappiè, Lodovici et al. 2004). Moreover, TPGS
was found to enhance the cytotoxicity of doxorubicin, vinblastine, paclitaxel and colchine
that are P-gp substrate agents in G185 cells but not for that of 5-FU which is not P-gp
substrate (Dintaman and Silverman 1999). TPGS can improve the permeability of a drug
across cell membranes by inhibiting P-gp, and thus enhance the bioavailability and
absorption of a drug.
43
2.4.5 Anticancer Enhancer
TPGS, a PEG-conjugated derivative of TOS, has been found possessing potent anticancer
activity since TOS has anticancer properties against wide spectra of cancers such as colon,
melanomas, breast, brain, lung and prostate cancers (Malafa and Neitzel 2000; Neuzil,
Weber et al. 2001; Barnett, Fokum et al. 2002; Zhang, Ni et al. 2002; Swettenham,
Witting et al. 2005). As a comprising moiety, α-tocopherol had the multidrug resistance
(MDR)-neutralizing activity of the chemosensitizers cyclosporine A, verapamil GF120918,
clofazimine and a derivative of clofazimine, B669 (Van Rensburg, Jooné et al. 1998). The
anticancer activity of TOS is related to its apoptosis-inducing characteristics, which
seemed to be mediated involving the generation of reactive oxygen species (ROS) which
can damage DNA, proteins and fatty acids in cells leading to apoptotic cell death (Ottino
and Duncan 1997; Wang, Witting et al. 2005). In xenograft studies, TOS was able to
suppress tumor growth either alone or in combination with other anticancer agents but
showed less antiproliferative potent in normal cell types including intestinal epithelial
cells, prostate cells and hepatocytes than that in tumor cells (Neuzil, Weber et al. 2001;
Barnett, Fokum et al. 2002; Weber, Lu et al. 2002; Zhang, Ni et al. 2002). However, the
poor water solubility of TOS limits its therapeutic efficacy. Through the conjugation to
PEG, the solubility was significantly increased, which was expected to show better
efficacy. Youk et al. have confirmed that TPGS possessed more effective inhibition on the
growth of human lung carcinoma cells in in vitro cells and in nude mice than that of TOS
(Youk, Lee et al. 2005). The superiority of TPGS over TOS in anticancer efficiency was
attributed to the increased apoptosis rather than increased cell uptake.
44
2.4.6 Drug Delivery Applications
Polymeric drug carriers, by which the drug can be entrapped, encapsulated, adsorbed,
attached or coupled, open a new era of drug delivery to provide sustained, controlled and
targeted effects. In drug delivery, TPGS can be used to improve the dissolution rate and
bioavailability by forming a solid dispersion or a solid solution, controlling or modifying
the release of the drug, and masking the bitter taste of a drug. Due to the bulky
amphiphilic structure and large surface area, TPGS is expected to act as a good emulsifier
or surfactant in drug delivery system, especially in fabricating micro/nanoparticles. As Ke
et al (Bogman, Zysset et al. 2005) indicated, TPGS can be a good surfactant in oral
delivery of protein drugs and in synthesis of biodegradable magnetic microspheres. TPGS
can also be surfactant coating material in fabrication of PLGA nanoparticles by modified
solvent extraction/evaporation method (Mu, Seow et al. 2004). Mu and Feng demonstrated
that TPGS acted as a more effective and safer emulsifier for fabrication and
characterization of polymeric nanoparticles in comparison with traditional PVA
(Riebeseel, Biedermann et al. 2002). TPGS can achieve far higher emulsification
efficiency with the amount of 0.015% (w/w) in fabrication whereas 1% PVA is required in
similar process. Co-polymerizing TPGS with a polymer such as PLA, PLGA or PCL can
enhance the emulsification efficiency and drug loading capability as demonstrated in the
studies on microencapsulated paclitaxel (Mu and Feng 2003; Zhang and Feng 2006). The
in vitro drug release was increased by 2-fold after one day and 3-fold after 31 days for the
PLGA and PLA/TPGS nanoparticles, respectively. It also showed to tailor the release rate
via increasing the entrapped drug release (Xie and Wang 2005). Besides, TPGS can be
used as a good additive in fabricating PLGA nanoparticles and discs for paclitaxel
45
formulation with up to 80% drug loading efficiency (Wang, Ng et al. 2003; Mu, Teo et al.
2005).
The further in vitro and in vivo investigation confirmed the great potential of TPGS in
drug delivery. Feng et al (Feng 2004) demonstrated that TPGS emulsified nanoparticles
could increase cancer cell mortality and enhance the cellular uptake of Caco-2 cells that
used as a model of the GI barrier. Nanoparticles coated by TPGS eliminated the acute side
effects caused by Cremophor EL which is an adjuvant in commercial Taxol. In in vivo PK
investigation of PLA-TPGS nanoparticles prepared by the dialysis method, 1.8-times
larger AUC and 11.6-times longer half-life were observed than that of Taxol after i.v.
administration (Feng 2006). As it is for TPGS emulsified PLGA nanoparticles, 4.9-times
increased AUC and 2.4-times prolonged therapeutic time in comparison of Taxol after i. v.
injection were reported (Win and Feng 2006).
Moreover, TPGS displayed promise as a matrix material in nasal delivery. Prepared by the
combination of double emulsion method and spray drying method, diphtheria taxoid
loaded PCL/TPGS microspheres exhibited high immune response than that achieved using
PCL microspheres alone, which showed favorable potential of TPGS as a novel adjuvant
either alone or with other appropriate delivery materials (Somavarapu, Pandit et al. 2005).
2.5 Doxorubicin
2.5.1 History
Doxorubicin, (8s, 10s)-10-(40amino-5hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,
8, 11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7, 8, 9, 10-tetrahydrotetracene-5, 12-
46
dione, is an anthracycline antibiotic. The history of its discovery can trace back to the
1950s, when a bright red pigment, later named daunorubicin, was isolated from
Streptomyces peucetius. The clinical use began in 1960s for treating acute leukemia and
lymphoma, although fatal cardiac toxicity was recognized in 1967 (Tan, Tasaka et al.
1967). In 1969, a strain of Streptomyces mutated by N-nitroso-N-methyl urethane
produced a different, red-colored antibiotic, doxorubicin (Scheme 2-17), which exhibited
better anticancer activity than daunorubicin but the cardiotoxicity remained (Arcamone,
Cassinelli et al. 1969). Doxorubicin is one of the most powerful chemotherapeutic drugs
for the treatment of some leukemias, hodgkin’s lymphoma, as well as cancers of the
bladder, breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiple myeloma,
etc.
Scheme 2-17 Chemical structure of doxorubicin
2.5.2 Mechanism of Action
47
Despite doxorubicin has been frequently used in the treatment of numerous human
malignancies, the exact mechanism of the action is still somewhat unclear. Several
mechanisms have been proposed and often subject to controversy.
One of the most popular explanations is the capability of doxorubicin to inhibit DNA
synthesis which may be due to (a) DNA intercalation or inhibition of DNA polymerase
activity such as Topoisomerase II (Tanaka and Yoshida 1980; Gewirtz 1999), (b) effects
on signaling issues of growth arrest and p53 function (Kastan, Onyekwere et al. 1991), (c)
induction of enzymatic or chemically activated DNA adducts (Cullinane, Cutts et al. 1994)
and DNA cross-linking (Skladanowski and Konopa 1994), (d) interference with DNA
strand separation and helicase activity (Fornari, Randolph et al. 1994).
Another widely accepted theory is that oxidative stress and the resultant free radicals that
can lead to direct oxidative injure to DNA are related to doxorubicin action (Gewirtz 1999)
through two different ways. As for the first way, a doxorubicin semiquinone free radical
were formed involving some NADPH-dependent reductases and the oxygen, redox
cycling of adriamycin-derived quinine-semiquinone produced superoxide radicals (Olson
and Mushlin 1990; Singal, Li et al. 2000). In the other way, doxorubicin free radicals may
be from the reaction with iron which is non-enzymatic. A Fe2+ doxorubicin free radical
complex could be produced after a redox reaction of doxorubicin and Fe3+ (De Beer,
Bottone et al. 2001). Besides, several other mechanisms have also been suggested such as
alterations in calcium metabolism (Singal and Panagia 1984), and non-physiological
mechanisms (Gewirtz 1999).
48
2.5.3 Side Effects and Limitations
Although doxorubicin is one of most effective chemotherapeutic agents with a wide
anticancer spectrum, its clinical use is still limited by the serious side effects including
nausea, vomiting, mild alopecia, palmarplantar erythrodysesthesia (Hand-Foot Syndrome),
neutropenia, and the most serious one, cardiotoxicity. Doxorubicin can not only cause
acute cardiovascular chances including hypotension, tachycardia and various arrhythmias
after minutes intravenous administration but also lead to life-threatening chronic effects
such as hypotension, tachycardia, cardiac dilation and ventricular failure after several
weeks or months and even years (Edward, Lefrak et al. 1973; Edward A. Lefrak, tcaron et
al. 1973; Singal, Deally et al. 1987). The cardiotoxicity confines the cumulative dose of
DOX to 500-600 mg/m2, which still can be increased for tumor but not for heart (Petit
2004). On the basis of mechanisms of the action, the cardiotoxicity mechanisms of
doxorubicin can be attributed to free radical induction, Ca2+ overload accumulation, toxic
doxorubicin metabolites, production of prostaglandins and platelet activating factor,
simulation of histamine release, and direct interaction with the actin-myosin contractile
system (De Beer, Bottone et al. 2001).
Besides these serious irreversible side effects, other drawbacks also limit the clinical use
of doxorubicin. The multi-drug resistance (MDR) is one of the serious limitations in the
treatment of cancers through overexpression of MDR transporter proteins such as P-
glycoproteins (P-gp) and multidrug resistance associated protein (MRP). P-gp and MRP
can be overexpressed in malignant cells, as well as liver, kidney, and colon cells, to pump
anticancer drugs out of the cancer cells, significantly restricting the intracellular level of
the drug for effective therapy. Doxorubicin is the very substrate of P-gp and MRP, which
49
results in its shot half-life in circulation and low therapeutic efficacy (Krishna and Mayer
2000).
2.5.4 Formulations
In order to impair the side effects and other drawbacks of doxorubicin in chemotherapy, a
number of studies on the drug formulations have been undertaken. Liposome entrapped
doxorubicin achieved nigericin-induced “remote release” which resulted in a subsequent 8
times more effective cytotoxicity than the free drug (Goren, Horowitz et al. 2000). Recent
report showed that doxorubicin-loaded heparin-liposomes possessed high drug levels for
up to 64 h after i.v. administration and the half-life was approximately 8.4-fold increased
than the control liposomes (Han, Lee et al. 2006). Jane et al. (Janes, Fresneau et al. 2001)
fabricated chitosan nanoparticles as colloidal carriers for doxorubicin, achieving a
minimal burst release and retained cytotoxicity but induced anti-proliferative activity.
Later, dextran-doxorubicin conjugate loaded chitosan nanoparticles exhibited enhanced
therapeutic effect and survival rate in vivo (Mitra, Gaur et al. 2001). With in vitro and in
vivo work, doxorubicin-loaded polyisohexylcyanoacrylate nanoparticles exhibited
superiority to overcome the MDR phenotype and lead to a higher anti-tumor efficacy on
hepatocellular carcinoma that is well known as chemoresistant (Barraud, Merle et al.
2005).
Numerous investigations involving polymeric prodrug strategy in delivery of doxorubicin
were also demonstrated and showed favorable results. In early 1995,
monomethoxypoly(ethylene glycol)-propionic acid N-hydroxysuccinimide ester
conjugated doxorubicin was designed and studied, which had controlled release via
50
enzyme dependent hydrolysis, less cytotoxicity in vitro, longer circulation time, greater
extent in tumor xenografts (Senter, Svensson et al. 1995). Influence of polymer structure
on drug release, in vitro cytotoxicity, biodistribution and antitumor activity in PEG-
doxorubicin conjugate were extensively investigated by Veronese et al. (Veronese,
Schiavon et al. 2005). The highest MW PEG showed the longest plasma residence time
and the greatest targeting but all conjugates were 10-fold less toxic. The doxorubicin
prodrug N-[4doxorubicin-N-carbonyl (oxymethyl) phenyl] O-β-glucuronyl carbonate
could achieve completely parent drug activation, 5-fold decreased peek concentration of
doxorubicin in heart and 10-fold increased AUC in normal tissues than those in tumor
tissue (Houba, Boven et al. 2001). In the HPMA-based prodrug with doxorubicin attached
to the carrier via oligopeptide spacer or amide bonds, some of “classic” prodrugs have
been developed to clinical test (Kopeček, Kopečková et al. 2001; Chytil, Etrych et al.
2006). Water soluble HPMA copolymer-doxorubicin prodrug with pH-controlled
activation promisingly displayed high cytostatic activity in vitro and superior anti-tumor
activity in vivo in comparison of free drug (Chytil, Etrych et al. 2006).
Although hundreds of studies have been reported in formulations of doxorubicin, the
problems in stability of liposomes, incomplete release of nanoparticles and reduced
cytotoxicity in vitro of most prodrugs require new better drug delivery systems emerge.
51
3 SYNTHESIS AND CHARACTERIZATION OF THE TPGS-DOX
CONJUGATE
3.1 Introduction
TPGS is the derivative of PEG 1000 by conjugation to α-tocopheryl succinate and thus the
strategy of TPGS-DOX conjugate was similar as PEGylation (Hazra, Golenser et al. 2002).
As to get the TPGS-DOX conjugate, the terminal hydroxyl group in TPGS was first
reacted with succinic anhydride through ring-opening polymerization in the presence of
DMAP as base. Then the carboxyl group needed to be activated by NHS using DCC as
catalyst. At last, the amine group in DOX interacted with the activated carboxyl group in
TPGS-SA to get the conjugate, TPGS-DOX. The mechanism for the reaction was shown
in scheme 3-1 and scheme 3-2. The synthesized conjugate was characterized by fourier
transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) for the
molecular structure, gel permeation chromatography (GPC) for the weight-averaged
molecular weight and polydispersity, microplate reader for drug loading efficiency. The
stability of the conjugate was studied in PBS at 4 °C.
3.2 Experiment Section
3.2.1 Materials
Vitamin E TPGS was obtained from Eastman Chemical Company (TN, USA).
Doxorubicin hydrochloride, N,N’-Dicyclohexylcarbodiimide (DCC),
dimethylaminopyridine (DMAP), N-hydroxysuccinimide (NHS), succinic anhydride (SA),
triethylanmine (TEA), diethyl ether and tetrahydrofuran (THF) were received from Sigma-
Aldrich (St. Louris, MO, USA). All solvents used are HPLC grade, which include
52
dichloromethane (DCM), dimethyl sulfoxide (DMSO, anhydrous) from Aldrich, and ethyl
acetate from Merck. All reagent water used in the laboratory was pretreated with Milli-Q
Plus System (Millipore Corporation, Bredford, USA).
3.2.2 Synthesis of TPGS-DOX
3.2.2.1 TPGS Succinoylation
TPGS was activated by succinic anhydride through ring-opening reaction mechanism in
the presence of DMAP (Sheme 3-1). In brief, 0.77 g (0.5 mmol) TPGS, 0.10 g (1mmol)
succinic anhydride and 0.12 g (1mmol) DMAP were mixed and heated at 100 °C under
nitrogen atmosphere for 24 h (Hazra, Golenser et al. 2002). The mixture was cooled to
room temperature, taken up in 5.0 mL cold DCM, filtered to remove excessive succinic
anhydride and then precipitated in 100 mL diethyl ether at -10 °C overnight. The white
precipitate was filtered and dried in vacuum to obtain TPGS succinate reagent with a yield
of 90%. To synthesize TPGS-SA as a precursor polymer for DOX conjugation, TPGS was
succinoylated with an excess of succinic anhydride because complete succinoylation is
essential to avoid polymer cross-linking during DOX conjugation (Tomlinson, Heller et al.
2003).
53
O
O
O n
COC
OO
O
O
+
Succinic AnhydrideVintamin E TPGS
CH2CH2O H
O
O
O n
COC
O
OHO
OCH2CH2O
TPGS-SA
100 OC
DCC, DMAP
Scheme 3-1 Scheme of TPGS succinoylation
3.2.2.2 TPGS-DOX Conjugation
The succinoylated TPGS (191.3 mg, 0.12 mmol) was reacted with DOX·HCl (102.5 mg,
0.18 mmol) with the presence of DCC (74.2 mg, 0.36 mmol), NHS (41.4 mg, 0.36 mmol)
and TEA (50 µL, 0.36 mmol ) in DMSO at room temperature under nitrogen atmosphere
for 24 h. In this process, the reaction can be separated into two steps. First, TPGS-SA was
esterized with NHS using DCC as coupling agent. Then the TPGS-NHS was interacted
with DOX to get the conjugate. The product was filtered to remove N,N-dicyclohexylurea
(DCU) and then dialyzed in DMSO for 24 h to remove excess reagents and unconjugated
DOX, and further dialyzed against Millipore water for 24 h to remove DMSO. The
resultant solution was freeze-dried to get the red powder with a yield of 83% and a purity
of 98%. The conjugate scheme is shown in Scheme 3-2.
54
O
O
O n
COC
O
OHO
OCH2CH2O
TPGS-SA
+ NOH
O
O
DCCO
O
O n
COC
O
OO
OCH2CH2O N
O
O
TPGS-NHS
NHS
O
O
OH
OH
O OH
OH
OO
O
NH2OH
Doxorubicin
O
O
O n
COC
O
O
O
O
O
OH
OH
O OH
OH
OO
O
HO NHCH2CH2O
TPGS-DOX
DMSO TEA DMSO
Scheme 3-2 Scheme of TPGS-DOX conjugation
3.2.3 Characterization of TPGS-DOX Conjugate
3.2.3.1 FT-IR
55
The chemical structure of TPGS-DOX was investigated by Fourier Transform Infrared
Spectroscopy (FT-IR) (Shimadzu, Japan). The FT-IR samples were prepared by mixing
99% KBr with 1% conjugate and then pressing to be a transparent tablet.
3.2.3.2 1H NMR
The molecular structure of TPGS-SA and TPGS-DOX were further confirmed by 1H
Nuclear Magnetic Resonance Spectroscopy (NMR) in DMSO-d6 at 300 MHz (Bruker
ACF300).
3.2.3.3 GPC
The molecular weight and the polydispersity of the conjugate were determined using gel
permeation chromatography (GPC, Aglient 1000 series GPC analysis system with RI-
G1362A refractive index detector). Agilent PL gel 5 µm mixed-C 300×7.5 mm column
was employed, which was eluted by THF as a mobile phase at a flow rate of 1mL/min.
The injection volume was 100 µL of sample solution with a concentration at 1 mg/mL in
mobile phase. Molecular weight of the conjugate was calculated using a series of
Polystyrene standards (Mw: 640, 7150, 111400, 1997000).
3.2.3.4 Drug Conjugate Efficiency
The DOX content conjugated to TPGS was measured using microplate reader (GENios,
Tean, Switzerland) in DMSO with fluorescence detection at λex=480 nm and λem=560nm.
A standard curve was generated by using free DOX at concentration range of 100 ng/mL -
500 µg/mL in DMSO.
56
3.2.3.5 Stability of the Conjugate
The stability of the conjugate was determined as follows. Five mg prodrug was suspended
in 5 mL PBS buffer (10 mM, pH 7.8) and sealed in a dialysis bag (M.W. cutoff: 1,000).
The dialysis bag was incubated in 20 mL PBS buffer at 4oC. The released DOX in the
incubation medium was collected at the designated time intervals and analyzed at 480 nm
fluorescence.
3.3 Results and Discussion
3.3.1 FT-IR Spectra
The spectra of DOX, TPGS and TPGS-DOX are shown in Fig 3-1. The FT-IR spectrum of
doxorubicin in Fig 3-1 exhibits multiple peaks at 3346, 2940, 2354, 1617, 1416 and 1072
cm-1, which corresponded to the different quinine and ketone carbonyls in DOX. However,
it cannot be delineated the specific bonds between specific quinine and ketone because
they both have carbonyl groups. The peaks at 872 and 806 cm-1 corresponded to the wag
of primary amine NH2 and deformation of N-H bonds, respectively. As for the spectrum
of TPGS, the peak at 1740 cm-1 was due to the carbonyl group. The peak region of 2870-
3000 cm-1 was generated by the aliphatic (C-H) functional group. From the spectrum of
TPGS-DOX conjugate, we can find that the peak at 872 and 806 cm-1 had disappeared and
instead, a peak at 1430 cm-1 turned up standing for N-H deformation in secondary amine
structure, which confirmed the secondary amine linkage between –COOH group in TPGS
and –NH2 group in doxorubicin. The conjugate showed significant signal from TPGS but
relative weak one of DOX, which was due to the low drug content in the conjugate.
57
Fig 3-1 FT-IR spectra of DOX (red line), TPGS (purple line) and TPGS-DOX (blue line)
3.3.2 1H NMR Spectra
1H NMR employing DMSO-d6 as solvent was used to confirm the product identity. The
spectrum of TPGS-SA demonstrated that the succinoyl methylene (CH2) gave signals at
2.3-2.4 ppm in comparison with that of TPGS as shown in Fig 3-2a, b. The typical 1H
NMR spectra of DOX and TPGS-DOX conjugate are shown in Fig 3-2c, d. The spectrum
of DOX exhibited typical peaks at 3.99 ppm (H3C-O-C-1), 5.45 ppm (HC-1’), 7.69 ppm
(HC-3) and 7.91 ppm (HC-2 and HC-4). The spectrum of TPGS showed an intense peak
at 3.51 ppm, which was the characteristic of methylene protons of the PEG part of TPGS.
The spectrum of TPGS-DOX contained both signals originating from DOX and TPGS,
58
but with much weaker intensity than the signals of DOX. This may be due to the small
molecular weight of DOX compared with that of TPGS.
3.3.3 GPC Results
The TPGS-DOX conjugate was analyzed by gel permeation chromatography (GPC) as
shown in Fig 3-3. The prodrug was eluted earlier than the unconjugated TPGS, which
demonstrated that DOX was conjugated to TPGS. The molecular weight of the conjugate
and the TPGS was 2,655 and 2,221, respectively. The ratio of the weight-averaged over
the-number molecular weight of the conjugate (Mw/Mn) was 1.16. The slight increase was
due to the DOX conjugation and the loss of the low molecular weight TPGS fraction in
the purification process. By GPC analysis, it can be confirmed that the TPGS-DOX
conjugate was synthesized successfully and the product was not a physical mixture of
TPGS and DOX.
59
60
Fig 3-2 1H NMR spectra of (a) TPGS, (b) TPGS-SA, (c) DOX, (d) TPGS-DOX with the insert for a magnification of the region between 6 and 10 ppm
-350
-300
-250
-200
-150
-100
-50
0
50
100
150
200
15.5 16.5 17.5 18.5 19.5 20.5 21.5 22.5
Retention time (min)
MV
TPGS
TPGS-DOX
Fig 3-3 Gel permeation chromatogram (GPC) of the TPGS-DOX conjugate and the unconjugated TPGS
3.3.4 Drug Loading Capacity
The DOX content in the TPGS-DOX conjugate was determined to be 8.0% by the
microplate reader using fluorescence at 480 nm. It is important to note that the DOX
loading capacity (8.0 wt%) of our TPGS-DOX conjugate is as favorable as that for other
polymer–DOX conjugates being tested clinically, which is, for example, 8.5 wt% for
HPMA copolymer-DOX conjugate (Vasey, Kaye et al. 1999), 2.5-5 wt% for PEG-DOX
conjugate (Rodrigues, Beyer et al. 1999), and 5-16 wt% for PGA-DOX conjugate (Hoes,
Grootoonk et al. 1993). The satisfactory drug loading capacity of TPGS-DOX conjugate
facilitates its clinical test in future.
61
3.3.5 In Vitro Stability
As the stability regards, the result is also satisfactory. It was found that after 24 h in PBS
buffer (pH 7.8) at 4 oC, the conjugate underwent only a small amount of hydrolysis
resulting in the release of approximately 2% of the bound DOX. Even after 5 months, only
12% of the conjugated drug was released from the conjugate. Therefore, most of the DOX
can be stably attached to the polymer under the test conditions, which enables its
relatively long time storage.
3.4 Conclusions
TPGS-DOX conjugate were synthesized via the interaction between the carboxyl group in
TPGS-SA and the primary amine group in DOX in the presence of the coupling agent.
The conjugate was characterized by FT-IR, 1H NMR and GPC to detect the molecular
structure, molecular weight and polydispersity, in which results confirmed the successful
synthesis of the conjugate. The TPGS-DOX also exhibited favorable drug loading
capacity in comparison with the clinical prodrug candidate. Besides, the in vitro stability
result showed the stable attachment between TPGS and DOX, which made the regular
storage feasible.
62
4 IN VITRO STUDIES ON DRUG RELEASE KINETICS,
CELLULAR UPTAKE AND CELL CYTOTOXICITY OF THE
TPGS-DOX CONJUGATE
4.1 Introduction
The release of the drug from the polymeric carrier is important for its further therapeutic
function. Usually, the release of drug can be mediated by simple hydrolysis. In this study,
the drug release was studied in PBS at 37 oC with different pH values. It is demonstrated
that TPGS can enhance the intracellular cell uptake of human colon carcinoma cells (Win
and Feng 2006). As for the in vitro study, MCF-7 breast adenocarcinoma cells and C6
glioma cells were utilized as in vitro model. The TPGS-DOX may also enhance the
cellular uptake of the doxorubicin through escaping from P-gp drug efflux pump, which
was investigated in this chapter. Cellular uptake of the conjugate was further visualized by
confocal laser scanning microscopy (CLSM) to indicate the intracellular distribution.
Cancer cell viability of TPGS-DOX conjugate was quantitatively determined by MTT
method. All the experiments were conducted in close comparison with the pristine DOX.
4.2 Materials and Methods
4.2.1 Materials
3-(4,5-cimethylthiazol-z-yl)-2,5-diphenyl chloroformate (MTT), phosphate buffered
saline (PBS), Dulbecco’s Modified Eagel Medium (DMEM), penicillin-streptomycin
solution, Trypsin-EDTA and Triton® X-100 were obtained from Sigma-Aldrich (St.
63
Louris, MO, USA). Fetal bovine serum (FBS) was received from Gibco (Life
Technologies, AG, Switzerland).
4.2.2 In Vitro Drug Release
The rate of DOX release from the conjugate was investigated in 10 mM PBS at pH 3.0,
5.0 and 7.4 at 37 oC, respectively. The conjugate solution of 200 µg/mL equivalent DOX
concentration was placed in a dialysis bag (MW cutoff: 1,000) and incubated in 20 mL of
the PBS solution with gentle shaking. The incubated solution was collected at designated
time points and equal volume of fresh medium was compensated. The released DOX
amount was determined by fluorescence detection at 480 nm.
4.2.3 Cell Culture
MCF-7 breast adenocarcinoma cells and C6 glioma cells (American Type Culture
Collection, VA) were employed as in vitro models. The cells were cultured in the DMEM
medium supplemented with 10% FBS, 1% penicillin-streptomycin solution, and incubated
in SANYO CO2 incubator at 37oC in humidified environment of 5.0% CO2. The medium
was replenished every other day until confluence was achieved. The cells were then
washed with PBS and harvested with 0.125% Trypsin-EDTA solution.
4.2.4 In Vitro Cell Uptake Efficiency
In the quantitative investigation, MCF-7 and C6 cells were seeded in 96-well black plates
(Costar, IL, USA) at a density of 3×104 cells/well. When the cells reached about 80%
confluence, as for the investigation of comparison of the prodrug and the free drug, the
medium was replaced by 100 µL TPGS-DOX or free DOX solution in medium at 1
64
µg/mL DOX concentration for 0.5, 1.5, 4, 6 h, respectively. Regarding the concentration
related effects in cell uptake, the medium was replaced by 100 µL TPGS-DOX or free
DOX at 1, 5, 25 µg/mL DOX for 4 h. For each sample, six wells were seeded for positive
control and six wells for sample wells. At the designated time interval, the sample wells
were washed three times with 50 µL PBS and then added 100 µL culture medium. All the
wells were then lysed by 50 µL 0.5% Triton 100 in 0.2 M NaOH. The fluorescence
intensity of each sample was detected by the microplate reader (Tecan, Männedorf,
Switzerland, λex = 480 nm, λem = 560 nm). Cellular uptake efficiency was expressed as the
percentage of the fluorescence associated with the cell vs that present in the positive
control (Zhang, Lee et al. 2007).
4.2.5 Confocal Laser Scanning Microscopy
As for the qualitative investigation, MCF-7 and C6 cells were incubated with TPGS-DOX
conjugate or free DOX medium solution at 1 µg/mL DOX concentration under 37oC for 4
h. Then the cells were rinsed with cold PBS for three times, fixed by 75% ethanol for 20
min, and then washed twice by PBS. Finally, the cells were mounted by the mounting
medium (DAKO® Fluorescent Mounting Medium) and observed under confocal laser
scanning microscopy (Zeiss LSM 510, Germany).
4.2.6 In Vitro Cytotoxicity
MCF-7 and C6 cells were seeded at a density of 5×103 cells/well in 96-well plates (Costar,
IL, USA) and incubated for 24 h. The medium was then replaced by the free DOX or
TPGS-DOX conjugate at various drug concentrations from 0.002 to 100 µM in the
medium. The cell viability was determined by the MTT assay. At the designated time
65
intervals 24, 48, 72 h, the medium was removed and the wells were washed twice with
PBS. Ten percent MTT (5 mg/mL in PBS) in medium was added and the cells were
incubated for 3-4 h. After that, the precipitant was dissolved in 100 µL isopropanol and
each well was finally analyzed by the microplate reader with absorbance detection at 570
nm. The cell viability was figured out using the following formula,
Cell viability (%) = (Abss / Absc) × 100
where Abss stands for the fluorescent absorbance of the wells of the drug samples and
Absc is that of the wells of the culture medium used as positive control.
The MCF-7 and C6 cell viability of TPGS was also investigated at various concentrations
equivalent to those from 0.008 to 400 µM for the conjugate experiment using the same
method.
4.2.7 Statistics
Statistical analysis was conducted by using the student’s t-test with p<0.05 as significant
difference.
4.3 Results and Discussion
4.3.1 In Vitro Drug Release
In vitro release of the drug from TPGS-DOX conjugate was measured at various pH
conditions. As shown in Fig 4-1, the DOX release showed no dramatic initial burst. It was,
however, significantly pH-dependent. The lower the pH value was, the faster the drug
released. Specifically, the drug released rapidly from the conjugate at pH 3.0, reaching
66
52.3±2.4% after 48 h and almost 100% within 10 days, whereas the DOX release at pH
5.0 was much slower, implying 27.1±1.4% and 43.6±2.8% in the same period,
respectively (p<0.01). As desired, only a small amount of drug (12.6±0.2%) was released
at pH 7.4 over the observed period.
The release of the free active drug from the carrier may play an important role, which is
considered as a prerequisite for the conjugate bearing drugs bound to the polymer via
degradable bonds. In most cases, release of anticancer drugs from the polymer carrier is
mediated by simple hydrolysis (Maeda, Seymore et al. 1992; Takakura and Hashida 1995).
The free DOX is known to be stable in the pH range 3.0-6.5 (Vigevani and Williamson
1980). In the TPGS-DOX conjugate, DOX might be released from the conjugate by
hydrolysis, which was sensitive to pH value. This effect may be involved to speed up the
drug release inside cancer cells as the proton concentration in lysosomes is usually at least
100-fold higher (<pH 5.0) than the outside of the cells (pH 7.4). The release profile
confirmed that the linkage was labile under mild acidic conditions anticipating endosomal
environment and sufficiently stable in blood circulation (pH 7.4) to allow the transport of
the prodrug to tumor cells.
67
0
20
40
60
80
100
0 50 100 150 200 250
Time (h)
Cum
ulat
ive
rele
ased
DO
X (%
)
pH 3.0
pH 5.0
pH 7.4
Fig 4-1 Release of DOX from TPGS-DOX conjugate incubated in phosphate buffer at 37 oC (mean ± SD and n=3)
4.3.2 In Vitro Cellular Uptake
It is known that the cellular internalization and sustained retention of the drug play
significant role in therapeutic effects. The in vitro investigation can give preliminary
information of the priority of the prodrug over the parent drug despite it cannot make sure
the definitely same results in vivo.
Fig 4-2 shows (a) MCF-7 and (b) C6 cellular uptake efficiency of the TPGS-DOX
conjugate compared to the pristine DOX after 0.5, 1.5, 4, 6 h cell culture. The same 1
µg/mL DOX concentration in the culture medium was applied in the experiment. It can be
seen from Fig 4-2 that the TPGS-DOX conjugate exhibited enhanced cellular uptake of
1.7- (69.8±8.8% for TPGS-DOX vs 39.9±2.4% for DOX), 1.3- (75.5±6.0% for TPGS-
68
DOX vs 56.7±3.4% for DOX), 1.2- (78.6±6.8% for TPGS-DOX vs 64.2±6.4% for DOX),
1.2-fold (86.0±5.1% for TPGS-DOX vs 72.5±2.0% for DOX) (p<0.05) for the MCF-7
cells and that of 5.4- (27.0±1.7% for TPGS-DOX vs 4.93±0.1% for DOX) , 5.9-
(34.0±2.7% for TPGS-DOX vs 5.70±0.2% for DOX), 1.3- (48.7±7.8% for TPGS-DOX vs
37.4±5.4% for DOX), 1.1-fold (73.6±1.8% for TPGS-DOX vs 69.7±3.5% for DOX)
(p<0.05) for the C6 cells after 0.5, 1.5, 4, 6 h cell culture, respectively, in comparison with
pristine DOX. Besides, the absolute cellular uptake efficiency for the MCF-7 cells is
generally higher than that for the C6 cells. Almost all of the TPGS-DOX samples in MCF-
7 cells displayed the cell uptake efficiency above 70%, whereas most of the TPGS-DOX
cell uptake efficiency in C6 cells was below 70%.
a
0
10
20
30
40
50
60
70
80
90
100
0.5 1.5 4 6
Time (h)
MC
F-7
cell
upta
ke e
ffic
ienc
y (%
)
DOX
DOX-TPGS
69
b
0
10
20
30
40
50
60
70
80
90
100
0.5 1.5 4 6
Time (h)
C6
cell
upta
ke e
ffic
ienc
y (%
)
DOX
DOX-TPGS
Fig 4-2 (a) MCF-7 and (b) C6 cell uptake efficiency cultured with the pristine DOX or the TPGS-DOX for 0.5, 1, 4, 6 h respectively at equivalent drug concentration 1 µg/mL (mean ± SD and n=6)
Fig 4-3 exhibits the cellular uptake efficiency of TPGS-DOX and free DOX in (a) MCF-7
and (b) C6 cells, respectively, which was assayed upon 4 h culture with different drug
concentration. It is obvious that both prodrug and free drug showed decreased cellular
uptake efficiency when the equivalent drug concentration was increased from 1 µg/mL to
25 µg/mL. The trend was found in both MCF-7 and C6 cancer cells, which indicated a
saturated and limited property of cellular uptake of the prodrug and free drug. Similarly as
the above results, the cell uptake of TPGS-DOX was higher than that of DOX, no mater in
which cell line and at which drug concentration. The cell uptake was enhanced to 1.2-
(78.6±6.8% vs 64.2±6.4%), 1.1- (30.6±1.2% vs 27.5±0.3%), 1.1-fold (26.4±0.6% vs
24.0±2.0%) (p<0.05) in MCF-7 cells and 1.3- (48.7±7.8% vs 37.4±5.4%), 1.4- (39.5±8.0%
70
vs 28.9±4.9%), 1.1-fold (25.4±6.9% vs 323.5±4.2%) (p<0.05) in C6 cells by TPGS-DOX
at 1, 5, 25 µg/mL, respectively.
a
0
10
20
30
40
50
60
70
80
90
100
0.5 1.5 4 6
Time (h)
MC
F-7
cell
upta
ke e
ffic
ienc
y (%
)
DOX
DOX-TPGS
b
0
10
20
30
40
50
60
70
80
90
100
0.5 1.5 4 6
Time (h)
C6
cell
upta
ke e
ffic
ienc
y (%
)
DOX
DOX-TPGS
Fig 4-3 Cell uptake of the TPGS-DOX and DOX in (a) MCF-7 and (b) C6 cells after 4 h incubation (mean ± SD and n=6)
71
The difference of the cell uptake between the conjugate and the free drug may be due to
the MDR effect, which is a major cause for the failure of anti-cancer chemotherapy
because of the overexpression of MDR transporter proteins such as P-glycoprotein (P-gp)
that mediate unidirectional energy-dependent drug efflux and thus reduce intracellular
drug levels. As the free DOX regards, most of the molecules would be effluxed out by P-
gp pump proteins except those that could bind to DNA after entering the cells (Zhang and
Feng 2006). In contrast, the TPGS has inhibitory effect on the P-gp, the multidrug
resistance transporter, so that less drug would thus be pumped out of the cells, resulting in
a higher cell uptake efficiency (Dintaman and Silverman 1999).
4.3.3 Confocal Laser Scanning Microscopy
The cellular uptake of TPGS-DOX conjugate was further investigated by confocal laser
scanning microscope (CLSM). The four pictures in Fig 4-4 show respectively the
confocal laser scanning microscopy of MCF-7 cancer cells after 4 h incubation with (a)
the pristine drug DOX and (b) with the TPGS-DOX conjugate, and that of C6 cancer cells
with (c) the DOX and (d) the TPGS-DOX conjugate at the same 1 µg/mL DOX
concentration. The cells in (b) and (d) had become unhealthy, which was agreeable with
the cellular uptake results as shown in Fig 4-3 and thus confirmed the advantages of the
TPGS-DOX conjugate over the original DOX. Moreover, it can be seen from Fig 4-4 that
the free DOX was mainly distributed within the nucleus (Fig 4-4 a, c) in both cancer cells,
while the conjugate could be observed around the nucleus resulting a broad intracellular
distribution in the cytoplasm (Fig 4-4 b, d). The change of intracellular distribution of the
72
drug by TPGS conjugation may play an important role, which is similar to that reported in
the literature for the PEG-DOX conjugate (Rodrigues, Beyer et al. 1999). This result
reveals that the conjugate may deliver the drug into the cells in a different way from the
simple diffusion of the free drug in the cells. Besides intercalation with DNA, the
conjugate might exert its cytotoxicity by a different mode of action.
(a)
(b)
73
(c)
(d)
Fig 4-4 Confocal laser scanning microscopy (CLSM) of MCF-7 cells after 4 h incubation with (a) the pristine drug DOX and (b) with the TPGS-DOX conjugate, and that of C6 cells with (c) the DOX and (d) the TPGS-DOX conjugate at the equivalent 1 µg/mL DOX concentration
4.3.4 In Vitro Cytotoxicity
The MCF-7 and the C6 cell line were employed as in vitro models to investigate the
cytotoxicity of the TPGS-DOX conjugate in close comparison with the pristine DOX as a
positive control. Fig 4-5 shows the cellular viability of (a) MCF-7 cells and (b) C6 cells
after 24, 48, 72 h culture respectively with the TPGS-DOX conjugate in comparison with
that of the pristine DOX at various DOX concentrations. It can be seen from the two
graphs that the TPGS-DOX conjugate exhibited significantly higher cytotoxicity
74
(equivalent lower cell viability) at low drug concentration (p<0.05) or at least, comparable
cytotoxicity at high drug concentration in comparison with the pristine DOX. This
advantage became more significant for longer time cell culture.
As the cytotoxicity of TPGS regards, no significant cell killing ability was found for C6
cancer cells and only slight cytotoxicity was observed for MCF-7 cancer cells (Fig 4-6).
However, the cell viability of TPGS in each sampling point is above 50%. This means that
the enhanced cytotoxicity of the TPGS-DOX came from the conjugation strategy but not
from the TPGS carrier.
a
0
20
40
60
80
100
120
0.002 0.02 0.2 2 20 100
Concentration (µM)
MC
F-7
cell
viab
ility
(%)
DOX-1DAY DOX-TPGS-1DAY DOX-2DAYSDOX-TPGS-2DAYS DOX-3DAYS DOX-TPGS-3DAYS
75
b
0
20
40
60
80
100
120
0.002 0.02 0.2 2 20 100
Concentration (µM)
C6
cell
viab
ility
(%)
DOX-1DAY DOX-TPGS-1DAY DOX-2DAYSDOX-TPGS-2DAYS DOX-3DAYS DOX-TPGS-3DAYS
Fig 4-5 Cellular viability of (a) MCF-7 breast cancer cells and (b) C6 glioma cells after 24, 48, 72 h culture with the DOX-TPGS conjugate respectively in comparison with that of the pristine DOX at various equivalent DOX concentrations (mean ± SD and n=6)
It is obvious that the cell viability decreased with increase of the drug concentration. The
IC50 value, i.e. the drug concentration at which 50% cells have been killed in a given
period, can thus be used as an quantitative index to evaluate in vitro the therapeutic
effects of a drug in that given period. Table 4-1 lists the IC50 values of the TPGS-DOX
conjugate and the free drug DOX after 24, 48, 72 h cell culture, respectively. It can be
concluded from this Table that the TPGS-DOX conjugate achieved much lower IC50
values than the free drug DOX in all the cases for MCF-7 and C6 cells at various periods.
The trends became more significant for longer time incubation. The IC50 of the MCF-7
cells was found to be 52.3, 0.357, 0.00916 µM for the TPGS-DOX conjugate vs 76.7,
0.1173, 0.0577 µM for the pristine drug DOX, which implied 31.8, 69.6, 84.1% more
76
effective in vitro, after 24, 48, 72 h culture, respectively. The IC50 of C6 cells was found to
be 20.8, 0.0712, 0.00468 µM for the TPGS-DOX conjugate vs 37.1, 0.568, 0.00810 µM
for the pristine drug DOX, which implied 43.9, 87.7, 42.2% more effective in vitro, after
24, 48, 72 h culture, respectively.
Table 4-1 IC50 values (in equivalent µM DOX level) of MCF-7 and C6 cancer cells cultured with the TPGS-DOX conjugate vs the pristine DOX in 24, 48, 72 h
IC50 (µM)
C6 cells MCF-7 cells Incubation time (h) DOX TPGS-DOX DOX TPGS-DOX
24 37.1 20.8 76.7 52.3
48 0.568 0.0712 0.1173 0.0357
72 0.00810 0.00468 0.0577 0.00916
It is thus evidenced at least in vitro that the TPGS conjugation greatly enhanced the
therapeutic effects of doxorubicin. What’s more, the IC50 values of TPGS in both cell lines
were larger than 400 µM. It is thus evidenced, once again, that the enhanced cytotoxicity
of the TPGS-DOX came from the conjugation strategy but not from the TPGS carrier.
77
0
20
40
60
80
100
120
0.008 0.08 0.8 8 80 800
TPGS concentration (µM)
Cell
viab
ility
(%)
C6-1DAY C6-2DAYS C6-3DAYSMCF7-1DAY MCF7-2DAYS MCF7-3DAYS
Fig 4-6 Cellular viability of MCF-7 breast cancer cells and C6 glioma cells after 24, 48, 72 h culture with TPGS respectively at various equivalent concentrations in the conjugate (mean ± SD and n=6)
It is well known that polymer-drug conjugation usually results in higher IC50 values in
vitro than the parent drug due to changes caused in cellular pharmacokinetics such as
slower endocytic capture compared with rapid transmembrane passage of the parent drug
(Duncan 1992). The DBM2-PEG4000-S-PEG3000-GFLG-DOX conjugate was found 10-20-
fold less toxic than free DOX against B16F10 murine melanoma in vitro (Andersson,
Davies et al. 2005). The PEG-DOX conjugates were demonstrated over 40-fold less toxic
than the free DOX toward H2981 human lung adenocarcinoma cells (Senter, Svensson et
al. 1995). On the contrary, our TPGS-DOX conjugate showed much lower IC50 values in
comparison with the parent drug, which indicated that TPGS-DOX is promising in
78
enhancing the therapeutic effects of the conjugated drug. Although TPGS itself showed
slight cytotoxicity for MCF-7 cells, its IC50 is much larger than that of the DOX and the
TPGS-DOX. This proves that the enhanced therapeutic effect mainly comes from the
conjugation strategy but not from the carrier itself. This great advantage may be attributed
to the unique pharmaceutical properties of TPGS, which is also involved after the
conjugation. First, it can induce apoptosis, a cell death mechanism shared by the majority
of anticancer drug, to exert anti-tumor effect (Porta 2004; Youk, Lee et al. 2005). Second,
TPGS is able to inhibit the MDR effect caused by P-gp efflux pump. The mechanism of
TPGS-DOX internalization may deliver DOX to intracellular compartments that are less
accessible to the P-gp. In this respect, the result of the cytotoxicity experiment was
consistent with that of the cellular uptake. Last but not least, as TPGS possesses great
ROS (reactive oxygen species)-generating ability (Youk, Lee et al. 2005), it is plausible
that TPGS shows selective cytotoxicity to facilitate cytotoxicity of the conjugated drug
toward cancer cells due to the disparities between normal and cancer cells (Renschler
2004).
4.4 Conclusions
In vitro release of DOX from the conjugate was found pH dependent with no significant
initial burst. TPGS-DOX conjugate displayed higher cellular uptake efficiency than that of
free DOX, no matter the drug concentration, incubation time nor MCF-7 or C6 cancer
cells. The in vitro confocal laser scanning microscope imaging confirmed that the TPGS-
DOX conjugate was found distributed around the nucleus and cytoplasm while the DOX
was mainly entrapped in the nucleus. The prodrug showed higher cytotoxicity and
achieved much lower IC50 values in comparison with the pristine DOX, especially for
79
MCF-7 cancer cells. Longer time treatment resulted in better cytotoxicity. Further in vivo
investigation is then required to confirm the preliminary and satisfactory results of in vitro
studies.
80
5 IN VIVO INVESTIGATION ON PHARMACOKINETICS AND
BIODISTRIBUTION OF THE TPGS-DOX CONJUGATE
5.1 Introduction
The in vivo pharmacokinetics (PK) and biodistribution (BD) of the TPGS-DOX conjugate
were investigated on rats in comparison with commercial DOX formulation. The
noncompartmental PK analysis, which estimates the exposure to a drug by the
determining the area under the curve of a concentration-time graph, was employed to test
the in vivo therapeutic effects of the TPGS-DOX conjugate. Quite a few methods have
been developed to determine the drug level in blood and tissues. Here HPLC detection
method was utilized due to its easy handling with acceptable sensitivity and selectivity.
The blood and organs collected from the rats after drug administration needed to be
treated through liquid-liquid extraction and no internal standard was needed in assay. And
then the samples were measured by HPLC using fluorescence detection.
5.2 Materials and Methods
5.2.1 Animal
Male Sprague-Dawley (SD) rate, which were 150-200 g and 4-5 weeks old, were provided
by the Laboratory Animals Centre of Singapore and maintained at the Animal Holding
Unit (AHU) of National University of Singapore. They were kept in well-ventilated rooms
at a temperature of 25±2 °C and a humidity of 50-60% under nature lighting conditions.
All rats caring and handling procedures and protocols were approved by Institutional
81
Animal Care and Use Committee (IACUC), Office of Life Science, National University of
Singapore under the authority of Animal Welfare Act (AWA).
5.2.2 In Vivo Pharmacokinetics
5.2.2.1 Drug Administration
The SD rats were randomly assigned to two groups with each of four rats; one group for
i.v. administration of free DOX and the other for that of TPGS-DOX. Before drug
administration, commercial DOX or TPGS-DOX conjugate were diluted in normal saline
containing 1.9% w/v NaCl to obtain an estimated injection volume of 1-1.5 ml.
Intravenous injection was given via the tail vein at a 5 mg/kg equivalent drug dosage. All
animals were observed for mortality, general condition and potential clinical signs.
5.2.2.2 Blood Collection and Sample Analysis
The blood samples were collected by heparinized tube at 0 (pre-dose), 10, 30 min, 1, 2, 4,
8, 12, 24, 48, 72 h post-treatment. Plasma samples were harvested by centrifugation at
1500×g for 10 min and stored at -20 oC until analysis. Liquid-liquid extraction was
performed prior to the HPLC analysis. Briefly, the plasma (100 µL) was mixed with 100
µL of 10 mM phosphate-buffered saline (pH 7.8). The drug was extracted by
dichloromethane-isopropanol (4:1, v/v) on a vortex-mixer for 90 s. Upon centrifugation at
2000×g for 15 min, the upper aqueous layer was removed by aspiration and the organic
layer was transferred to a glass tube and evaporated under nitrogen at room temperature
overnight. The residue was dissolved in 100 µL of the HPLC mobile phase (1/15 M
KH2PO4/CH3CN=75:25 v/v, pH 4.16, adjusted with H3PO4) by vortex and transferred to
auto sampler vials containing limited-volume inserts (100 µL). The standards needed to be
82
prepared using blank plasma with a series concentration of commercial DOX (0.05 µg/ml-
100µg/ml), followed by the same treatment as the samples done. The drug concentration
in samples was calculated using the standard calibration curve.
For the HPLC analysis, the drug concentration in plasma was determined using Agilent®
1100 Series installed with Agilent® Eclipse XDB-C18 column with 5 µm pore size. The
mobile phase was delivered at a rate of 1 mL/min. Twenty µL of sample were injected and
the column effluent was detected with a fluorescence detector (Ex 470 nm, Em 585 nm)
(Watson, Stewart et al. 1985; Park, Lee et al. 2006).
5.2.2.3 Pharmacokinetic Parameters
Non-compartmental Analysis (NCA), done by Kinetica Software (Thermo Electron
Corporation, USA), provides an estimate of the kinetic parameters of a drug based on
statistical moment theory. As for the specific parameters, the maximum drug
concentration (Cmax) and the corresponding time (tmax) can be observed from the plasma
concentration vs time curve. The elimination half-life (t1/2), an important index, can be
calculated as㏑ 2/λn, in which λn is the elimination constant obtained via log-linear
regression analysis of the terminal phase of the profile. The area under the curve (AUC)
and area under the first moment (AUMC) can be figured out using log-linear trapezoid
rule. The mean residence time (MRT) is calculated as AUMC/AUC. Apparent volume of
distribution at steady state (Vss) and plasma clearance (CL) were obtained as Dosage ×
AUMCinf/(AUCinf)2 and Dosage/AUCinf, respectively, in which AUMCinf and AUCinf
mean the corresponding value from 0 to infinity.
83
5.2.3 In Vivo Biodistribution
5.2.3.1 Drug Administration
The SD rats were randomly assigned to two groups, i.e. Group A with i.v. injection of the
pristine DOX suspension and Group B of the TPGS-DOX conjugation at the equivalent 5
mg/kg, respectively. Each group has 4 sets corresponding to 4 time points, and each set
having 3 rats. Before drug administration, commercial DOX or TPGS-DOX conjugate
were diluted in normal saline containing 1.9% w/v NaCl to obtain an estimated injection
volume of 1-1.5 ml. Intravenous injection was given via the tail vein at a 5 mg/kg
equivalent drug dosage. All animals were observed for mortality, general condition and
potential clinical signs.
5.2.3.2 Tissues Collection and Samples Analysis
Animals in each set were sacrificed by cardiac stick exsanguinations at 0.5, 2, 8 and 24 h
respectively after the injection and tissues (heart, spleen, stomach, lung, intestine, kidney
and liver) were collected. The tissues were then washed with saline and stored at -80oC
prior to analysis.
For the analysis, the tissues were freeze-dried, homogenized. After that, 30 mg organ for
each was mixed with 300 µL PBS, followed by extraction and HPLC analysis as the blood
samples done. The standards of different organs needed to be prepared using the blank
tissues collected from the rats without any drug administration. A series of commercial
DOX was added in the different blank organs respectively and then the standards were
84
treated in the same way as the samples done. The drug level in organs was figured out
using the standard calibration curve.
5.2.3.3 Statistics
Statistical analysis was conducted by using the student’s t-test with p<0.05 as significant
difference.
5.3 Results and Discussion
5.3.1 Pharmacokinetics
1
10
100
1000
10000
100000
0 10 20 30 40 50 60 70 80
Time (h)
DO
X in
pla
sma
(ng/
ml)
DOX-TPGS DOX LOWEST EFFECTIVE LEVEL PEAK LEVE OF MTD
Fig 5-1 Pharmacokinetic profile of the pristine DOX and the DOX-TPGS conjugate after intravenous injection in rats at a single equivalent dose of 5 mg/kg (mean ± SD and n=4)
85
The plasma concentration level of DOX was determined after a single intravenous
injection of the commercial DOX or the TPGS-DOX conjugate at a dose of 5 mg/kg DOX
equivalent in male SD rats, which is shown in Fig 5-1 in a period up to 72 h. The free
DOX rapidly disappeared from the circulation due to its short half-life. In contrast, the
TPGS-DOX conjugate showed a much longer circulation time. Both formulations reached
the highest level 10 min after the injection, which is 199.5±38.0 ng/mL for the pristine
DOX and 4,110±1,419 ng/mL (p<0.01) for the TPGS-DOX conjugate. The drug
concentration of the TPGS-DOX conjugate achieved much higher drug concentration in
the plasma than the pristine DOX did all the time in the experiment (p<0.05), but lower
than the peak concentration (16.4 µM) with the MTD (maximum tolerated dose)
administration (8mg/kg) of free DOX (Houba, Boven et al. 2001).
Table 5-1 Pharmacokinetic parameters of the TPGS-DOX conjugate vs the pristine DOX i.v. injected in the SD rats at the equivalent 5 mg/kg dose
Parameter DOX TPGS-DOX
t1/2 a (h) 2.53±0.26 9.65±0.94
teffectb (h) 9.90±0.51 62.1±2.53
MRTc (h) 2.86±0.39 10.9±1.87
AUC0-∞d (h·ng/mL) 288±54 6812±2149
CLtote (L/h/kg) 17.3±3.08 0.734±0.164
Vdssf (L/kg) 49.6±6.13 8.02±0.91
a half-life time; b therapeutic effective period; c mean residence time; d area under the curve; e total clearance; f volume of distribution at steady state.
86
The pharmacokinetic parameters are summarized in Table 5-1. It can be seen from the
Table 5-1 that the half-life of the drug was 9.65±0.94 h for the TPGS-DOX conjugate and
2.53±0.26 h for the original DOX, 3.81 times longer (p<0.01). Our measurement of the
half-life of the doxorubicin at the 5 mg/kg dose was in good agreement with that found
from the literature (Gao, Lee et al. 2005). The mean residence time of the drug in the
plasma was 10.9±1.87 h for the TPGS-DOX conjugate vs 2.86±0.39 h for the pristine
DOX, also 3.81 times longer (p<0.05) since it has the same physical meaning as the half-
life. The therapeutic effective period was 62.1±2.53 h for the TPGS-DOX conjugation and
9.90±0.51 h for the DOX alone, 6.27 times longer (p<0.05) (the minimum effective level
10 nM was found from the literature (Gavenda, Ševčík et al. 2001)). This may be due to
the enhanced permeability and retention effect (ERF) by the TPGS conjugation. As
prolonged plasma circulation is the driving force for increased tumor targeting (Seymour,
Miyamoto et al. 1995), the conjugate was supposed to show improved therapeutic efficacy.
Indeed, the area-under-the-curve (AUC), which is a key therapeutic index of a drug, was
6,810±2149 h·ng/mL for the TPGS-DOX conjugate and 288±54 h·ng/mL for the DOX
alone, 23.6 times larger (p<0.05). The volume distribution at steady state was 8.02±0.91
L/kg for the TPGS-DOX conjugate and 49.6±6.13 L/kg for the DOX alone, which was
6.18 times smaller (p<0.05), and the total clearance of the drug from the plasma was
0.763±0.164 L/h/kg for the TPGS-DOX conjugate vs 17.3±3.08 L/h/kg for the DOX alone,
22.7 times longer (p<0.05). All these results showed the significant enhancement of the
drug’s pharmacokinetics through TPGS conjugation. It should be emphasized that,
although polymer-drug conjugation enhanced the pharmacokinetics of the drug in general,
our TPGS-DOX conjugate showed the most significant effects compared with other
polymer-DOX conjugates in the literature (Senter, Svensson et al. 1995; Veronese,
87
Schiavon et al. 2005). Although both increased AUC and retention time were
demonstrated but they were not as significant as the TPGS-DOX had achieved, compared
to the pristine DOX.
5.3.2 Biodistribution
The distribution of DOX in solid tissues such as heart, lung, spleen, liver, gastric, intestine
and kidney was investigated at four time points: 0.5, 2, 8 and 24 h. The results are shown
in Fig 5-2 with graph (a) for pristine DOX and graph (b) for the TPGS-DOX
administration of 5 mg/kg doxorubicin dose. It can be seen that TPGS conjugation
significantly changed the biodistribution of the drug. The peak concentrations were
detected at 0.5 or 2 h for DOX administration while most peak concentrations were found
at 2 or 8 h for the TPGS-DOX conjugate. TPGS conjugation delayed the time of the peak
concentrations and reduced their magnitude, which may imply reduced side effects and
thus have clinical significance. Such an advantage of TPGS conjugation may be attributed
to the slower penetration of the prodrug into the tissues. For DOX, the highest
concentration was found in heart (50.08±5.27 µg/g at 2h) followed by stomach
(46.97±12.61 µg/g at 2 h), lung (44.44±12.85 µg/g at 2 h), kidney (42.63±9.27 µg/g at 0.5
h) and intestine (41.71±4.92 µg/g at 2 h). On the contrary, the peak concentration in these
tissues for the TPGS-DOX conjugate dropped by 5.4-, 7.3-, 2.2-, 2.4- and 5.3-fold, which
was 9.22±6.69 µg/g at 2 h, 6.44±5.93 µg/g at 0.5 h, 20.19±11.2 µg/g at 8 h, 17.66±9.56
µg/g at 2 h and 7.83±4.66 µg/g at 2 h, respectively (p<0.05). The conjugate presented a
little higher DOX peak levels than the free drug in spleen (22.52±8.50 vs 12.68±0.74 µg/g)
and liver (17.57±3.24 vs 14.70±6.13 µg/g) at 2 and 8 h, respectively (p>0.05).
88
Table 5-2 summarizes the AUC values of DOX in various tissues after intravenous
injection of the TPGS-DOX conjugate vs the free DOX. In comparison with that of the
DOX, the AUC of the TPGS-DOX conjugate was a bit higher in liver (262±47 vs 228± 20
µg·h/g, p=0.31) and spleen (335±43 vs 127.0±32.6 µg·h/g, p<0.05), comparable in lung
(313±50 vs 304±86 µg·h/g, p=0.88) and kidney (169.4±39.6 vs 166.3±21.6 µg·h/g,
p=0.91), and lower in heart (155.2±33.1 vs 307±64 µg·h/g, p<0.05), stomach (114.3±38.6
vs 313±14 µg·h/g, p<0.01) and intestine (86.7±20.1 vs 246±51 µg·h/g, p<0.01). The
TPGS-DOX showed great superiority in decreasing the AUC in heart by over 5 times,
which was indicated only 3 times decline for prodrug DOX-GA3 (Houba, Boven et al.
2001) and other polymer-DOX conjugate (Tomlinson, Heller et al. 2003; Veronese,
Schiavon et al. 2005).
a
0
10
20
30
40
50
60
heart lung spleen liver stomach intestine kidney
Tissues
µg D
OX/
g or
gan
for
DO
X
0.5h2h8h24h
89
b
0
10
20
30
40
50
60
heart lung spleen liver stomach intestine kidney
Tissues
µg D
OX/
g or
gan
for
DO
X-T
PG
S0.5h2h8h24h
Fig 5-2 The DOX levels (µg/g) in heart, lung, spleen, liver, stomach, intestine and kidney after i.v. administration at 5 mg/kg equivalent drug of (a) the free DOX, (b) the TPGS-DOX conjugate (mean ± SD and n=3) DOX is a lipophilic molecule and can rapidly penetrate into normal tissues (Houba, Boven
et al. 2001), which may account for unfavorable side-effects. The hydrophilic TPGS
moiety of the TPGS-DOX would prevent the rapid diffusion of the prodrug into tissue
cells. This is consistent with the delayed time and decreased magnitude of the peak drug
concentration in normal tissues of the TPGS-DOX conjugate. Significantly higher kidney
level of the free drug was observed at 0.5 and 2 h due to its on-going renal elimination,
which was also consistent with the faster clearance of the free drug. High accumulation of
the prodrug in liver and spleen may suggest that these two organs be the major ones for
the clearance, in which the prodrug was preferentially uptaken by the reticuloendothelial
90
system. More importantly, in contrast with the increased level of DOX in liver and spleen
after TPGS-DOX conjugate administration, the concentrations of the drug in heart,
stomach and intestine for the TPGS-DOX administration were reduced much more. As the
major side effect of DOX is considered as serious cardiac and gastrointestinal toxicity
(Mishra and Jain 2000), the TPGS-DOX prodrug significantly impaired such limitations
and showed great superiority over the free DOX.
Table 5-2 AUC values (µg·h/g) of biodistribution in various organs after intravenous injection of the free DOX and the TPGS-DOX conjugate to rats at 5 mg/kg equivalent dose
heart lung spleen liver stomach intestine kidney
DOX 307±64 304±86
127.0±32.6
228±20 313±14 246±51 166.3±21.
6
TPGS-DOX
155.2±33.1
313±50 335±43 262±4
7 114.3±38.6
86.7±20.1
169.4±39.6
5.4 Conclusions
The in vivo pharmacokinetics investigation showed that the TPGS-DOX resulted in much
higher AUC and much longer half-life than the original DOX, which implied enhanced
therapeutic effects. The peak value of the drug concentration in the tissues was reduced
and the time at which the peak concentration was delayed, which implied reduced side
effects. The biodistribution investigation showed that the drug accumulation for the
TPGS-DOX administration was higher in liver and spleen, and lower in heart, stomach
91
and intestine than that for the pristine DOX administration, which indicated the reduced
systemic toxicity, especially impaired the cardiotoxicity.
92
6 CONCLUSIONS AND RECOMMENDATIONS
6.1 Conclusions
The main objective of this project is to develop a novel polymeric anti-cancer drug
conjugate, TPGS-Doxorubicin, for cancer chemotherapy, which comprises prodrug
synthesis and confirmation, in vitro characterization of release, cytotoxicity and cellular
uptake, in vivo investigations in pharmacokinetics and biodistribution. There has been a
lot of work on polymer-drug conjugation, specifically on PEG-DOX conjugation. To our
knowledge, however, TPGS conjugation represents a novel idea in polymer-drug
conjugation strategy since TPGS is a PEGylated vitamin E, which can further improve the
cellular uptake and half-life and thus the therapeutic effects of the drug.
As chapter 3 indicated, TPGS-DOX conjugate was successfully synthesized by chemical
attachment between the carboxyl group in TPGS-SA and the primary amine group in
DOX in the presence of the coupling agent. 1H NMR, FT-IR and GPC were employed to
determine the chemical structure and molecular weight, which confirmed the success of
the DOX conjugation onto TPGS. The drug loading efficiency of the conjugate was
detected using microplate reader, implying 8%. Moreover, the stability study showed that
only 12% drug was released after 5 months as stored at 4 oC, indicating DOX could be
stably attached to TPGS. In chapter 4, in vitro investigation demonstrated that the prodrug
could release DOX for more than 10 days without initial burst in a controlled way under
different pH values. Compared to free DOX, the conjugate displayed higher cellular
uptake and better cytotoxicity in both C6 and MCF-7 cell lines. The lower IC50 values of
93
the TPGS-DOX showed its great superiority over the existing polymeric DOX conjugates.
Besides, the conjugate was found to be distributed differently in cancer cells from the free
DOX, which may imply the different mechanism of drug delivery. At last, the in vivo
pharmacokinetics and biodistribution were further demonstrated in chapter 5. The TPGS-
DOX conjugate resulted in controlled release in blood and tissues as well as less toxicity
in comparison with pristine DOX. On one hand, the TPGS-DOX conjugate resulted in
much higher AUC and much longer half-life in plasma than the original DOX, which
implied greatly enhanced therapeutic effects. On the other hand, the peak value of the drug
concentration in normal tissues after TPGS-DOX administration was reduced and the time
of the peak concentration was delayed, which implied reduced side effects.
All in all, the work above demonstrated that TPGS could carry doxorubicin by
conjugation and realize controlled and sustained release under mild acidic conditions that
allowed the transport of the prodrug to tumor cells and facilitate drug release to exert its
therapeutic function there. The higher in vitro cellular uptake efficiency, cytotoxicity and
much lower IC50 values of the conjugate in comparison with the pristine DOX or other
reported polymer-DOX conjugate approved the great clinical promise of TPGS for drug
conjugation. The in vivo pharmacokinetics index further revealed that the significant
enhancement of the drug’s pharmacokinetics by TPGS conjugation. As for the
biodistribution, the drug accumulation for the TPGS-DOX administration was much lower
in heart, gastric and intestine than that for the pristine DOX administration, which greatly
impaired the limitation of commercial doxorubicin. The TPGS-DOX prodrug showed
great potential to become a novel dosage form of doxorubicin and the methodology can
also be applied to other anticancer drugs.
94
6.2 Recommendations
To further improve the TPGS-drug conjugate in cancer chemotherapy, the following work
is recommended:
To develop TPGS prodrug for other hydrophobic drug such as Docetaxel, hydrophilic
drug or protein drug;
To transplant different tumor models in animals and evaluate the inhibition effect of
TPGS-drug conjugate (Xenograft);
To apply the TPGS-drug conjugate in clinic phase I test for further investigation in
therapeutic and side effects;
To conjugate targeting agent on to the prodrug for targeted delivery of the drug, for
example folate targeted TPGS-DOX conjugate.
95
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