AN ABSTRACT OF DISSERTATION OF
Nijaporn Yanasarn for the degree of Doctor of Philosophy in Pharmacy presented on
August 4, 2011.
Title: Lipid Based Nanocarriers for Chemotherapeutic Drug Docetaxel and Vaccine
Delivery
Abstract approved:
_____________________________________________________________________
Zhengrong Cui
Nanoscale drug delivery systems have a great impact in current medical field.
These carriers have the potential to improve the efficacy and reduce the toxicity of
various medicinal products. A broad variety of different lipid based carriers had been
developed and used as delivery systems in the past decades. This dissertation focused
on the development of solid lipid nanoparticles (SLN) as delivery systems for a
chemotherapeutic agent, docetaxel, and the use of liposomes as a carrier for
recombinant protein vaccines.
Docetaxel is a potent anticancer drug. However, there continues to be a need
for alternative docetaxel delivery systems to improve its efficacy. Docetaxel
nanoparticles comprised of lecithin as the main component were engineered using two
methods, the emulsion precursor method and the solvent emulsification/evaporation
method. Docetaxel in nanoparticles were more effective in killing tumor cells in
culture than docetaxel solution. The intravenously injected docetaxel-nanoparticles
increased the accumulation of docetaxel in tumors in mice. When administered by
intravenous injection or oral routes, docetaxel-nanoparticles showed antitumor activity
in tumor-bearing mice. The lecithin-based nanoparticles have the potential to be a
novel biocompatible and efficacious delivery system for docetaxel.
Liposomes, a well-known lipid based carrier, have been investigated
extensively as a vaccine delivery system. The adjuvant activities of liposomes with
different net surface charges (neutral, positive, or negative) were evaluated when
simply admixed with protein antigens. Immunization study in mice after
subcutaneously injection of different net charged liposomes showed different antibody
responses, depending on the protein antigens. Antigens (OVA, PA) admixed with the
negatively charged liposomes prepared with phospholipid, DOPA, induced a strong
and functional antibody response comparable to the positively charged liposomes
prepared with DOTAP lipid. The negatively charged DOPA liposomes admixed with
OVA also induced OVA-specific CD8+ cytotoxic T lymphocyte responses and
significantly delayed the growth of OVA-expressing B16-OVA melanoma in a mouse
model. The adjuvant activity of the negatively charged liposomes may be related to
the liposome‟s ability (i) to upregulate the expression of molecules related to the
activation and maturation of antigen-presenting cells and (ii) to slightly facilitate the
uptake of the antigens by antigen-presenting cells. Simply admixing certain negatively
charged liposomes with certain protein antigens of interest may represent a novel
platform for vaccine development.
©
Copyright by Nijaporn Yanasarn
August 4, 2011
All Rights Reserved
Lipid Based Nanocarriers for Chemotherapeutic Drug Docetaxel
and Vaccine Delivery
by
Nijaporn Yanasarn
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented August 4, 2011
Commencement June 2012
Doctor of Philosophy dissertation of Nijaporn Yanasarn presented on August 4, 2011
APPROVED:
Major Professor, representing Pharmacy
Dean of the College of Pharmacy
Dean of the Graduate School
I understand that my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of my
dissertation to any reader upon request.
Nijaporn Yanasarn, Author
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my advisor, Dr. Zhengrong Cui,
for the opportunity to be one of his research team both at Oregon State University and
The University of Texas at Austin, his helpful guidance in scientific perspective,
encouragement, and financial support. I would like to thank Dr. J. Mark Christensen,
Dr. Rosita Rodriguez Proteau, Dr. Virginia Lesser, and Dr. Mahfuzur Sarker for their
kindness to serve as my committee members and provide useful knowledge. I also
would like to thank Dr. Daniel D. Rockey for his valuable time as my graduate
representative in the first committee meeting and the preliminary examination.
I am very thankful to Ms. Teresa Sawyer for her assistance to obtain the TEM
images, Dr. Nikki Marshall and Mr. Richard A. Salinas (University of Texas at
Austin) for their helpful assistance in flow cytometry experiments.
It is a pleasure to thank all current students in Dr. Cui‟s lab, Ms. Letty
Rodriguez, Ms. Xinran Li, Mr. Michael Sandoval, and Mr. Amit Kumar for their help
and friendship throughout the years. I also would like to thank Dr. Brian R. Sloat, Dr.
Woongye Chung, Dr. Melisande Holzer, Dr. Dharmika Lansakara-P, Dr. Saijie Zhu,
Dr. Piyanuch Wonganan, Dr. Rebecca De Angel, Dr. Gang Xiao, and Dr. Gang Zhao,
former or current postdocs in Dr. Cui‟s lab, for their useful advice and excellent
collaborations. I am very grateful to Dr. Uyen Minh Le, former graduate students, for
her friendship, kindness, and lab advice.
I highly appreciate Payap University, Chiang Mai, Thailand, for the fellowship
and financial support in my Ph.D. program in 2006-2011. I also would like to thank
my colleges in the Department of Pharmaceutics, OSU College of Pharmacy for their
friendship and helpful advice.
Lastly, I owe my deepest gratitude to my family and friends, especially my
wonderful parents, Mr. Surapong and Mrs. Supaporn Yanasarn for their love, support,
and encouragement throughout my study, and definitely, to God who made all things
possible.
CONTRIBUTION OF AUTHORS
Dr. Brian R. Sloat (College of Pharmacy, Oregon State University; College of
Pharmacy, University of Texas at Austin) assisted with some nanoparticle
formulation in chapter 2 and experiments in chapter 4.
TABLE OF CONTENTS
Page
CHAPTER 1. GENERAL INTRODUCTION .............................................................. 1
1.1 Lipid based delivery systems .............................................................................. 1
1.2 Lipid based delivery systems for anticancer drugs ............................................. 2
1.2.1 Docetaxel as a chemotherapeutic agent ....................................................... 6
1.2.2 Engineering of nanoparticles for docetaxel.................................................. 7
1.2.2.1 Nanoparticles engineered from lecithin-in-water emulsions .............. 9
1.2.2.2 Nanoparticles engineered from solvent emulsification/evaporation . 10
1.3 Lipid based delivery systems for vaccines ........................................................ 10
1.3.1 Vaccines ..................................................................................................... 11
1.3.2 Immunological adjuvants ........................................................................... 14
1.3.3 Liposomes as an adjuvant .......................................................................... 18
CHAPTER 2. NANOPARTICLES ENGINEERED FROM LECITHIN-IN-WATER
EMULSIONS AS A POTENTIAL DELIVERY SYSTEM FOR DOCETAXEL .. 20
2.1 Abstract ............................................................................................................. 21
2.2 Introduction ....................................................................................................... 21
2.3 Materials and Methods ...................................................................................... 23
2.3.1 Materials ..................................................................................................... 23
2.3.2 Mice and cell lines ..................................................................................... 24
2.3.3 Preparation of nanoparticles ....................................................................... 24
2.3.4 Transmission electron microscopy (TEM)................................................. 25
2.3.5 In vitro release of docetaxel from nanoparticles ........................................ 25
2.3.6 The uptake of nanoparticles by tumor cells in culture ............................... 26
2.3.7 In vitro cytotoxicity assays ........................................................................ 27
2.3.8 Biocompatibility of docetaxel-nanoparticles ............................................. 27
2.3.9 In vivo plasma ALT determination ............................................................ 28
TABLE OF CONTENTS (Continued)
Page
2.3.10 The accumulation of docetaxel in tumors in mice ................................... 29
2.3.11 Data and statistical analyses ..................................................................... 29
2.4 Results and Discussion ...................................................................................... 30
2.4.1 Preparation of nanoparticles from lecithin-in-water emulsions ................. 30
2.4.2 In vitro release of docetaxel from nanoparticles ........................................ 36
2.4.3 The uptake of nanoparticles by tumor cells in culture ............................... 37
2.4.4 In vitro cytotoxicity .................................................................................... 37
2.4.5 Biocompatibility of docetaxel-nanoparticles ............................................. 40
2.4.6 Nanoparticles increased the accumulation of docetaxel in tumors
in mice ........................................................................................................ 44
2.5 Conclusions ....................................................................................................... 48
CHAPTER 3. ENHANCED IN VITRO AND IN VIVO ANTITUMOR ACTIVITY
OF DOCETAXEL BY SOLID LIPID NANOPARTICLE CARRIER................... 49
3.1 Abstract ............................................................................................................. 50
3.2 Introduction ....................................................................................................... 51
3.3 Materials and Methods ...................................................................................... 53
3.3.1 Materials ..................................................................................................... 53
3.3.2 Mice and cell lines .................................................................................... 54
3.3.3 Preparation and characterization of docetaxel-nanoparticles .................... 54
3.3.4 Transmission electron microscopy (TEM)................................................. 55
3.3.5 In vitro stability of docetaxel-nanoparticles .............................................. 55
3.3.6 In vitro release of docetaxel from nanoparticles ....................................... 56
3.3.7 In vitro uptake of the nanoparticles by tumor cells ................................... 56
3.3.8 In vitro cytotoxicity assays ........................................................................ 57
3.3.9 Pharmacokinetics studies ........................................................................... 58
TABLE OF CONTENTS (Continued)
Page
3.3.10 In vivo tumor uptake study ..................................................................... 59
3.3.11 In vivo therapeutic study ......................................................................... 59
3.3.12 HPLC analysis of docetaxel .................................................................... 60
3.3.13 Data and statistical analysis .................................................................... 60
3.4 Results and discussion ...................................................................................... 61
3.4.1 Preparation and characterization of docetaxel-nanoparticles from
solvent emulsification/evaporation ............................................................ 61
3.4.2 In vitro release of docetaxel from the nanoparticles .................................. 62
3.4.3 In vitro uptake of docetaxel by tumor cells ................................................ 66
3.4.4 In vitro cytotoxicity .................................................................................... 66
3.4.5 Pharmacokinetics studies ........................................................................... 71
3.4.6 In vivo tumor uptake study ........................................................................ 73
3.4.7 In vivo therapeutic studies ......................................................................... 74
3.5 Conclusions ....................................................................................................... 76
CHAPTER 4. NEGATIVELY CHARGED LIPOSOMES SHOW POTENT
ADJUVANT ACTIVITY WHEN SIMPLY ADMIXED WITH PROTEIN
ANTIGENS ............................................................................................................. 78
4.1 Abstract .............................................................................................................. 79
4.2 Abbreviations .................................................................................................... 80
4.3 Introduction ...................................................................................................... 80
4.4 Materials and Methods ..................................................................................... 84
4.4.1 Materials .................................................................................................... 84
4.4.2 Cells and cell lines .................................................................................... 85
4.4.3 Preparation and characterization of liposomes ......................................... 85
TABLE OF CONTENTS (Continued)
Page
4.4.4 Immunization studies ................................................................................ 88
4.4.5 Enzyme-linked immunosorbent assay (ELISA) ........................................ 88
4.4.6 Anthrax lethal toxin neutralization assay ................................................... 89
4.4.7 In vivo CTL assay and mouse tumor prevention study ............................. 90
4.4.8 In vitro uptake of OVA admixed with liposomes by DC2.4 cells ............. 91
4.4.9 CD80 and MHC II expression on DC2.4 cells after in vitro stimulation... 91
4.4.10 Mouse dendritic cell and antigen-presenting cell PCR array ................... 92
4.4.11 Reverse transcription PCR ....................................................................... 93
4.4.12 Determination of cytokine concentration ................................................. 93
4.4.13 Statistics ................................................................................................... 94
4.5 Results ............................................................................................................... 94
4.5.1 Preparation and characterization of OVA-liposome mixtures ................... 94
4.5.2 OVA admixed with net negatively charged liposomes was as
immunogenic as OVA admixed with net positively charged liposomes ... 95
4.5.3 Net negatively charged DOPA liposomes admixed with anthrax
protective antigen protein induced a functional anti-PA antibody
response ...................................................................................................... 98
4.5.4 The specific antibody responses induced by liposomes with different
charges were not significantly different when cationized OVA was
used as the antigen ................................................................................... 102
4.5.5 Net negatively charged liposomes prepared with different lipids had
different adjuvant activities ...................................................................... 106
4.5.6 Immunization with OVA admixed with the negatively charged DOPA
liposomes significantly delayed the growth of B16-OVA tumors
in mice ...................................................................................................... 110
4.5.7 OVA admixed with the DOPA liposomes induced OVA-specific CTL
responses .................................................................................................. 113
4.5.8 The negatively charged DOPA liposomes only slightly enhanced the
uptake of the OVA by DC2.4 cells in culture .......................................... 113
TABLE OF CONTENTS (Continued)
Page
4.5.9 The negatively charged DOPA liposomes up-regulated the expression
of genes related to DC activation and maturation .................................... 117
4.6 Discussion ........................................................................................................ 119
4.7 Conclusions ..................................................................................................... 130
CHAPTER 5. GENERAL CONCLUSIONS ............................................................. 131
BIBLIOGRAPHY ...................................................................................................... 133
LIST OF FIGURES
Figure
Page
1.1 The structure of docetaxel ........................................................................................ 6
1.2 The sizes of pathogenic agents and adjuvant delivery systems ............................. 11
2.1 Preparation of DTX-NPs ........................................................................................ 32
2.2 Nanoparticles were stable and lyophilizable .......................................................... 35
2.3 The release of docetaxel from DTX-PEG-NPs ...................................................... 36
2.4 The uptake of nanoparticles by TC-1 cells in culture ............................................ 38
2.5 Docetaxel in nanoparticles was more effective in killing tumor cells in culture ... 41
2.6 Nanoparticles have a favorable biocompatibility profile ....................................... 45
3.1 Preparation and characterization of solid lipid docetaxel-nanoparticles
(DTX-NPs) ............................................................................................................. 63
3.2 In vitro release profile of docetaxel from docetaxel-nanoparticles ........................ 65
3.3 Uptake of docetaxel in nanoparticles (DTX-NPs) or docetaxel in solution
(DTX) by TC-1 cells in culture .............................................................................. 67
3.4 Docetaxel in nanoparticles was more effective than in solution in killing
tumor cells in culture.............................................................................................. 68
3.5 Plasma pharmacokinetics of docetaxel in mice ..................................................... 72
3.6 Nanoparticles increased the accumulation of docetaxel in tumors in mice ........... 73
3.7 In vivo antitumor efficacy after intravenous injection ........................................... 74
3.8 In vivo antitumor efficacy after oral administration .............................................. 76
4.1 Preparation of OVA-liposome mixtures ................................................................ 96
LIST OF FIGURES (Continued)
Figure
Page
4.2 Immunization of mice with OVA admixed with net positively or net
negatively charged liposomes induced a strong OVA-specific serum IgG
response .................................................................................................................. 99
4.3 Immunization of mice with PA admixed with liposomes with different net
charges induced strong and functional PA-specific antibody responses ............. 103
4.4 When admixed with cationized OVA, liposomes with different net charges
showed comparable adjuvant activities ................................................................ 107
4.5 Net negatively charged liposomes prepared with different lipids had different
adjuvant activities.................................................................................................. 111
4.6 Immunization of mice with OVA admixed with DOPA liposomes significantly
delayed the growth of B16-OVA tumors in mice and induced a specific CTL
response ................................................................................................................ 114
4.7 In vitro uptake of OVA admixed with liposomes with different charges by
DC2.4 cells ............................................................................................................ 117
4.8 The expression of MHC II and CD80 on DC2.4 cells after in vitro stimulation .. 118
4.9 Effect of the negatively charged DOPA liposomes on DC2.4 cells .................... 120
4.10 DOPA and DOPS liposomes up-regulated the expression of IL-6 and
CCL-17 in mouse BMDC ................................................................................... 124
LIST OF TABLES
Figure
Page
3.1 Pharmacokinetic parameters after intravenous administration of DTX and
DTX-NPs to mice .................................................................................................... 72
LIPID BASED NANOCARRIERS FOR CHEMOTHERAPEUTIC DRUG
DOCETAXEL AND VACCINE DELIVERY
Chapter 1
GENERAL INTRODUTION
1.1 Lipid based delivery systems
Particulate drug carriers include traditional systems such as emulsions to
innovative systems such as liposomes, micelles, polymeric micro- and nanoparticles,
solid lipid nanoparticles, cyclodextrins, dendrimers, and bioconjugated nanoparticles,
to name a few, and have been investigated intensively in the past several decades.
These carrier systems provide several advantages to overcome drug therapy failure,
which is mainly related to an insufficient drug concentration at the desired area and /
or drug distribution to other tissues combined with high drug toxicity, resulting in
more side effects (Mehnert and Mader, 2001). In addition, new biologics such as
protein and nucleic acids require novel delivery systems to administer and to improve
their efficacy (El-Aneed, 2004; Kefalides, 1998). The size of particulate carriers
ranges from few nanometers to the micrometer range, depending on the desired route
of administration. The submicron size particulate carriers (nanoscale carriers) have
gained increased attention during recent years (Hughes, 2005) due to their feasibility
for parenteral administration, specifically intravenous injection, and drug targeting,
2
which can reduce drug toxicity and improve drug distribution (Lobenberg and Kreuter,
1996). Other advantages of nanoscale delivery systems include the ability to overcome
various anatomic features such as the blood-brain barrier, the branching pathway of
the pulmonary systems (Courrier et al., 2002), and the tight epithelial junctions of the
skin. Nanoscale delivery systems are also able to penetrate tumors due to the
discontinuous nature of the tumor vasculature (Matsumura and Maeda, 1986). Among
various kinds of particulate carriers, lipid based carriers have increased attention from
many researchers due to the well tolerability of lipids, which leads to a less toxicity
and a high biocompatibility.
Lipid based delivery systems have been applied in various fields including
cancer chemotherapy and vaccine delivery. My research focused on the development
of nanoscale carriers for a chemotherapy agent, docetaxel, to increase the effectiveness
and to avoid or minimize adverse reactions of the current approved formulation.
Besides the use of lipid based delivery systems for cytotoxic drugs, we also
investigated the use of liposomes as a carrier for recombinant protein vaccines, which
have significant advantages over traditional vaccines and have been greatly developed
in recent years.
1.2 Lipid based delivery systems for anticancer drugs
Cytotoxic drugs are still the major form of chemotherapy for the treatment of
cancers. The actions of these drugs are primarily related to the cell cycle. In general,
3
mammalian cell cycle is composed of four phases: G1, S, G2, and M. In G1 phase,
preliminary synthetic cellular processes occur to prepare cells for the DNA synthesis
(S phase). After the completion of the S phase, cells enter the pre-mitosis (G2) phase
prior to undergoing mitosis (M phase), in which one cell divides into two daughter
cells (Airley, 2009). Chemotherapy agents are a diverse class of compounds that effect
the cell cycle and can be classified according to the phase of the cell cycle, in which
they are active. Cancer cells often undergo rapid growth and proliferation, and thus are
preferentially killed by these agents. However, fast-growing normal cells such as
blood cells, cells in the digestive tract, hair follicles, and reproductive systems, are
also affected by these cytotoxic drugs, resulting in common undesirable side effects
such as immunosuppression, anemia, fatigue, nausea, vomiting, and hair loss, etc.
(Airley, 2009). Due to the poor side effect profile, the therapeutic value of the
cytotoxic drugs has been greatly diminished.
Conventionally, cytotoxic drugs are administered systemically by intravenous
bolus or infusion in the form of free drug solutions. However, most of these drugs are
poorly water-soluble and often bind extensively to body tissues and serum proteins,
which leads to only a small fraction of drugs reaching the tumor tissues. This may
reduce the therapeutic efficacy and increase the risk of side effects and toxicity of the
drugs (Ratain, 1996). In addition, clinical drug resistance has been recognized as a
major obstacle in cancer chemotherapy. The causes of drug resistance can be
categorized into two groups, the inadequate drug exposure (non-cellular drug
4
resistance) and the alterations in the cancer cell itself (cellular drug resistance) (Baird
and Kaye, 2003). Factors leading to the inadequate drug exposure are associated with
the poor pharmacokinetic profile of the drugs, the impaired diffusion of the drugs to
tumor cells, and the tumor-induced environmental changes (e.g. high interstitial
pressures, hypoxia, and lower pH). For the cellular drug resistance, cancer cells
develop a variety of mechanisms at cellular level to diminish the toxicity of
chemotherapeutic agents. These mechanisms include the increase of drug efflux
mediated by membrane-associated drug transporters (e.g. P-glycoprotein and
multidrug resistance associated protein), apoptosis evasion, activation of drug
detoxification, and alterations of the drug targets (Baird and Kaye, 2003; Gieseler et
al., 2003). These obstacles lead to disappointing clinical outcomes even though an
anticancer drug may have a strong in vitro efficacy.
As mentioned earlier, nanoparticles are one of the innovative particulate
delivery systems which have been intensively developed as carriers for chemotherapy
compounds. These nanoscale delivery systems have unique properties that can
potentially improve the efficacy of anticancer drugs (Allen and Cullis, 2004). For
example, nanoparticles may be used to enhance the solubility of poorly-water soluble
anti-cancer drugs and to modify their pharmacokinetics (Gabizon et al., 2003).
Nanoparticles may also be engineered to reduce the uptake of the drugs by the
reticuloendothelial system and / or to target the drugs to specific tumor cells (Allen,
2002; Emerich and Thanos, 2006). Importantly, submicron-sized particles may
5
preferentially penetrate into tumors due to the defective, leaky vascular structure of the
tumors and retain there, which is often referred as the „enhance permeability and
retention‟ (EPR) effect (Fang et al., 2010; Matsumura and Maeda, 1986; Yuan et al.,
1995). Additionally, there was data showing that nano-sized drug carriers can
overcome the multi-drug resistance in many cancer cells (Chavanpatil et al., 2007a;
Jabr-Milane et al., 2008; Wong et al., 2006).
In the past two decades, a variety of nanoparticles have been developed for
cytotoxic drugs, such as protein-based nanoparticles (Dreis et al., 2007; Langer et al.,
2003; Weber et al., 2000), polymeric nanoparticles (Fonseca et al., 2002; Hwang et al.,
2008; Kalaria et al., 2009; Musumeci et al., 2006; Ong et al., 2009), solid lipid
nanoparticles (SLN) (Cavalli et al., 2000; Harivardhan Reddy et al., 2005; Xu et al.,
2009; Yang et al., 1999b; Yang and Zhu, 2002; Zara et al., 2002), polymer-surfactant
nanoparticles (Chavanpatil et al., 2007b), and polymer-lipid hybrid nanoparticles
(PLN) (Wong et al., 2004), to name a few. Additionally, in 2005, the first commercial
protein-based nanoparticles for cytotoxic drug, paclitaxel (nab-paclitaxel; Abraxane),
was approved by the Food and Drug Administration (FDA) for the treatment of breast
cancer. Clinical data revealed that the nab-paclitaxel has improved efficacy and safety
compared with conventional solvent-based formulation (Hawkins et al., 2008). The
nab-paclitaxel represents a promising advance in using particulate delivery systems
for the delivery of various anticancer compounds.
6
1.2.1 Docetaxel as a chemotherapeutic agent
Docetaxel is a semi-synthetic compound belonging to the taxane family of
anticancer drugs. The first taxane, paclitaxel, was identified as the cytotoxic
compound from the crude extract of the North American pacific yew tree (Wani et al.,
1971). However, the scarce supply of the pacific yew tree and the difficulties of
paclitaxel formulation led to the discovery of a taxane derivative, docetaxel, which is
produced from 10-deacethylbaccatin-III found in the European yew tree (Gelmon,
1994). Docetaxel is a molecule with an anhydrous molecular weight of 807.9 and a
chemical formula of C43H53NO14 (Figure 1.1).
Figure 1.1 The structure of docetaxel
The taxanes are a unique class of cytotoxic drugs that promote the
polymerization of microtubules, which leads to the disruption of mitosis, cell cycle
arrest at G2/M, and cell death (Schiff et al., 1979). Docetaxel has a significant
7
antitumor activity against various human malignancies and is approved by U.S. FDA
(Food and Drug Administration) for the treatment of breast cancer, ovarian cancer,
non-small-cell lung cancer, and prostate cancer.
In spite of its high efficiency, side effects limit the clinical use of the
docetaxel. Due to its highly lipophilicity and practically insolubility in water (3
µg/mL) (Engels et al., 2007), the current formulation of docetaxel contains 40 mg/mL
docetaxel and 1040 mg/mL polyoxyethylated surfactant, polysorbate 80 (Tween 80),
which requires further dilution with 13% ethanol before addition to intravenous
infusion solution. Adverse reactions due to either the drug itself (e.g., neurotoxicity,
musculotoxicity, and neutropania) (Persohn et al., 2005) or the solvent system (e.g.,
hypersensitivity and fluid retention) (ten Tije et al., 2003) have been reported.
Therefore, many alternative docetaxel formulations such as liposomes (Immordino et
al., 2003), emulsion (Gao et al., 2008; Yin et al., 2009; Zhao et al., 2010), polymeric
nanoparticles (Hwang et al., 2008; Musumeci et al., 2006), micelles (Liu et al., 2008;
Mu et al., 2010), and solid lipid nanoparticles (Xu et al., 2009) have been investigated
to eliminate the Tween 80-based vehicle.
1.2.2 Engineering of nanoparticles for docetaxel
As an alternative carrier system to traditional colloidal carriers, solid lipid
nanoparticles (SLN) were first introduced more than a decade ago (Muller et al.,
1995). Since then, they have gained attention from many research groups due to their
8
combined advantages over other carrier systems (Mehnert and Mader, 2001) such as
high drug loading, increased drug physical stability, controlled drug release,
possibility of drug targeting, and feasibility of lipophilic and hydrophilic drugs
incorporation. Moreover, SLN minimize the problems associated with other carriers
such as the toxicity of the carriers and difficulty for the large scale production and
sterilization. SLN have also been studied as carriers for anticancer drugs, and a
number of positive outcomes have been reported (Wong et al., 2007). SLN are capable
of improving the stability of cytotoxic compounds, improving pharmacokinetics and
drug biodistribution, and significantly enhancing the anticancer activity of
encapsulated drugs.
General ingredients of SLN are composed of solid lipid(s), emulsifier(s), and
water. The lipid matrix is generally made from physiological lipids (e.g., triglycerides,
glyceryl monostearate, stearic acid, and palmitic acid), which are biocompatible and
biodegradable. The emulsifier can be chosen depending on the route of administration,
which is more limited to parenteral administrations. There are several techniques used
for SLN preparation: high pressure homogenization using hot or cold homogenization
(Muller et al., 2000), emulsion/microemulsion-based technique (Gasco, 1993), solvent
emulsification and evaporation (Siekmann and Westesen, 1996; Sjostrom and
Bergenstahl, 1992), and more recently, supercritical fluid (SCF) technology has been
studied as an alternative method for drying protein formulation to produce solvent-free
particulate carriers (Almeida and Souto, 2007; Ribeiro Dos Santos et al., 2002).
9
1.2.2.1 Nanoparticles engineered from lecithin-in-water emulsions
The nanoparticles used in my research are composed of lecithin and
polysorbate 20 (Tween 20) and prepared via the emulsion precursor (Cui et al., 2006).
This technique provides several advantages. It avoids the use of organic solvent, the
high-torque mechanical mixing or the homogenization. The resultant nanoparticles
are uniform and reproducible. Lecithin is a complex mixture of phosphatides
consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol and other substances such as triglycerides and fatty acids, which
are components of cell membranes and regularly consumed as part of a normal diet
(Jimenez et al., 1990; Wade, 1994). It is also used extensively in pharmaceutical
applications as emulsifying, dispersing, and stabilizing agents and is included in
intramuscular and intravenous injectables and other parenteral nutrition formulations
(Lixin et al., 2006; Williams et al., 1984). In addition, lecithin is GRAS (generally
regarded as safe) listed and accepted in the FDA Inactive Ingredients Guide for
parenterals (e.g., 0.3-2.3% for intramuscular injection) (Wade, 1994). Tween 20 is a
polyoxyethylene derivative of sorbitan monolaurate. It is also GRAS listed and
included in the FDA Inactive Ingredients Guide for parenterals (Wade, 1994).
Therefore the lecithin-based nanoparticles are relatively safe and feasible as a carrier
for cytotoxic drug, docetaxel.
10
1.2.2.2 Nanoparticles engineered from solvent emulsification/evaporation
SLN can also be prepared by a precipitation method similar to the production
of polymeric nanoparticles. This method is different from other methods for SLN
preparation by the need of solvents. In general, a lipophilic drug is dissolved in a
water-immiscible organic solvent (e.g., chloroform, cyclohexane, or dichloromethane)
and is emulsified in an aqueous phase containing surfactant. After evaporation of the
solvent, nanoparticle dispersion is formed by precipitation of lipid in the aqueous
phase (Siekmann and Westesen, 1996; Sjostrom and Bergenstahl, 1992; Vitorino et
al., 2011; Zhu et al., 2009). The particles obtained from this method are small and
homogeneous.
1.3 Lipid based delivery systems for vaccines
Particulate delivery systems (e.g. emulsions, liposomes, micro- or nanoparticles, virosomes, and virus-like particles) have also been evaluated for vaccine
delivery for decades. These carriers have comparable size to the pathogens (Figure
1.2), therefore they are normally passive targeted and efficiently internalized by the
antigen-presenting cells (APCs) within the innate immune system (O'Hagan, 2001). It
is generally accepted that many particle carriers have adjuvant activity, which is likely
due to the particle‟s ability to facilitate the uptake of antigens by APCs. In addition,
enhancement of antigen stability, presentation of multiple copies of antigens, and the
11
ability to include immunomodulators are also the advantages of using particulates as a
vaccine delivery system (O'Hagan et al., 2006).
Figure 1.2 The sizes of pathogenic agents and adjuvant delivery systems (adopted
from Bachmann and Jennings, 2010). ISCOMs, immunostimulating complexes; VLPs,
virus-like particles.
1.3.1 Vaccines
Vaccination was first developed more than 200 years ago, and today it remains
the most effective strategy for preventing and controlling many infectious diseases
(Plotkin, 2005). Recently, vaccines are also been studied as immunotherapeutic agents
against non-communicable diseases such as hypertension, Alzheimer‟s disease,
obesity, cancer, and autoimmune diseases (Acres et al., 2004; Rohn and Bachmann,
12
2010). Several animal studies and clinical trials using these vaccines have
demonstrated them as affordable and effective therapeutic options for non infectious
diseases (Rohn and Bachmann, 2010). Vaccines can be classified into three general
categories: live, killed/inactivated, or subunit (Wilson-Welder et al., 2009).
Live vaccines contain modified live organisms that can replicate in the host or
infected cells and induce the most potent and lasting immune response in a fashion
similar to that elicited by the natural infection. In addition, these vaccines usually
induce both humoral immunity (antibodies) and cell mediate immunity (CMI) (Ellis,
2001). However, the major drawback of live vaccines is that they are capable of
reverting to virulent wild-types, especially in the immunosuppressed individuals, and
can cause severe adverse reactions ranging from simple headache or inflammation to
systemic disease (Huang et al., 2004; Wilson-Welder et al., 2009). This safety issue
limits the use of live vaccines and drives the development of safer vaccines.
In contrast to live vaccines, killed or inactivated vaccines are composed of
whole organisms that have been treated with either heat or chemicals and no longer
replicate in the host (Wilson-Welder et al., 2009). Killed or inactivated vaccines also
induce strong humoral immunity, but do not carry the same risks of reverting to a
virulent form as live vaccines. Generally, these vaccines require multiple doses for the
induction of long-term protective immunity (Ellis, 2001), and the induction of CMI by
killed or inactivated vaccines may be weak. However, killed or inacctivated vaccines
still contain cellular components of the pathogen and are highly reactogenic, which
13
can cause side effects such as fever, pain, redness and swelling at the site of injection
(Sidey et al., 1989). Due to the high potency of live or killed/inactivated vaccines, a
large proportion of currently licensed vaccines for human use are still based on these
vaccines (e.g., polio, smallpox, measles, mumps, rubella, rotavirus, rabies,
tuberculosis
(BCG),
and
Japanese
encephalitis
vaccines)
(http://www.fda.gov/BiologicsBloodVaccines/Vaccines/default.htm).
New approaches of vaccine development to overcome the safety problems of
traditional vaccines include subunit and nucleic acid-based vaccines. Subunit vaccines
contain only a portion of the organism that lack the components that trigger innate
immune recognition, e.g., bacterial DNA or lipopolysaccharides (LPS). Thus subunit
vaccines offer improved safety (Wilson-Welder et al., 2009). Subunit vaccines include
toxoids (inactivated bacteria toxins), recombinant protein subunits, synthetic peptides,
and protein polysaccharide conjugates (Singh and O'Hagan, 2002). Another type of
subunit vaccines being developed in recent years is plasmid DNA vaccine, which does
not include protein or other components of a pathogen (Wilson-Welder et al., 2009).
DNA vaccine contains the DNA sequence encoding the gene of the antigen of interest
from a pathogen. It can be taken up by cells and directs the synthesis of antigen(s) it
encodes (Seder and Gurunathan, 1999). It has been established that DNA vaccines
offer significant potential for the induction of cytotoxic T lymphocytes (CTL)
responses, which can kill cells infected with intracellular organisms (Liu, 1997; Wang
et al., 1998). CTL responses play an important role for the development of vaccine
14
against more challenging and difficult pathogens such as human immunodeficiency
virus (HIV), hepatitis C virus (HCV), tuberculosis, and malaria, as well as
immunotherapies for cancer (Sheng and Huang, 2011). Currently, there are several
DNA-based vaccines in clinical trials (e.g., Dengue virus, HIV, hepatitis B virus, and
melanoma cancer (Wilson-Welder et al., 2009). As mentioned earlier, subunit
vaccines have significant advantages over traditional vaccines. However, these
vaccines alone are often poorly immunogenic or nonimmunogenic and thus require
multiple doses and/or potent adjuvants to enhance the resultant immune responses
(Singh and O'Hagan, 2002). Therefore, new and improved vaccine adjuvants are being
actively sought to develop subunit vaccines that are safe and can induce long-lasting
immune responses.
1.3.2 Immunological adjuvants
Adjuvants are defined as substances that stimulate the immune system and
increase immune response to a co-administered vaccine (O'Hagan et al., 2001). The
three major functions of adjuvants are 1) to provide a “depot” for the antigen to allow
its slow release, 2) to facilitate the delivery and targeting of the antigen to immune
cells (APC), enhance antigen uptake, and activate the APC, and 3) to modulate and
enhance the type of immune responses induced by the antigen (e.g., antibody isotype
switching, stimulating CTL) (Wilson-Welder et al., 2009). The key issue in adjuvant
development is the safety profile of the adjuvants. Many experimental adjuvants have
15
been studied and shown high potency, but most have proven too toxic for clinical use,
such as water-in-oil emulsions (e.g., incomplete Freund‟s adjuvant, syntax adjuvant
formulation), and immunostimulatory molecules (e.g., lipopolysaccharides (LPS)
(Singh and O'Hagan, 2002; Wilson-Welder et al., 2009). In addition to safety issue,
other characteristics for successful adjuvants include stability, biodegradability, simple
synthetic pathway, cost, and compatibility with many different kinds of antigens
(Singh and O'Hagan, 2002). The first FDA approved adjuvant for human is aluminum
salts (commonly called alum), which were identified in the 1920s and are used in
vaccines across the world for more than 90 years (Clements and Griffiths, 2002).
Alum enhances the production of antibody immune response to traditional vaccines,
but not as effective for small peptide vaccines, recombinant proteins, and DNA-based
vaccines (Hunter, 2002). In addition, alum fails to stimulate CTL response and has
various limitations such as local reactions and the induction of hypersensitivity
(Baylor et al., 2002).
Currently, there are more information and understanding in the mechanisms of
action of adjuvants, which have been classified in different ways. The broadest
classification of adjuvants based on the mechanisms of action separates adjuvants into
two types: immune stimulation and vaccine delivery systems (Singh and O'Hagan,
2002). Immunostimulatory adjuvants are derived from pathogens that display
pathogen-associated molecular patterns (PAMPs) modules such as LPS and CpG
DNA, which activate cells of the innate immune systems. The activation of APC by
16
these modules through the Toll-like receptors (TLR) has been established as a key
regulator of both innate and adaptive immune responses (Pandey and Agrawal, 2006).
TLRs are type I transmembrane proteins that mediate the initial recognition of
microbial components and expressed in diverse cell types such as epithelial cells, B
cells, mast cells, natural killer (NK) cells, dendritic cells (DC), regulatory T cells,
macrophages, monocytes, neutrophils, basophils and endothelial cells (Hopkins and
Sriskandan,
2005).
Examples
of
immunostimulatory
adjuvants
include
monophosphoryl lipid A (MPL), unmethylated CpG dinucleotides, saponins, and
cytokines (Singh and O'Hagan, 2002). Another class of adjuvants based on
mechanisms of action is vaccine delivery systems. There is a proposed geographical
concept of immune reactivity, in which antigens that do not reach the local lymph
nodes do not induce immune responses (Zinkernagel et al., 1997). This concept
supports the adjuvanticity of some adjuvants that are particulate carriers. DCs that
circulate in peripheral tissues are thought to be the key cells, which are efficient for
antigen uptake and the transfer of antigens to lymph nodes. Thus, the uptake of
antigen by DCs, the trafficking of them to lymph nodes, and the maturation of DCs are
thought to be the key components to induce immune responses (Singh and O'Hagan,
2002). One of many important factors that influent adjuvant efficacy is the size of
vaccine delivery systems (Bachmann and Jennings, 2010). As shown in Figure 1.2, the
particle sizes of vaccine delivery systems should be within the range that allows the
most efficient uptake by APCs. In addition, particulates of 20 - 200 nm can efficiently
17
enter the lymphatic system and promote the trafficking of antigens. Various particulate
adjuvants have been evaluated for many years such as emulsions (e.g., MF59 and
montanide), microparticles, liposomes, immunostimulating complexes (ISCOMs) (a
mixture of cholesterol, phospholipids and Quillaja saponins), virosomes, virus-like
particles (VLPs), and nanoparticles.
Most recently, the classification of adjuvants based on the mechanisms of
action has been reclassified because of the observation that some delivery systems are
also immune stimulator (Seubert et al., 2008). There is an attempt to classify adjuvants
in terms of different generation (O'Hagan and De Gregorio, 2009). The first generation
adjuvants comprise of one component used as adjuvant in vaccines. Examples of first
generation adjuvants which are used and tested in human include alum, o/w emulsion
(MF59), liposomes, saponin (QS21), and polymeric microparticles (PLG) (O'Hagan
and De Gregorio, 2009). The second generation adjuvants, which comprise of more
than one adjuvant components, have been developed intensively. Generally, immune
stimulator components are added to the first generation adjuvants to increase antibody
titers or to induce a more potent T cell response. An example of successful second
generation adjuvants used in humans is the MPL co-adjuvanted with alum (AS04) ,
which has been licensed for human use as an adjuvant in human papillomavirus
vaccine
(HPV)
in
the
United
States
in
October,
2009
(http://www.fda.gov/BiologicsBloodVaccines/Vaccines/default.htm). Several other
18
second generation adjuvants tested in humans are also in phase I and II clinical trials
(O'Hagan and De Gregorio, 2009).
1.3.3 Liposomes as an adjuvant
Liposomes, a well-known lipid-based particulate delivery system, are spherical
vesicles composed of lipid bilayers separated by aqueous compartments. Liposomes
are prepared from phospholipids similar to cell membranes that make them
biocompatible and biodegradable, and therefore are generally regarded as nontoxic.
Liposomes can be used as a delivery system for a variety of molecules. Water soluble
substances such as drugs, proteins, nucleic acids, and dyes, can be encapsulated into
the aqueous compartments of the liposomes. For hydrophobic substances, they can be
entrapped in the lipid bilayers of liposomes. In addition, the advantages of liposomes
as a delivery system include the protection of the encapsulated molecules in the blood
stream, being capable of targeting the molecules to the site of action, and ease of
surface modification. Liposomes have been evaluated as vaccine adjuvant as well. The
adjuvant activity of liposomes was first reported by Allison and Gregoriadis in 1974
using diphtheria toxoid as an antigen (Allison and Gregoriadis, 1974). Since then the
adjuvant activity of liposomes has been extensively studied (Alving, 1991; Davis et
al., 1987; Gregoriadis and Panagiotidi, 1989; Jaafari et al., 2006; Richards et al.,
1998). Many parameters that may affect the adjuvant activity of liposomes have been
investigated. It was found that a variety factors such as particle size, surface charge,
19
lipid composition, and method of antigen loading, to name a few, in a liposome-based
vaccine formulation can potentially shape the resultant immune responses against the
antigen of interest (Latif and Bachhawat, 1984b).
Liposomes have been commonly used in combination with MPL or other
immunostimulators to enhance the induced immune responses. For example,
liposomes co-adjuvanted with MPL and QS21 (AS01) has been tested currently in
phase II clinical trials for malaria and tuberculosis (TB) vaccines (O'Hagan and De
Gregorio, 2009).
20
Chapter 2
NANOPARTICLES ENGINEERED FROM LECITHIN-IN-WATER
EMULSIONS AS A POTENTIAL DELIVERY SYSTEM FOR DOCETAXEL
Nijaporn Yanasarn, Brian R. Sloat, and Zhengrong Cui
International Journal of Pharmaceutics, 2009; 379: 174-180
21
2.1 Abstract
Docetaxel is a potent anti-cancer drug. However, there continues to be a need for
alternative docetaxel delivery systems to improve its efficacy. We reported the
engineering of a novel spherical nanoparticle formulation (~270 nm) from lecithin-inwater emulsions. Docetaxel can be incorporated into nanoparticles, and the resultant
docetaxel-nanoparticles were stable when stored as an aqueous suspension. The
release of docetaxel from nanoparticles was likely caused by a combination of
diffusion and Case II transport. The docetaxel-in-nanoparticles were more effective in
killing tumor cells in culture than free docetaxel. Moreover, docetaxel-nanoparticles
did not cause any significant red blood cell lysis or platelet aggregation in vitro, nor
did they induce detectable acute liver damage when injected intravenously into mice.
Finally, compared to free docetaxel, the intravenously injected docetaxel-nanoparticles
increased the accumulation of docetaxel in a model tumor in mice by 4.5-fold. These
lecithin-based nanoparticles have the potential to be a novel biocompatible and
efficacious delivery system for docetaxel.
2.2 Introduction
Recently, nanoparticles have gained much attention as a delivery system for
anticancer drugs, primarily due to their unique properties that can potentially improve
the efficacy of these drugs (Allen and Cullis, 2004). For example, nanoparticles may
be used to enhance the solubility of poorly-water soluble anti-cancer drugs and to
22
modify their pharmacokinetics (Gabizon et al., 2003). Nanoparticles may also be
engineered to reduce the uptake of the drugs by the reticuloendothelial system and / or
to target the drugs to specific tumor cells (Allen, 2002; Emerich and Thanos, 2006).
Additionally, there were data showing that nano-sized drug carriers can overcome the
multi-drug resistance in many cancer cells (Jabr-Milane et al., 2008).
Docetaxel is a semi-synthetic anticancer agent in the taxane class. It is potent
against many solid tumors such as breast cancer, non-small cell lung cancer, ovarian
cancer, and prostate cancer (Clarke and Rivory, 1999). In spite of its high efficiency,
side effects limit the clinical use of docetaxel. The current formulation of docetaxel
contains a non-ionic surfactant, polysorbate 80 (Tween 80), which requires further
dilution with 13% ethanol before addition to intravenous infusion solution. Adverse
reactions due to either the drug itself or the solvent system have been reported in
patients (e.g., hypersensitivity, fluid retention) (ten Tije et al., 2003). Therefore, many
alternative docetaxel formulations such as liposomes (Immordino et al., 2003),
polymeric nanoparticles (Hwang et al., 2008), micelles (Liu et al., 2008), and solid
lipid nanoparticles (Xu et al., 2009) have been investigated.
Previously, our laboratory reported the preparation of a nanoparticle formulation
from lecithin-in-water emulsions (Cui et al., 2006). Lecithin is a component of cell
membranes and is regularly consumed as part of a normal diet (Jimenez et al., 1990;
Wade, 1994). It is used extensively in pharmaceutical applications as emulsifying,
dispersing, and stabilizing agents and is included in intramuscular and intravenous
23
injectables and other parenteral nutrition formulations (Lixin et al., 2006; Williams et
al., 1984). In the present study, we investigated the feasibility of using the
nanoparticles as a carrier for docetaxel. Our data showed that docetaxel can be
incorporated in nanoparticles, and the docetaxel in the nanoparticles was more
effective in killing tumors cells in culture than free docetaxel. Our data also showed
that docetaxel-loaded nanoparticles were biocompatible and tended to increase the
accumulation of docetaxel in tumors pre-established in mice.
2.3 Materials and Methods
2.3.1 Materials
Docetaxel was purchased from LC Laboratories (Woburn, MA). Lecithin (soy,
refined) was from MP Biomedicals, LLC (Santa Ana, CA). The 1,2-dioleoyl-snglycero-3-phosphoethanoamine-N-[methoxy (polyethylene glycol)-2000] (DSPEPEG-2000)
and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-
(carboxyfluorescien) (FITC-DOPE) were from Avanti Polar Lipids, Inc (Alabaster,
AL). Sepharose
®
4B, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) kit, sodium dodecyl sulfate (SDS), and epinephrine were from SigmaAldrich (St. Louis, MO). Cellulose dialysis tubes (MWCO 50,000 Da) were from
Spectrum Chemicals & Laboratory Products (New Brunswick, NJ). Citrated whole
mouse blood was from Pel-Freez Biologicals (Rogers, AR). Alanine Aminotransferase
(ALT) kit was from Teco Diagnostic (Anaheim, CA).
24
2.3.2 Mice and cell lines
Female BALB/c and C57BL/6 mice, 6-7 weeks of age, were from Simonsen
Laboratories, Inc. (Gilroy, CA). The TC-1 cells (a mouse lung cancer cell line) and
PC-3 cells (a human prostate cancer cell line) were from American Type Culture
Collection (ATCC) and grown in RPMI 1640 medium supplemented with 10% fetal
bovine serum, 100 U/mL of penicillin (Invitrogen), and 100 g/mL of streptomycin
(Invitrogen).
2.3.3 Preparation of nanoparticles
Nanoparticles were prepared from emulsions (Cui et al., 2006). Briefly, 4 mg of
lecithin was weighed into a 7 mL glass scintillation vial. One mL of de-ionized,
filtered (0.2 μm) warm water was added into the vial, followed by heating on a hot
plate to 50 – 55 °C with stirring until a milky dispersion was formed. Tween 20 was
added in a step-wise manner to a final concentration of 2% (v/v). The milky dispersion
was stirred until a smooth, less opaque, but not clear system was formed, which was
then allowed to cool down to room temperature while stirring. The particle size was
determined by photon correlation spectroscopy (PCS) using a Coulter N4 Plus
Submicron Particle Sizer (Beckman Coulter Inc., Fullerton, CA).
To prepare docetaxel (DTX)-loaded nanoparticles (DTX-NPs), various amount
of docetaxel (50 to 250 μg) in chloroform was added into a glass vial containing 4 mg
of lecithin, and the chloroform was evaporated. The remaining steps were similar to
25
the preparation of the blank nanoparticles. Similarly, to incorporate DSPE-PEG-2000
into nanoparticles, DSPE-PEG-2000 (5% of the lecithin (m/m), or 10% where
mentioned) was added into a glass vial containing lecithin prior to the nanoparticle
preparation. Unloaded docetaxel was separated from nanoparticles using gel
permeation chromatography (GPC, Sepharose 4B, 6 x 150 mm). The eluted fractions
were analyzed for docetaxel by measuring the absorption at 230 nm.
Nanoparticles were freeze-dried using a FreeZone 6 Liter Freeze Dry System
(Labconco, Kansas City, MI). Dextrose (5%, w/v) was used as the lyoprotectant.
2.3.4 Transmission electron microscopy (TEM)
The size and morphology of the nanoparticles were examined using a
transmission electron microscope (TEM, Philips CM12 TEM/STEM) in the Oregon
State University Electron Microscope Facility as previously described (Cui and
Mumper, 2002b).
2.3.5 In vitro release of docetaxel from nanoparticles
Lyophilized docetaxel-loaded, PEG-2000-containing nanoparticles (DTX-PEGNPs, 10% of DSPE-PEG-2000, m/m) were suspended in 1 mL of de-ionized water
(final docetaxel concentration, 100 µg/mL) and placed into a 1 mL cellulose ester
dialysis tube (MWCO 50,000 Da). The tube was incubated in a 13 mL of release
medium (0.5% of SDS in PBS, 10 mM, pH 7.4) at 37°C under horizontal shaking. At
predetermined time points, the dialysis tube was taken out and was re-placed into a
26
new 13 mL fresh medium. In order to detect docetaxel in the release medium, the
docetaxel molecules have to be released from the nanoparticles and then diffuse across
the cellulose ester dialysis membrane. To make sure that the diffusion of the docetaxel
molecules across the membrane was not the rate-limiting step, docetaxel was
dissolved in Tween 80 (20 mg/mL) and diluted with 13% ethanol to the concentration
of 5 mg/mL. It was then diluted with de-ionized water to a final docetaxel
concentration of 100 μg/mL and placed into a dialysis tube as described above. The
amount of docetaxel released was determined by an HPLC method with the following
conditions: Zorbax Eclipse Plus® C18 column (150 mm x 4.6 mm, pore size 5 μm,
Aligent), the mobile phase: CH3CN:H2O (1:1, v/v), flow rate: 1.0 mL/min, and
measured wavelength: 230 nm (Xu et al., 2009).
2.3.6 The uptake of nanoparticles by tumor cells in culture
Nanoparticles were prepared without docetaxel to avoid its cytotoxicity. FITCDOPE was incorporated into the nanoparticles for fluorescent detection. TC-1 cells (5
× 105 cells/well) were seeded in a 24-well plate and incubated overnight at 37 °C, 5%
CO2. The cells were then incubated with 500 μL of FITC-labeled nanoparticles (FITCNPs) or FITC-labeled, PEG-2000-containing nanoparticles (FITC-PEG-NPs) (1.5
times dilution) at 4 °C or 37 °C for 2 h or longer. At 4 oC, the internalization of the
nanoparticles was inhibited, and thus, a comparison of the data at 4 °C and 37 °C was
informative of the internalization of the particles. After the incubation, the cells were
27
washed 3 times with cold PBS (10 mM, pH 7.4) and lysed with a lysis buffer (0.5%
Triton X-100). The fluorescence intensity was measured using a BioTek Synergy HT
Multi-Mode Microplate Reader (BioTek Instruments, Inc. Winooski, VT).
2.3.7 In vitro cytotoxicity assays
Cells were seeded in a 96-well plate (3,000/well) and incubated with the DTXPEG-NPs or free docetaxel dissolved in DMSO for 24 or 48 h. The final concentration
of the docetaxel in the cell culture medium was adjusted to 100 nM. At this
concentration, both the nanoparticles without docetaxel and the DMSO did not cause
any significant cell death. The number of surviving cells was determined using the
MTT assay (Le et al., 2008).
2.3.8 Biocompatibility of docetaxel-nanoparticles
The red blood cell (RBC) lysis assay was carried out as described with slight
modifications (Koziara et al., 2005). Citrated whole mouse blood was mixed at a 1:1
(v/v) ratio with water as a positive control or with normal saline as a negative control.
Freshly prepared DTX-PEG-NPs (50 μg/mL docetaxel) were diluted 1, 10, 100, or
1000-fold with 0.9% NaCl prior to mixing with the blood. Samples were incubated at
37 °C for 30 min in a shaker followed by centrifugation at 600 × g for 5 min to
remove the undamaged erythrocytes. Supernatant was collected and incubated at room
temperature for 30 min. Oxyhemoglobin was measured at 540 nm. To evaluate the
28
effect of the incubation time on the RBC lysis in the presence of the nanoparticles,
DTX-PEG-NPs (5 μg/mL docetaxel) were incubated with the blood for up to 6 h.
The platelet aggregation assay was also completed as previously described
(Oyewumi et al., 2004). Briefly, citrated whole mouse blood was centrifuged to obtain
platelet-rich plasma (PRP) and platelet-poor plasma (PPP) (Yang et al., 1999a).
Freshly prepared DTX-PEG-NPs (50 μg/mL docetaxel) were diluted and incubated for
10 min with the PRP containing 106 platelets in a final volume of 1 mL at 37 °C in an
incubator shaker. Platelet aggregation was measured at 500 nm. The absorptions of the
PRP, PPP, and the DTX-PEG-NPs alone were also measured as controls. Epinephrine
(10 mM) was used as a positive control.
2.3.9 In vivo plasma ALT determination
Healthy BALB/c mice were injected intravenously via the tail vein with normal
saline or the DTX-PEG-NPs (with 10 μg of docetaxel). Polyinosinic-cytidylic acid
(pI:C) (50 µg/mouse), which was previously shown to increase plasma ALT activity,
was used as a positive control. After 24 h, the mice were euthanized. Plasma ALT
activity was measured using the ALT kit. A high plasma ALT level is an indication of
liver damage. Generally, an ALT increase of 3 - 5-fold higher than that in the
untreated control mice is considered toxic.
29
2.3.10 The accumulation of docetaxel in tumors in mice
TC-1 cells were subcutaneously implanted in the flank of C57BL/6 mice (n = 4 5, 5 x 105 cells/mouse). Seventeen days after the implantation, the mice were injected
intravenously with free docetaxel or DTX-PEG-NPs (PEG-2000, 10% m/m, docetaxel,
1 mg per kg of body weight in 200 µL). Tumor samples were collected 4 h later and
homogenized. Docetaxel was extracted from the tumor samples with methanol and
quantified using HPLC as described above (Xu et al., 2009). The data were
normalized to the sample weight.
2.3.11 Data and statistical analyses
The in vitro release of the docetaxel from the PEG-2000-containing
nanoparticles (DTX-PEG-NPs) was modeled using the equation M = k tn, where M is
the % of total docetaxel released and t is time (Ritger and Peppas, 1987). The
parameter k is the kinetic constant, incorporating the structural and geometric
characteristics of the release system. From the value of exponential component (n), the
mechanism of the in vitro release was determined. The computer software program
MATLAB® (Natick, MA) was used to fit the data. Statistical analyses were completed
using ANOVA followed by the Fischer‟s protected least significant difference
procedure. A p-value of ≤ 0.05 (two-tail) was considered statistically significant.
30
2.4 Results and discussion
2.4.1 Preparation of nanoparticles from lecithin-in-water emulsions
Lecithin is an integral part of the cell membrane. It is a complex mixture of
phosphatides
consisting
of
phosphatidylcholine,
phosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol and other substances such as triglycerides
and fatty acids. It is GRAS (generally regarded as safe) listed and accepted in the FDA
Inactive Ingredients Guide for parenterals (e.g., 0.3 - 2.3% for intramuscular injection)
(Cui and Qiu, 2005). Tween 20 is a polyoxyethylene derivative of sorbitan
monolaurate. It is also GRAS listed and included in the FDA Inactive Ingredients
Guide for parenterals (Cui and Qiu, 2005). Thus, the nanoparticles were prepared from
emulsions using lecithin as the oil phase and Tween 20 as the surfactant. With 4
mg/mL of lecithin and 2% Tween 20, nanoparticles of around 270 nm were generated
(Fig. 2.1A). Docetaxel can be incorporated into the nanoparticles, and it seemed that
the incorporation of the docetaxel did not significantly affect the size of the resultant
nanoparticles (Figure 2.1A) (p = 0.12, ANOVA). The nanoparticles were spherical
(Figure 2.1B). As expected, the average size of the nanoparticles derived from the
micrograph was apparently smaller than that determined using the particle sizer
(photon correlation spectroscopy, PCS). The particle size from the PCS measurement
was the size of the particles plus an aqueous layer that surrounds the particles and
moves together with the particles. Data in Figure 2.1C showed that free docetaxel can
be separated from the nanoparticles using a Sepharose 4B column. When 50 µg/mL of
31
docetaxel was added during the nanoparticle preparation, close to 100% of the
docetaxel was incorporated into the nanoparticles (Figure 2.1D). When more
docetaxel was added into the preparation, an increasing amount of it was found in the
micelle fractions (Figure 2.1D, fractions 11 - 20). Therefore, the nanoparticles
prepared with 50 µg/mL of docetaxel were used for further study without purification.
Data from a short-term stability study showed that the nanoparticles, with or without
PEG-2000, were stable when stored at room temperature in an aqueous suspension
(Figure 2.2A). The polyoxyethylene chains of the Tween 20 on the surface of the
nanoparticles may have prevented the nanoparticles from aggregating. Similarly, no
aggregation was observed when the nanoparticles were co-incubated in a simulated
biological medium (10% fetal bovine albumin in PBS, 10 mM, pH 7.4) (data not
shown), suggesting that it is very unlikely that the nanoparticles will aggregate after
intravenous injection. Interestingly, the PEG-2000-containing nanoparticles, with or
without docetaxel, were smaller than their corresponding PEG-2000-free nanoparticles
(Figure 2.2A). We speculate that this may be due to the significant change in the
nanoparticle composition when the DSPE-PEG-2000 was included into the
nanoparticles. The nanoparticles were also successfully lyophilized using dextrose
(5%) as the lyoprotectant (Figure 2.2B). If needed, the final optimized docetaxelnanoparticle formulation may be stored long-term in a lyophilized form.
32
Figure 2.1 Preparation of DTX-NPs. (A) The size of nanoparticles prepared with
different concentrations of docetaxel (mean  S.D., n = 3). (B) The micrograph of the
nanoparticles prepared without docetaxel (NPs). (C) The gel permeation
chromatograph of DTX-NPs prepared with different concentrations of docetaxel.
DTX-NPs suspension was applied to Sepharose 4B column. The absorbance of the
elution fractions (0.25 mL) at 230 nm was measured. (D) Area under the curve (AUC)
of the GPC fractions 11 - 20 in C calculated by trapezoidal method.
33
(A)
Particle size (nm)
300
200
100
0
0
(B)
Figure 2.1
50 100 150 200 250
[DTX] (g/mL)
34
(C)
0.5
50
100
0.4
OD 230
150
200
0.3
250
0.2
0.1
0 0
5
10 15 20 25
Fraction of 0.25 m L
(D)
AUC (f11-20)
0.1
0
50
Figure 2.1 (Continued)
100 150 200
[DTX] (g/m L)
250
35
(A)
DTXPEG-NP
Day 6
PEG-NP
Day 0
DTX-NP
NP
0
100
200
Particle size (nm )
300
(B)
Particle size (nm)
300
p = 0.003
p = 0.62
200
100
0
DTX- FD, 5% FD, 0%
PEG-NP Dex
Dex
Figure 2.2 Nanoparticles were stable and lyophilizable. (A) Docetaxel nanoparticles
(DTX-NP) with or without PEG-2000 were freshly prepared and stored at room
temperature for 6 days. (B) The size of the lyophilized DTX-PEG-NPs was
comparable to that of the freshly prepared DTX-PEG-NPs (FD, freeze-dried; Dex,
dextrose). The values reported were mean  S.D. (n = 3).
36
2.4.2 In vitro release of docetaxel from nanoparticles
The release of the docetaxel from nanoparticles was slow (Figure 2.3). It took
3 days for 80% of the docetaxel to be released. The diffusion of the free docetaxel out
of the dialysis tube was much faster (Figure 2.3), demonstrating that the diffusion of
the docetaxel across the semi-permeable dialysis membrane was not rate-limiting.
When the first 60% of the release data of the DTX-PEG-NPs were fitted into the
equation of M = k tn, a n-value of 0.72 ± 0.20 (R2 = 0.9908) was obtained. Thus, it is
likely that both the diffusion of the docetaxel out of the nanoparticles and the process
of the erosion of the nanoparticles have influenced the docetaxel release (Ritger and
Peppas, 1987).
DTX
100
DTX-PEG-NP
% released
80
60
40
20
0
0
24
48
Tim e (h)
72
Figure 2.3 The release of docetaxel from DTX-PEG-NPs. The values reported were
mean  S.D. (n = 4).
37
2.4.3 The uptake of nanoparticles by tumor cells in culture
The uptake of the nanoparticles by tumor cells in vitro was studied using a model
mouse lung cancer cell line, the TC-1 cells. The nanoparticles were labeled with FITC
for fluorescent detection. As expected, after a 2 h incubation at 37 °C, the uptake of
the PEG-2000-containing nanoparticles (PEG-NPs) by the TC-1 cells was less than the
uptake of the PEG-2000-free nanoparticles (NPs) (Figure 2.4A). However, the uptake
of the PEG-NPs by the TC-1 cells was significantly increased by prolonging the
incubation time to 18 h (Fig. 2.4B). Clearly, the polyethylene glycol groups of the
Tween 20 and the DSPE-PEG-2000 do not have the same activity. When the TC-1
cells and the nanoparticles were incubated together at 4 °C, the amount of
nanoparticles associated with the TC-1 cells did not significantly increase even after a
prolonged incubation period (p = 0.132, ANOVA), indicating that at 37 °C, the
nanoparticles were internalized by the cells, not just simply bound on the cell surface.
2.4.4 In vitro cytotoxicity
The cytotoxicity of the docetaxel-loaded, PEG-2000-containing nanoparticles
(DTX-PEG-NPs) was evaluated using the TC-1 cells and PC-3 cells. After 24 h of
incubation, close to 100% of the TC-1 cells incubated with free docetaxel in DMSO
were still alive, while only about 60% of the TC-1 cells incubated with the DTX-PEGNPs were alive (Figure 2.5A).
38
Figure 2.4 The uptake of nanoparticles by TC-1 cells in culture. (A) The uptake of
NPs and PEG-NPs by TC-1 cells at 37 °C. Control was from cells incubated with
culture medium alone. Fluorescence intensity was determined after 2 h of incubation.
(B) The effect of the incubation time on the uptake of PEG-NPs by TC-1 cells. Cells
were incubated at 4 °C or 37 °C for up to 18 h. The three values at 4 oC (white bars)
were not different from one another (p = 0.13, ANOVA), whereas the three values at
37 oC were different from one another (p = 0.0003, ANOVA). Data reported were
mean  S.D. (n = 3).
39
(A)
Mean Fluo Unit
60
p = 0.04
40
20
0
Control
NP
PEG-NP
(B)
150
4C
Mean Fluo Unit
37 C
100
50
0
2
4
18
Incubation tim e (h)
Figure 2.4
40
The cell death generated by the DTX-PEG-NPs was very likely due to the
docetaxel in the nanoparticles, rather than the nanoparticles themselves, because the
same concentration of the docetaxel-free PEG-NPs failed to cause any significant cell
death (Figure 2.5A). Increasing the incubation time from 24 h to 48 h led to more cell
death (Figure 2.5B). Moreover, when the incubation time was extended to 48 h, death
was also detected in the cells treated with the free docetaxel (Figure 2.5B). A similar
trend was observed when the PC-3 cells were used (Figure 2.5C). Taken together, it
appeared that the docetaxel in the nanoparticle form was more effective in killing the
tumor cells than the free docetaxel. Our data in Figure 2.4 showed that the DTX-PEGNPs were taken up by the TC-1 cells, especially after a prolonged incubation period.
Thus, the improved cytotoxicity from the DTX-PEG-NPs may be due to the
nanoparticles‟ ability to facilitate or increase the uptake of the docetaxel by the tumor
cells.
2.4.5 Biocompatibility of docetaxel-nanoparticles
The docetaxel-nanoparticles were engineered for potential intravenous
infusion. Thus, it is important to evaluate the biocompatibility of the nanoparticles
because the nanoparticles may interact with the erythrocytes and cause anemia,
jaundice, and other pathological conditions (De Jong and Borm, 2008; Zbinden et al.,
1989). An evaluation of the compatibility of our docetaxel-nanoparticles (DTX-PEGNPs) with blood was carried out in vitro.
41
Figure 2.5 Docetaxel in the nanoparticles was more effective in killing tumor cells in
culture. (A) In vitro cytotoxicity against TC-1 cells. DMSO alone and DTX-free PEGNPs were used as controls. (B) The effect of the incubation time on the cytotoxicity of
the DTX-PEG-NPs and free DTX in TC-1 cells (24 h vs. 48 h, p = 0.01 for the free
DTX; p = 0.005 for the DTX-PEG-NP). (C) In vitro cytotoxicity against PC-3 cells.
Data reported were mean  S.D. (n = 3).
42
(A)
140
w /o DTX
p = 0.09
p = 0.009
120
% TC-1 cells alive
w / DTX
100
80
60
40
20
0
DMSO
PEG-NP
DTX form ulation
(B)
120
% TC-1 cells alive
100
80
60
40
DTX
20
DTX-PEG-NP
0
24 h
48 h
Incubation tim e
Figure 2.5
43
(C)
60
DTX
DTX-PEG-NP
p = 0.0008
p = 0.08
% PC-3 died
50
40
30
20
10
0
24 h
48 h
Incubation tim e
Figure 2.5 (Continued)
Incubation of the blood with an equal volume of the freshly prepared DTXPEG-NPs (50 μg/mL docetaxel) resulted in a slight RBC lysis as compared to the
osmotic cell lysis caused by water (Figure 2.6A). At the dilution of 10, 100, or 1000fold, no significant RBC lysis was observed after 30 min of incubation (Figure 2.6A).
No significant RBC lysis was detectable even after the DTX-PEG-NPs (diluted 10fold) were incubated with the blood for up to 6 h (Figure 2.6B). In fact, there was not a
significant difference in the RBC lysis values between the DTX-PEG-NPs and the
normal saline at all time points examined (Figure 2.6B). A platelet aggregation assay
was also carried out in the presence of the DTX-PEG-NPs at various dilutions (starting
from 50 μg/mL DTX). Data in Fig. 2.6C showed that the DTX-PEG-NPs at the
44
dilutions of 10, 100, or 1000-fold did not cause any significant platelet aggregation.
Finally, no significant change in the plasma ALT level was observed 24 h after the
DTX-PEG-NPs were injected intravenously into healthy mice (Figure 2.6D), which is
another indication of the potentially favorable biocompatibility profile of our
docetaxel-loaded nanoparticles.
2.4.6 Nanoparticles increased the accumulation of docetaxel in tumors in mice
Finally, to preliminarily investigate whether the DTX-PEG-NPs can improve the
accumulation of the docetaxel into tumors in vivo, the DTX-PEG-NPs were injected
intravenously into C57BL/6 mice with pre-established TC-1 tumors. Four hours after
the injection, the amount of docetaxel recovered from the tumors in mice injected with
the DTX-PEG-NPs was 3.38 ± 1.94 µg/g of tumor (mean ± S.D., n = 5), 4.5-fold
higher than that in mice injected with the free docetaxel (0.76 ± 0.17 µg/g of tumor, n
= 4). Another number in the free docetaxel group was identified as an outlier using the
Grubb‟s extreme studentized deviate (ESD) method. The data suggested the feasibility
of using the nanoparticles to target the docetaxel into solid tumors.
45
Figure 2.6 Nanoparticles have a favorable biocompatibility profile. (A) DTX-PEGNPs (diluted by 1, 10, 100, or 1000-fold) were incubated with mouse blood for 30 min
at 37°C. The % RBC lysis was determined. Docetaxel in ethanol and Tween 80 (E/T)
preparation was included as a control. * The value of the 1 x group is different from
that of the saline (p = 0.001) and that of the E/T (10 x) (p = 0.0003). (B) The effect of
the incubation time on the RBC lysis in the presence of the DTX-PEG-NPs.
Nanoparticles were diluted 10-fold. (C) The effect of DTX-PEG-NPs on platelet
aggregation. DTX-PEG-NPs (50 μg/mL DTX) at various dilutions were incubated
with mouse platelet-rich plasma, and the platelet aggregation was measured.
Epinephrine (Epi) was used as a positive control. ** The value of the 1 x group is
different from those of the other dilutions (p = 0.019, ANOVA). (D) DTX-PEG-NPs
did not cause any significant change in mouse plasma ALT level. DTX-PEG-NPs (10
µg of DTX/mouse) was i.v. injected into mice and the plasmid ALT level was
measured 24 h later. The pI:C (50 µg/mouse) was used as a positive control. The
values from the Control (normal saline) and DTX-PEG-NP were comparable (p =
0.84). *** The value of the pI:C was different from that of the other two (p = 0.014,
ANOVA). All data reported were mean ± S.D. (n = 3).
46
(A)
E/T 10x
Dilution factor
1000 x
100 x
10 x
1x
*
Saline
0
20
40 60 80 100
% RBC lysis
(B)
4.5
Saline
% RBC lysis
DTX-PEG-NP
3
1.5
0
30
Figure 2.6
120
180
Tim e (m in)
360
47
(C)
Epi
Dilution factor
1000 X
100 X
10 X
1X
**
0
0.05
0.1
OD 500 nm
0.15
(D)
300
Plasma ALT (IU/L)
***
200
100
0
Control
Figure 2.6 (Continued)
DTXPEG-NP
pI:C
48
2.5 Conclusions
We have engineered a new docetaxel formulation by incorporating the docetaxel
into a lecithin-based nanoparticle carrier. The docetaxel in nanoparticles was more
effective in killing tumor cells in culture. The nanoparticles were shown to be
biocompatible and may be used to target the docetaxel into solid tumors to improve
the resultant anti-tumor activity.
49
Chapter 3
ENHANCED IN VITRO AND IN VIVO ANTITUMOR ACTIVITY OF
DOCETAXEL BY SOLID LIPID NANOPARTICLE CARRIER
Nijaporn Yanasarn and Zhengrong Cui
In preparation for the manuscript
50
3.1 Abstract
An alternative docetaxel formulation, a solid lipid nanoparticle of docetaxel,
was prepared by solvent evaporation/emulsification method. It was comprised of
biocompatible and biodegradable component, lecithin. The resultant docetaxelnanoparticles with the particle size about 120 nm were stable when stored as an
aqueous dispersion and were compatible with physiological environment. The in vitro
release study of docetaxel-nanoparticles revealed that about 80% of docetaxel was
released from nanoparticles within 48 h and the release of docetaxel from the
nanoparticles was likely caused by a combination of diffusion and erosion of the
particles. The docetaxel-nanoparticles were more cytotoxic against tumor cells in
culture than the commercial docetaxel formulation. In vivo pharmacokinetics and
antitumor activity in mice after intravenous injection of docetaxel-nanoparticles and
docetaxel solution were comparable. However, the in vivo tumor uptake data showed
that docetaxel in nanoparticles increased the accumulation of docetaxel in tumors by
1.8-fold compared with docetaxel solution. Moreover, oral administration of
docetaxel-nanoparticles showed a stronger antitumor activity than docetaxel in
solution in tumor bearing mice models. These results present the feasibility of this
docetaxel-nanoparticle formulation as an alternative delivery system for docetaxel.
51
3.2 Introduction
Docetaxel is a semi-synthetic compound belonging to the taxane family of
anticancer drugs, which is produced from 10-deacethylbaccatin-III found in the
European yew tree (Gelmon, 1994). The antitumor activity of docetaxel is due to the
promoting of the polymerization of microtubules, which leads to the disruption of
mitosis, cell cycle arrest at G2/M, and cell death (Schiff et al., 1979). Docetaxel has a
significant antitumor activity against various human malignancies and is approved by
U.S. FDA (Food and Drug Administration) for the treatment of breast cancer, ovarian
cancer, non-small-cell lung cancer, and prostate cancer. However, due to its highly
lipophilicity and practically insolubility in water (3 µg/mL) (Engels et al., 2007), the
current formulation of docetaxel contains a non-ionic surfactant, polysorbate 80
(Tween 80), which requires further dilution with 13% ethanol before addition to
intravenous infusion solution. Adverse reactions due to either the drug itself (e.g.,
neurotoxicity, musculotoxicity, and neutropania) (Persohn et al., 2005) or the solvent
system (e.g., hypersensitivity and fluid retention) (ten Tije et al., 2003) have been
reported.
Several nanoscale carriers have been evaluated as delivery systems for
anticancer drugs such as micelles, liposomes, polymer-based nanocarriers, and lipidbased nanocarries. These nanoscale delivery systems have unique properties that can
potentially improve the efficacy and minimize the toxicity of anticancer drugs (Allen
and Cullis, 2004). They provide several advantages including drug solubility
52
enhancement, controlled drug release, capable of passive and active tumor targeting,
and overcoming the multi-drug resistance (Vijayaraghavalu et al., 2007). Among the
various drug delivery systems, the solid lipid nanoparticles (SLN) has gained attention
from many research groups due to their combined advantages over other carrier
systems (Mehnert and Mader, 2001) such as high drug loading, increased drug
physical stability, controlled drug release, possibility of drug targeting, and feasibility
to the incorporation of lipophilic and hydrophilic drugs.
Currently, oral chemotherapy has gained much attention from many
researchers. Oral chemotherapy can provide a prolonged and continuous exposure of
the cancer cells to anticancer drugs, resulting in much better efficacy and fewer side
effects. Moreover, oral administration is convenient and preferred by the patients
(DeMario and Ratain, 1998; Terwogt et al., 1999). However, low oral bioavailability
is a major obstacle for many anticancer drugs that are very hydrophobic and waterinsoluble. One of possible solutions for oral delivery of anticancer drugs is to apply
the multidrug efflux pump P-glycoprotein (P-gp) inhibitors. Many studies showed that
co-administration of the P-gp inhibitors such as cyclosporine A and ritonavir could
increase the oral bioavailability of anticancer drugs (Bardelmeijer et al., 2002;
Malingre et al., 2001a; Malingre et al., 2001b; Malingre et al., 2000). However, P-gp
inhibitors also suppress the immune system and thus may lead to more side effects.
In the present study, we formulated the docetaxel-nanoparticles, which mainly
consisted of biodegradable and biocompatible components, soy lecithin and sodium
53
deoxycholate. Docetaxel-nanoparticles (DTX-NPs) were prepared by solvent
emulsification/evaporation method, and the physiochemical characteristics of DTXNPs were determined. In vitro uptake and cytotoxicity were evaluated on a mouse
lung cancer cell line (TC-1) and other human cancer cell lines, PC-3 (a human prostate
cancer cell line), A431 (a human epidermal squamous cancer cell line), and MDAMB-468 (a human breast cancer cell line). The pharmacokinetics, tumor uptake, and
antitumor activity of DTX-NPs were investigated in mice. We also evaluated the
feasibility of using docetaxel-nanoparticles for docetaxel oral delivery. The oral
administration of docetaxel is still limited because of its low oral bioavaiability (< 5%
in mice) (Kuppens et al., 2005), which is due to its poorly solubility and its high
affinity to the P-gp (Wils et al., 1994). The use of docetaxel in nanoparticles may
result in an improvement of oral bioavailability and increase of the efficacy.
3.3 Materials and Methods
3.3.1 Materials
Docetaxel was purchased from LC Laboratories (Woburn, MA). Lecithin (soy,
refined) was from MP Biomedicals, LLC (Santa Ana, CA). The 1,2-dioleoyl-snglycero-3-phosphoethanoamine-N-[methoxy (polyethylene glycol)-2000] (DSPEPEG-2000) was from Avanti Polar Lipids, Inc (Alabaster, AL). 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Sodium deoxycholate,
and sodium dodecyl sulfate (SDS) were from Sigma-Aldrich (St. Louis, MO).
54
3.3.2 Mice and cell lines
Female C57BL/6 mice, 6 - 8 weeks of age, were from Charles River
Laboratories (Wilmington, MA). Culture medium, fetal bovine serum (FBS), and
antibiotics were from Invitrogen (Carlsbad, CA). TC-1 cells (a mouse lung cancer cell
line) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum,
100 U/mL of penicillin, and 100 g/mL of streptomycin. PC-3 cells (a human prostate
cancer cell line), A431 cells (a human epidermal squamous cancer cell line), and the
MDA-MB-468 cells (a human breast cancer cell line) were from American Type
Culture Collection (ATCC) and grown in DMEM medium supplemented with 10%
fetal bovine serum, 100 U/mL of penicillin, and 100 g/mL of streptomycin.
3.3.3 Preparation and characterization of docetaxel-nanoparticles
Docetaxel-nanoparticles
(DTX-NPs)
were
prepared
from
solvent
emulsification/ evaporation method. Briefly, lecithin, DSPE-PEG-2000, and docetaxel
(2 mg) were dissolved in dichloromethane and mixed well. An aqueous phase
containing sodium deoxycholate was added to the organic phase and homogenized
(Ultra-Turrax, IKA, Wilmington, NC) at 15,000/min for 5 min to obtain a
homogenous emulsion. The organic solvent was then evaporated by heating in a water
bath (75 °C) under magnetic stirring. The DTX-NPs in aqueous dispersion was formed
when cooled down to room temperature.
55
The size, size distribution, and zeta potential of DTX-NPs were measured by
photon correlation spectroscopy (PCS) (Zetasizer, Malvern Instruments Ltd.,
Worcestershire, England) after the dispersion was diluted in de-ionized water (50
times). The concentration of docetaxel was determined by HPLC after the appropriate
dilution with methanol.
3.3.4 Transmission electron microscopy (TEM)
The size and morphology of the nanoparticles were examined using TEM (FEI
Tecnai Transmission Electron Microscope) in the Institute for Cellular and Molecular
Biology Microscopy and Imaging Facility at The University of Texas at Austin. A
carbon-coated 400-mesh copper specimen grid (Ted Pella, Inc., Redding, CA) was
glow-discharged for 2 min. DTX-NP dispersion deposition on the grid and uranyl
acetate staining were completed as described previously (MacLaughlin et al., 1998).
3.3.5 In vitro stability of docetaxel-nanoparticles
The short-term stability of DTX-NPs was monitored at 4 °C for a period of 7
days. The size, size distribution, and zeta potential were measured over time. The
stability in simulated biological medium (10% FBS in phosphate buffered saline, PBS,
0.01 M, pH 7.4) was also determined after incubation for 30 min at 37 °C.
56
3.3.6 In vitro release of docetaxel from nanoparticles
The dialysis method was used to monitor the release of docetaxel from
nanoparticles. One milliliter of DTX-NPs (diluted in PBS at the final docetaxel
concentration of 200 µg/mL) was placed in the cellulose ester dialysis device (MWCO
50 KDa) (Spectrum Laboratories, Inc., Rancho Dominguez, CA). The device was
incubated in a 13 mL of release medium (0.5% of SDS in PBS, 10 mM, pH 7.4) at 37
°C under horizontal shaking (150 rpm). At predetermined time points (0.5, 1, 2, 4, 6,
10, 25, and 48 h), the dialysis tube was taken out and was re-placed into a new 13 mL
fresh medium to provide the sink condition. The amount of docetaxel released in the
medium was determined by HPLC after two-time dilution in methanol.
3.3.7 In vitro uptake of the nanoparticles by tumor cells
TC-1 cells (5 × 105 cells/well) were seeded in a 6-well plate and incubated
overnight at 37 °C, 5% CO2. The medium was then replaced with 3 mL medium
containing 1 µM of docetaxel in DTX-NPs or in the Taxotere®, docetaxel solution
(DTX). Docetaxel solution was prepared by dissolving docetaxel in Tween 80 (40
mg/mL), which was diluted with 13% (w/w) ethanol to the concentration of 10 mg/mL
and then further diluted with the medium. Cells were incubated at 37 °C, 5% CO2 for
0.5 and 4 h. The medium was removed and cells were washed three times with cold
PBS. Cells were collected using cell scraper, and docetaxel was extracted with 0.5 mL
acetonitrile by vigorous mixing for 2 min. After centrifugation at 14,000 rpm for 10
57
min, the clear supernatant was collected and evaporated under vacuum at 37 °C
(CentriVap, Labconco Corporation, Kansas City, MO). The residue was then mixed
with 0.1 mL of methanol for 2 min. The solution was centrifuged at 14,000 rpm for 10
min, and 80 µL of the supernatant was analyzed by HPLC.
3.3.8 In vitro cytotoxicity assays
Cells were seeded in 96-well plates at a density of 3,000 cells per well and
incubated at 37 °C in a humidified atmosphere with 5% CO2 until 80% confluence
was reached. The medium was then replaced with 200 µL medium with an equivalent
dose of docetaxel in DTX-NPs or in docetaxel solution (DTX). Nanoparticles without
docetaxel (NPs) or Tween 80/ethanol vehicle (T80/EtOH) were used as controls. One
row of plate was used as reference, which was incubated with 200 µL culture medium
only. After 24, 48, or 72 h incubation, the medium was replaced with 200 µL fresh
culture medium, and 20 µL of MTT solution (5 mg/mL) was added. Cells were
incubated for an additional 3 h. After the removal of the MTT containing medium, 200
µL of dimethyl sulphoxide (DMSO) was added to dissolve the crystals formed by the
living cells. The absorbance was measured at 570 nm using a BioTek Synergy HT
Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT). Cell
viability was determined by the following formula: % cell viability = (Ab test
Abreference cells) × 100%.
cells
/
58
3.3.9 Pharmacokinetics studies
All animal studies were carried out following the National Institutes of Health
guidelines for animal care and use. Animal protocols were approved by the
Institutional Animal Care and Use Committee at the University of Texas at Austin.
Female C57BL/6 mice were randomly assigned to two groups (n = 18) and injected
intravenously through the tail vein with DTX-NPs diluted in 5% dextrose or docetaxel
solution diluted in normal saline (0.9% NaCl solution) at the docetaxel dose of 10
mg/kg mouse. In each group, mice were sacrificed at 5, 15 min, 1, 2, 4, and 6 h after
drug administration (n = 3 at each time point). Blood samples were put into tubes
containing heparin (33 IU) and centrifuged at 8000 ×g for 10 min to obtain plasma
samples. Plasma samples were then transferred to new tubes and stored at -80 °C until
analyzed by HPLC.
Plasma samples were extracted with ethyl ether prior to analyze. Briefly, 100
µL of plasma was spiked with 20 µL of paclitaxel (20µg/mL in methanol) as the
internal standard. One milliliter of ethyl ether was added and vortexed for 5 min. After
a centrifugation at 14,000 rpm for 10 min, the organic phase was collected and dried
under vacuum at 37 °C. The residue was then dissolved in 0.1 mL methanol and
mixed for 2 min. The solution was centrifuged at 14,000 rpm for 10 min, and 80 µL of
the supernatant was analyzed by HPLC.
59
3.3.10 In vivo tumor uptake study
Female C57BL/6 mice bearing mouse lung cancer (TC-1) were randomly
assigned to two groups (n = 5 or 6) and injected intravenously through the tail vein
with DTX-NPs or docetaxel solution (DTX) at the docetaxel dose of 10 mg/kg mouse.
Mice were scarified at 12 h after later. Tumors were collected, washed with PBS, and
dried on tissue paper. Tumor samples were stored at -80 °C until analyzed by HPLC.
About 100 mg tumor was homogenized using the bead beater (Biospec Products, Inc.,
Bartlesville, OK) at 4,800 rpm for 2 min. The homogenized samples were then spiked
with paclitaxel and extracted similar to plasma samples except that acetronitrile was
used instead of ethyl ether.
3.3.11 In vivo therapeutic study
Antitumor activity was evaluated in mice by parenteral or oral route of
administration. Female C57BL/6 mice were subcutaneously injected in the flank with
a suspension of TC-1 cells at the concentration of 5 × 105 cells/150 µL. Tumor were
allowed to grow approximately to a volume of 50 – 70 mm3. Mice were randomly
assigned to four groups (n = 5 – 7): control (PBS or normal saline), nanoparticles
without docetaxel (NPs), docetaxel solution (DTX), or DTX-NPs. For parenteral
route, mice were administered on day 5 and 10 after tumor implantation at 20 mg
docetaxel/kg body weight by intravenous injection through the tail vein. For oral
gavage, mice were treated on day 9 and 12 at the docetaxel dose of 10 mg/kg through
60
a blunt needle that was passed down the esophagus into the stomach. The body weight
and tumor size were monitored. Tumor size and tumor volume were calculated based
on the following equation: tumor size (mm) = ½ (width + length) and tumor volume
(mm3) = ½ [length × (width)2], respectively.
3.3.12 HPLC analysis of docetaxel
The concentration of docetaxel was determined by HPLC method. Samples
were directly injected (20 µL) into the HPLC system after appropriate dilution with
methanol. The HPLC system (1260 Infinity, Agilent, Santa Clara, CA) was used as
following
experimental
conditions;
column:
Zorbax
Eclipse
Plus
C18
(150 mm x 4.6 mm i.d., pore size 5 μm, Agilent), mobile phase: acetonitrile:water
(50:50 v/v), flow rate: 1 mL/min, and measured wavelength: 230 nm (Xu et al., 2009).
For the plasma and tissue samples, the mobile phase was acetonitrile:water (53:47
v/v).
3.3.13 Data and statistical analysis
The in vitro release of the docetaxel from the nanoparticles was modeled using
the equation M = ktn, where M is the % of total docetaxel released and t is time (Ritger
and Peppas, 1987). The parameter k is the kinetic constant, incorporating the structural
and geometric characteristics of the release system. From the value of exponential
component (n), the mechanism of the in vitro release was determined. The computer
61
software program GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA) was
used to fit the data.
Pharmacokinetic parameters were determined using WinNonlin® Professional
Version 2.1 from Pharsight Corporation (Mountain View, CA) using noncompartmental modeling (NCA Model 201) and the linear trapezoidal method.
Statistical analyses were completed using ANOVA followed by Fisher‟s protected
least significant difference procedure. A p-value of ≤ 0.05 (two-tail) was considered
statistically significant.
3.4 Results and discussion
3.4.1 Preparation and characterization of docetaxel-nanoparticles from solvent
emulsification/evaporation
Docetaxel-nanoparticles were prepared by solvent emulsification/evaporation
method comprised of soy lecithin and sodium deoxycholate as lipid and surfactant,
respectively. Lecithin is an integral part of the cell membrane. It is a complex mixture
of phosphatides consisting of phosphatidylcholine, phosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol and other substances such as triglycerides
and fatty acids. It is GRAS (generally regarded as safe) listed and accepted in the FDA
Inactive Ingredients Guide for parenterals (e.g., 0.3 - 2.3% for intramuscular injection)
(Wade, 1994). Sodium deoxycholate (bile salt) is also a natural surfactant which is
approved for parenteral product (Liu, 2008). Docetaxel-nanoparticles prepared with
this method were spherical (Figure 3.1A) with a particle dimension of 120 nm and zeta
62
potential of -53.67 ± 3.46 mV (Figure 3.1B). Data from a short-term stability study
showed that the DTX-NPs were stable for at least 7 days when stored at 4 °C as an
aqueous dispersion (Figure 3.1B). The particle size and zeta potential did not change
significantly during the storage period (p > 0.9, ANOVA). The negatively charged
phospholipids components in lecithin and the anionic surfactant, sodium deoxycholate,
result in the high negative zeta potential of the particles, which may help prevent the
aggregation due to electric repulsion and it has been known that dispersions with zeta
potential above 30 mV (absolute value) are physically stable (Muller et al., 2001).
Similarly, no aggregation was observed when the nanoparticles were co-incubated in a
simulated biological medium (10% FBS in PBS, 10 mM, pH 7.4) (Figure 3.1C),
suggesting that it is very unlikely that the nanoparticles will aggregate after
intravenous injection.
3.4.2 In vitro release of docetaxel from the nanoparticles
The in vitro release profile of docetaxel from the DTX-NPs was shown in
Figure 3.2. Docetaxel slowly released from nanoparticles, and about 80% of docetaxel
was released within 48 h. When the first 60% of the release data were fitted into the
equation of M = ktn, a n-value of 0.55 ± 0.02 (R2 = 0.9940) was obtained. Thus, it is
likely that both the diffusion of the docetaxel out of the nanoparticles and the process
of the erosion of the nanoparticles have influenced the docetaxel release (Ritger and
Peppas, 1987).
63
Figure 3.1 Preparation and characterization of solid lipid docetaxel-nanoparticles
(DTX-NPs). (A) The transmission electron micrograph of DTX-NPs. (B) The particle
size and zeta potential of DTX-NPs after a short-term storage at 4°C (mean  S.D., n =
3). (C) Overlaying of the dynamic light scattering spectra of DTX-NPs after coincubated in a simulated biological medium (10% FBS in PBS) for 0.5 h.
64
(A)
(B)
140
-20
100
80
-40
60
40
-60
20
0
-80
0 min
1h
24 h
Time
Figure 3.1
48 h 7days
Zetapotential (mV)
Particle diameter (nm)
120
65
(C)
FBS
DTX-NPs dispersion
0 min incubation
30 min incubation
Figure 3.1 (Continued)
Accumulative release (%)
100
80
60
40
20
0
0
10
20
30
40
50
60
Time (h)
Figure 3.2 In vitro release profile of docetaxel from docetaxel-nanoparticles. The
values reported were mean  S.D. (n = 3).
The use of dialysis membrane was to eliminate the possibility of the dispersion
of nanoparticles in the release medium, and since the molecular weight cut off was 50
KDa, corresponding to 6-7 nm, (http://www.spectrumlabs.com/dialysis/PoreSize.html)
66
it is unlikely that the nanoparticles could penetrate out of the membrane. Thus the
release of docetaxel into the release medium likely represented the actual release of
docetaxel from the nanoparticles.
3.4.3 In vitro uptake of docetaxel by tumor cells
The uptake of the nanoparticles by tumor cells in vitro was studied using a
model mouse lung cancer cell line, the TC-1 cells. After cells were incubated with
DTX-NPs or docetaxel solution (DTX) for 0.5 or 4 h, the amount of docetaxel taken
up by the cells was determined by HPLC. The amount of docetaxel in cells incubated
with docetaxel in solution was about 2-fold higher than in cells incubated with DTXNPs (Figure 3.3). Free docetaxel may transport across the cell membrane through
diffusion pathway due to its highly lipophillic property, while the docetaxel in
nanoparticles may enter the cells via the endocytosis, which is an energy-dependent
process. In addition, other factors may include the electrostatic repulsion forces
between the negatively charged particles and the cell membranes, and the pegylation
of the nanoparticles.
3.4.4 In vitro cytotoxicity
The cytotoxicity of docetaxel-nanoparticles (DTX-NPs) was evaluated by
using MTT assay in four different tumor cell lines, TC-1, PC-3, A431 and MDA-MB468.
67
80
DTX
Docetaxel content (ng)
p = 0.001
DTX-NP
p < 0.001
60
40
20
0
0.5
4
Incubation time (h)
Figure 3.3 Uptake of docetaxel in nanoparticles (DTX-NPs) or docetaxel in solution
(DTX) by TC-1 cells in culture (docetaxel equivalent to 1 µM) at 37 °C. Comparison
between 0.5 h and 4 h revealed the p values of 0.17 and 0.02 for DTX and DTX-NPs,
respectively. Data reported were mean  S.D. (n = 3).
After 48 h of incubation, at the docetaxel concentration of 10 nM or higher, the
cytotoxicity of DTX-NPs and docetaxel solution (DTX) was observed in the TC-1
cells (Figure 3.4A). At low docetaxel concentration (10 nM), the viability of the TC-1
cells treated with DTX-NPs was lower than treated with DTX (p = 0.046). Similar
results were observed with other three cell lines (Figure 3.4B) at the same docetaxel
concentration after 48 h of incubation for A431 cells and 72 h of incubation for PC-3
cells and MDA-MB-468 cells. The effect of incubation time on the cytotoxicity of PC3 cells and MDA-MB-468 cells were shown in Figure 3.4C and 3.4D, respectively.
Increasing the incubation time from 24 h to 72 h led to more cell death.
68
Figure 3.4 The docetaxel in nanoparticles was more effective than in solution in
killing tumor cells in culture. (A) In vitro cytotoxicity against TC-1 cells.
Nanoparticles without docetaxel (NPs) and Tween 80/ethanol vehicle (T80/EtOH)
were used as controls. Cells were incubated at different docetaxel concentration for 48
h, and cell viability was determined using MTT assay. *, p = 0.046 and **, p = 0.036
compared with DTX. (B) In vitro cytotoxicity against PC-3, A431, and MDA-MB-468
cells. Cells were treated at the docetaxel concentration of 10 nM for 48 h (A431) or 72
h (PC-3 and MDA-MB-468). (C, D) The effect of the incubation time on the
cytotoxicity of the DTX-NPs and DTX in PC-3 cells (C) and MDA-MB-468 cells (D).
Data reported were mean  S.D. (n = 3).
69
(A)
120
T80/EtOH
NP
DTX
DTX-NP
TC-1 cell viability (%)
100
80
60
*
40
**
20
1
10
100
Docetaxel concentration (nM)
1000
(B)
60
DTX
Cell viability (%)
p = 0.02
DTX-NP
p = 0.01
p = 0.01
40
20
PC-3
Figure 3.4
A431
MDA-MB-468
70
(C)
80
PC-3 Cell viability (%)
DTX
DTX-NP
60
40
20
24h
48h
Incubation time
72h
(D)
MDA-MB-468 Cell viablity (%)
100
DTX
DTX-NP
80
60
40
20
24h
Figure 3.4 (Continued)
48h
Incubation time
72h
71
Taken together, it appeared that docetaxel in nanoparticle form was more
effective in killing the tumor cells than docetaxel solution. Other mechanisms rather
than enhancing the docetaxel uptake by tumor cells might have an effect on the
cytotoxicity of docetaxel-nanoparticles because it was shown that the tumor cells took
up docetaxel in nanoparticles less efficiently than docetaxel in solution (Figure 3.3).
3.4.5 Pharmacokinetics studies
The plasma pharmacokinetic profile of docetaxel-nanoparticles (DTX-NPs)
was evaluated in mice by determining the concentration of docetaxel in plasma over
time after intravenous bolus administration at the dose of 10 mg docetaxel /kg body
weight. Docetaxel solution (DTX) was used as control. The mean plasma
concentration-time profiles of DTX-NPs and DTX were shown in Figure 3.5. In the
first 2 h after administration, a rapid decline in plasma concentration was observed,
likely representing the distribution phase, which was followed by the elimination
phase. At the initial time point of 5 min after dosing, docetaxel in nanoparticles
yielded about 2-fold higher plasma concentration than docetaxel in solution (p =
0.018). A non-compartment model was used to fit the data, and the main
pharmacokinetic parameters were listed in Table 3.1. The DTX-NPs group showed
higher AUC and lower volume of distribution (Vss) than DTX group. However,
elimination half-life (t½), clearance (Cl), and mean resident time (MRT) of DTX and
DTX-NPs were likely similar.
72
DTX
Docetaxel concentration (ng/mL)
10,000
DTX-NP
1,000
100
0
2
4
6
Time (h)
Figure 3.5 Plasma pharmacokinetics of docetaxel in mice after bolus intravenous
injection of DTX-NPs, or docetaxel solution (DTX) at the docetaxel dose of 10 mg/kg.
Each data point represents the mean ± S.D. from three mice.
Table 3.1 Pharmacokinetic parameters after intravenous administration of DTX and
DTX-NPs to mice (10 mg/kg of docetaxel).
Pharmacokinetic parameters
DTX
DTX-NP
AUC0-6 h (ng·h/mL)
1974.97
2559.44
AUC0-∞ (ng·h/mL)
3953.54
4689.01
t½ (h)
4.76
4.89
Cl (mL/h)
50.59
42.65
MRT (h)
6.13
5.64
Vss (mL)
309.97
240.63
The mean plasma concentration from three mice at each time point was used to obtain
pharmacokinetic parameters.
AUC, area under the concentration-time curve; t½, elimination half-life; Cl, clearance;
MRT, mean resident time; Vss, volume of distribution at steady state.
73
3.4.6 In vivo tumor uptake study
To preliminarily investigate whether the docetaxel in nanoparticles can
improve the accumulation of the docetaxel into tumors in vivo, DTX-NPs or DTX
were injected intravenously into C57BL/6 mice with pre-established TC-1 tumors. As
shown in Figure 3.6, twelve hours after the injection, the amount of docetaxel
recovered from the tumors in mice injected with the DTX-NPs was 1.13 ± 0.28 mg/g
of tumor (mean ± S.D., n = 6), 1.8-fold higher than that in mice injected with the
docetaxel in solution (0.63 ± 0.3 mg/g of tumor, n = 5).
Docetaxel concentration (mg/g tumor)
2
p = 0.02
1.5
1
0.5
0
DTX
DTX-NP
Figure 3.6 Nanoparticles increased the accumulation of the docetaxel in tumors in
mice. Female C57BL/6 mice bearing TC-1 tumors were injected intravenously of
docetaxel-nanoparticles (DTX-NPs) (n = 6) or docetaxel solution (DTX) (n = 5) at the
docetaxel dose of 10 mg/kg. Twelve hours after injection, mice were sacrificed and
docetaxel accumulated in tumors were analyzed by HPLC. Data were given as mean ±
S.D.
74
The higher accumulation of docetaxel in DTX-NPs tissue might be due to the
enhanced permeability and retention (EPR) effect (Fang et al., 2010).
3.4.7 In vivo therapeutic studies
After the pharmacokinetics and tumor uptake evaluation, it can be expected
that docetaxel-nanoparticles may improve the antitumor activity compared to
docetaxel solution. Antitumor activity after intravenous injection was evaluated at a
docetaxel dose of 20 mg/kg. Mice were treated on day 5 and 10 after tumor
implantation, and the tumor growth was monitored and shown in Figure 3.7.
14
PBS
DTX
Tumor diameter (mm)
12
DTX-NP
NP
10
8
*
6
4
2
5
7
9
11
13
15
17
19
21
Time (days)
Figure 3.7 In vivo antitumor efficacy after intravenous injection. Antitumor effects of
control (PBS), nanoparticles without docetaxel (NPs), docetaxel solution (DTX), and
docetaxel-nanoparticles (DTX-NPs) on TC-1 tumor bearing mice after dosing on day
5 and 10. * p = 0.02, DTX-NPs compared with PBS. Data were shown as mean ± SD
(n = 5)
75
By the end of the study (day 21), both docetaxel solution (DTX) and
docetaxel-nanoparticles (DTX-NPs) treated groups showed significant antitumor
activities compared to control (p = 0.01 and 0.02 for DTX and DTX-NPs,
respectively). However, there was no difference between DTX and DTX-NPs (p =
0.17), which may be due to their similar pharmacokinetic behaviors.
We further evaluated the antitumor activity of DTX-NPs after given orally. As
mentioned earlier, oral chemotherapy can provide many benefits in current regimen
chemotherapy. Mice were treated at a docetaxel dose of 10 mg/kg on day 9 and 12
after tumor implantation, and the tumor growth was monitored and shown in Figure
3.8. Docetaxel solution (DTX) and docetaxel-nanoparticles (DTX-NPs) tended to slow
the tumor growth. Importantly at the end of the study (day 15), the tumor volume in
mice treated with DTX-NPs was significantly smaller than that treated with DTX (p =
0.04) (Figure 3.8).
There was no significant body weight loss in both docetaxel treatment groups
compared with negative control indicating that there was no acute toxicity after oral
administration of docetaxel-nanoparticles at the given dose. However, in our
preliminary antitumor activity study, two out of five mice were found dead after
receiving four doses of docetaxel solution. This may due to the toxicity of docetaxel in
Tween 80/ethanol formulation.
76
350
Saline
Tumor volume (mm3)
NP
DTX-NP
DTX
250
*#
150
50
7
9
11
13
Time (days)
15
Figure 3.8 In vivo antitumor efficacy after oral administration. Antitumor effects of
control (saline), nanoparticles without docetaxel (NPs), docetaxel solution (DTX), and
docetaxel-nanoparticles (DTX-NPs) on TC-1 tumor bearing mice after oral
administration on day 9 and 12. * p = 0.04 and # p = 0.03 compared with DTX and
saline, respectively. Data were shown as mean ± SEM (n = 5 - 7).
3.5 Conclusions
Solid lipid docetaxel-nanoparticles of around 120 nm were successfully
engineered by solvent emulsification/evaporation method. The resultant docetaxel
nanoparticles were stable when stored as an aqueous dispersion and also likely
compatible with the physiological condition. The docetaxel in nanoparticles was more
effective in killing tumor cells in culture even though it was taken up less effective by
the tumor cells. Docetaxel-nanoparticle formulation enhanced the accumulation of
77
docetaxel in tumor after intravenous injection, and showed potential for oral
administration of docetaxel.
78
Chapter 4
NEGATIVELY CHARGED LIPOSOMES SHOW POTENT ADJUVANT
ACTIVITY WHEN SIMPLY ADMIXED WITH PROTEIN ANTIGENS
Nijaporn Yanasarn, Brian R. Sloat, and Zhengrong Cui
Molecular Pharmaceutics, 2011; in press
79
4.1 Abstract
Liposomes have been investigated extensively as a vaccine delivery system.
Herein the adjuvant activities of liposomes with different net surface charges (neutral,
positive, or negative) were evaluated when admixed with protein antigens, ovalbumin
(OVA, pI = 4.7), Bacillus anthracis protective antigen protein (PA, pI = 5.6), or
cationized OVA (cOVA). Mice immunized subcutaneously with OVA admixed with
different liposomes generated different antibody responses. Interestingly, OVA
admixed with net negatively charged liposomes prepared with DOPA was as
immunogenic as OVA admixed with positively charged liposomes prepared with
DOTAP. Immunization of mice with the anthrax PA protein admixed with the net
negatively charged DOPA liposomes also induced a strong and functional anti-PA
antibody response. When the cationized OVA was used as a model antigen, liposomes
with net neutral, negative, or positive charges showed comparable adjuvant activities.
Immunization of mice with the OVA admixed with DOPA liposomes also induced
OVA-specific CD8+ cytotoxic T lymphocyte responses and significantly delayed the
growth of OVA-expressing B16-OVA tumors in mice. However, not all net negatively
charged liposomes showed a strong adjuvant activity. The adjuvant activity of the
negatively charged liposomes may be related to the liposome‟s ability (i) to upregulate
the expression of molecules related to the activation and maturation of antigenpresenting cells and (ii) to slightly facilitate the uptake of the antigens by antigen-
80
presenting cells. Simply admixing certain negatively charged liposomes with certain
protein antigens of interest may represent a novel platform for vaccine development.
4.2 Abbreviations
Aluminum hydroxide (Alum), antigen presenting cells (APC), bone marrowderived dendritic cells (BMDC), bovine serum albumin (BSA), 5-(and-6-)carboxylfluorescein diacetate succinimidyl ester (CFSE), cholesterol (Chol), cytotoxic
T lymphocyte (CTL), dendritic cells (DC), dicetyl phosphate (DCP), 1,2-dioleoyl-3trimethylammonium-propane (chloride salt) (DOTAP), 1,2-dioleoyl-sn-glycero-3phosphate (sodium salt) (DOPA), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DOPG), 1,2dioleoyl-sn-glycero-3-phospho-L-serine
(sodium
salt)
(DOPS),
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide methiodide (EDC), fetal bovine serum (FBS),
fluorescein-5(6)-isothiocyanate (FITC), hexamethylene diamine (HMD), incomplete
Freund‟s adjuvant (IFA), lipopolysaccharides (LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), ovalbumin (OVA), protective antigen (PA),
phosphate buffered saline (PBS), polymerase chain reaction (PCR), isoelectric point
(pI), type 1/2 CD4+ T helper (Th1/2), 3,3‟,5,5‟-tetramethylbenzidine solution (TMB).
4.3 Introduction
New approaches to vaccine development using recombinant protein antigens
have significant advantages over traditional vaccines consisted of live attenuated
81
pathogens, whole inactivated organisms, or inactivated toxins. However, recombinant
protein antigens alone are often poorly immunogenic or nonimmunogenic and thus,
need a potent vaccine adjuvant to enhance the resultant immune responses.(Liu and
Cepica, 1990) In recent years, particulate carriers have been extensively researched as
vaccine delivery systems. It is generally accepted that many particle carriers have
adjuvant activity, which is likely due to the particle‟s ability to facilitate the uptake of
antigens by APC. Moreover, enhancement of antigen stability, presentation of multiple
copies of antigens, and the ability to include immunomodulators are also the
advantages of using particulates as a vaccine delivery system.(O'Hagan et al., 2006)
Liposomes are a well-known lipid-based particulate vaccine delivery system.
The adjuvant activity of liposomes was first reported by Allison and Gregoriadis in
1974 using diphtheria toxoid as an antigen.(Allison and Gregoriadis, 1974) Since then
the adjuvant activity of liposomes has been extensively studied.(Alving, 1991; Davis
et al., 1987; Gregoriadis and Panagiotidi, 1989; Jaafari et al., 2006; Richards et al.,
1998) Many parameters that may affect the adjuvant activity of liposomes have been
investigated. It was found that a variety factors such as particle size, surface charge,
lipid composition, and method of antigen loading, to name a few, in a liposome-based
vaccine formulation can potentially shape the resultant immune responses against the
antigen of interest. There were numerous studies defining the adjuvant activity of
negatively and positively charged liposomes and examining the effect of the charge of
liposomes on their adjuvant activities, but the results were rather conflicting and
82
inconclusive.(Afrin and Ali, 1997; Afrin et al., 2002; Allison and Gregoriadis, 1974;
Aramaki et al., 1995; Badiee et al., 2009; Chen et al., 2008; Christensen et al., 2009;
Cui et al., 2005; Galdiero et al., 1994; Guy et al., 2001; Hartikka et al., 2001; Hartikka
et al., 2009; Jiao et al., 2003; Nakanishi et al., 1999; Vangasseri et al., 2006; Walker et
al., 1992; Yan et al., 2007; Yan and Huang, 2009; Yotsumoto et al., 2004; Yotsumoto
et al., 2007) For example, Allison and Gregoriadis (1974) reported that diphtheria
toxoid in negatively charged liposomes elicited significantly higher antibody levels
than in positively charged liposomes.(Allison and Gregoriadis, 1974) However,
Nakanishi et al. reported that positively charged liposomes containing a protein
antigen were more potent inducers of antigen-specific CTL responses than negatively
charged and neutral liposomes.(Nakanishi et al., 1999) Similarly, Afrin et al. showed
that Leishmania donovani antigens encapsulated in positively charged liposomes
induced the strongest level of protection against experimental visceral leishmaniasis in
mice, followed by neutral and negatively charged liposomes, respectively.(Afrin et al.,
2002) On the contrary, in another study using the recombinant glycoprotein of
Leishmania antigen,(Badiee et al., 2009) it was shown that antigens entrapped in
neutral liposomes conferred a significantly higher protection and Th1 type immune
responses than antigens entrapped in positively charged liposomes, whereas the
negatively charged liposomes favored the induction of a Th2 type immune response.
In the present study, the adjuvant activities of liposomes with different net
surface charges (i.e., neutral, positively charged, or negatively charged) were further
83
investigated. Very often, protein antigens were either entrapped inside liposomes or
covalently conjugated onto the surface of liposomes when liposomes were used as a
vaccine delivery system.(Allison and Gregoriadis, 1974; Antimisiaris et al., 1993;
Bhowmick et al., 2010; Ohno et al., 2009; Shek and Heath, 1983; Shek and Sabiston,
1982b; Snyder and Vannier, 1984; Steers et al., 2009; Takagi et al., 2009) Entrapment
of protein antigens into liposomes has many advantages. For example, it improves the
stability of the proteins by protecting it from degradation after injection.(Shek and
Sabiston, 1982b) Using BSA as a model antigen, Shek and Sabiston (1982) showed
that trypsinization of liposomes with entrapped BSA did not reduce the anti-BSA
immune response induced by the liposomes, but trypsinization of liposomes with
surface adsorbed BSA significantly reduced the anti-BSA response induced.(Shek and
Sabiston, 1982b) Moreover, it is thought that liposomes with antigens entrapped inside
can act as a depot and allow the slow and persistent release of the antigen. Similarly,
conjugation of antigens onto liposomes has its advantages as well. For example, it was
shown that conjugation of lysozyme as an antigen onto neutral liposomes induced
significantly stronger antibody responses than entrapment of it in the liposomes.(Latif
and Bachhawat, 1984a) However, a significantly more convenient approach is to
simply mix the antigen of interest with preformed liposomes, similar to the mixing of
protein antigens with aluminum hydroxide or aluminum phosphate in suspensions,
which are used in many human vaccines. Using model antigens OVA and the PA
protein of Bacillus anthracis with a pI value of 4.7 and 5.6, respectively, the adjuvant
84
activities of net neutral, net positively charged, and net negatively charged liposomes
were evaluated by simply mixing the antigens with the liposomes before injecting
them into mice. It was found that net negatively charged liposomes prepared with
certain negatively charged lipids have a potent adjuvant activity when admixed with
protein antigens.
4.4 Materials and Methods
4.4.1 Materials
DOTAP, DOPA, DOPC, DOPS, and DOPG were from Avanti Polar Lipids, Inc.
(Alabaster, AL). BSA, horse serum, Chol, DCP, OVA (Cat. # A5503), HMD, EDC,
TMB, FITC, and MTT assay kit were from Sigma-Aldrich (St. Louis, MO). B.
anthracis PA protein and lethal factor were from List Biological Laboratories, Inc.
(Campbell, CA). Goat anti-mouse immunoglobulin (IgG, IgG1, and IgG2a) were from
Southern Biotechnology Associates, Inc. (Birmingham, AL). Class I (Kb)-restricted
peptide epitope of OVA (SIINFKEL) was from Ana Spec Inc. (Fremont, CA).
Phycoerythrin (PE)-labeled anti-I-A[b] and FITC-labeled anti-CD80 antibodies were
from BD Biosciences (San Diego, CA). RT2 First Strand Kit, RT2 SYBR Green/ROX
qPCR Master Mix, RT2 Profiler Mouse Dendritic and Antigen Presenting Cell PCR
Array were from SABioscience (Frederick, MD). CFSE and SuperScript III FirstStrand Synthesis SuperMix for qRT-PCR were from Invitrogen (Carlsbad, CA). Taq
DNA polymerase was from New England Biolabs (Ipswich, MA).
85
4.4.2 Cells and cell lines
Culture medium, FBS, and antibiotics were from Invitrogen. The DC2.4 cells (a
mouse DC line) were grown in RPMI 1640 medium supplemented with 10% FBS, 100
U/mL of penicillin, and 100 g/mL of streptomycin. The OVA-expressing B16-OVA
cells were kindly provided by Dr. Edith M. Lord and Dr. John Frelinger from the
University of Rochester Medical Center (Rochester, NY) (Lugade et al., 2005) and
grown in RPMI 1640 medium with 5% FBS and 400 µg/mL of Geneticin (G418). The
J774A.1 cells (mouse macrophages) were grown in DMEM medium supplemented
with 10% FBS, 100 U/mL of penicillin and 100 g/mL of streptomycin. BMDC of
C57BL/6 female mice were from Astarte Biologics (Redmond, WA) and grown in
DMEM high glucose medium with 10% FBS, 100 U/mL of penicillin, and 100 g/mL
of streptomycin.
4.4.3 Preparation and characterization of liposomes
Liposomes were prepared using the thin film hydration method.(Szoka and
Papahadjopoulos, 1980) Briefly, a thin film of net neutral phospholipids (DOPC) and
Chol (1:1 molar ratio, 20 mg total lipid) was formed in the bottom of 7 mL glass
scintillation vial by chloroform evaporation. The lipid thin film was then hydrated and
dispersed in 1 mL of PBS (10 mM, pH 7.4) by vigorous mixing at room temperature.
The resultant liposomes were extruded through 400 and 100 nm filters, sequentially,
using the Mini-Extruder (Avanti Polar Lipids, Inc.). To prepare net positively or
86
negatively charged liposomes, DOPC was replaced by DOTAP or DOPA,
respectively. For the preparation of different negatively charged liposomes, DOPS,
DOPG, or DCP was used as follow: DOPS:Chol (1:1, m/m), DOPG:DOPC:Chol
(1:2:1, m/m/m), and DCP:DOPC:Chol (1:2:1, m/m/m), all with 20 mg of total lipids.
The endotoxin level in the liposome preparations (measured in liposomes prepared
with DOTAP, DOPA, and DOPG) was estimated to be 0.11 - 0.35 EU/mL using a
ToxinSensor Chromogenic LAL Endotoxin Assay Kit from GenScript (Piscataway,
NJ).
An equal volume of liposomes and protein in solution (OVA or PA in PBS, 10
mM, pH 7.4) were mixed. The mixture was then vortexed for 3 - 5 s and incubated at
room temperature for at least 15 min prior to further use.
Cationized OVA (cOVA) was prepared as previously described.(Cui and
Mumper, 2002a) Briefly, 20 mg of OVA was dissolved into 1 mL of PBS. HMD (2
mL, 2 M, pH 6.8) and EDC (54 mg) were added, and the mixture was stirred at room
temperature for 2 h. The reaction was stopped by adding 1 mL of glycine solution (2
M) followed by another hour of stirring. The cOVA was purified using a PD-10
column (GE Healthcare, Piscataway, NJ) and concentrated using centrifugation filter
tubes (30 kDa cutoff) (Millipore, Billerica, MA). Protein concentration was
determined using the Bradford reagent (Sigma-Aldrich). The cOVA-liposome
mixtures were prepared as described above.
87
The particle size of liposomes and liposome-protein mixtures was determined
using a Coulter N4 Plus Submicron Particle Sizer (Beckman Coulter Inc., Fullerton,
CA). The turbidity was determined by measuring the absorbance at 655 nm using a
BioTek Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Inc.,
Winooski, VT).
The percent of protein associated with the liposomes was determined by
ultracentrifugation. Briefly, the antigen-liposome mixture was centrifuged at 150,000g
for 1 h using a Beckman-Coulter Optima TLX Benchtop Ultracentrifuge (Brea, CA),
and the protein content in the supernatant was determined using a CBQCA protein
quantitation kit (Molecular Probes, Inc., Eugene, OR). The amount of proteins
associated with the liposomes was derived by subtracting the amount of proteins in the
supernatant from the total amount of proteins added. Sucrose gradient centrifugation
was used for the protein-DOTAP liposome mixture because their mixture did not
precipitate after simple centrifugation. Sucrose gradient was prepared with the
following layering: 60% sucrose (0.4 mL), protein-DOTAP liposome mixture (0.1
mL), 25% sucrose (0.4 mL), and 10% sucrose (0.2 mL). After 1 h of centrifugation
(200,000g), 0.1 mL fractions (11 total) were collected. Protein concentration in each
fraction was quantified using the CBQCA kit. The liposome-associated protein peaked
at fraction 2, while the protein alone peaked at fraction 8. The % of protein associated
with liposomes was calculated using the trapezoidal rule. All experiments were
repeated as least 3 times.
88
4.4.4 Immunization studies
All animal studies were carried out following the National Institutes of Health
guidelines for animal care and use. Animal protocols were approved by the
Institutional Animal Care and Use Committee at the University of Texas at Austin and
at Oregon State University. Female BALB/c or C57BL/6 mice, 6-7 weeks of age, were
from Simonsen Laboratories, Inc. (Gilroy, CA) or Charles River Laboratories
(Wilmington, MA). The vaccine formulations were administrated by subcutaneous
(sc) injection once in every two weeks, three times for OVA-liposome mixtures (10 µg
of OVA/mouse/injection) or two times for PA-liposome mixtures (5 µg of
PA/mouse/injection) and cOVA-liposome mixtures (10 µg of cOVA/mouse/injection).
Proteins admixed with Alum (30 µg (or 50 for PA)/mouse/injection, USP grade from
Spectrum Chemicals & Laboratory Products, Cardena, CA) were used as a positive
control. Mice in the negative control group were injected with sterile PBS. Two weeks
after the last injection, mice were euthanized to collect blood.
4.4.5 Enzyme-Linked Immunosorbent Assay (ELISA)
Antigen-specific IgG, IgG1 and IgG2a levels were determined using
ELISA.(Sloat et al.) Briefly, polystyrene, medium binding 96-well plates (BD
Biosciences) were coated with 100 ng of OVA, cOVA, or PA in 100 µL of carbonate
buffer (pH 9.6) overnight at 4°C. For anti-OVA or anti-cOVA antibody determination,
the plates were washed with PBS/Tween 20 (10 mM, pH 7.4, 0.05% Tween 20) and
89
blocked with blocking solution (5% horse serum in PBS/Tween 20, v/v) for 1 h at
37°C. Serum samples were diluted 100, 1,000, and 10,000 times (or 2-fold serially for
titers) in blocking solution and then added to the plates following the removal of the
blocking solution. After 2 h of incubation at 37 °C, the samples were removed, and the
plates were washed five times with PBS/Tween 20. Horseradish peroxidaseconjugated goat anti-mouse immunoglobulins (IgG, IgG1, or IgG2a, 5000-fold
dilution in 1.25% horse serum in PBS/Tween 20, v/v) were added into the plates,
followed by another hour of incubation at 37 °C. The plates were again washed five
times with PBS/Tween 20. After 15 min of incubation at room temperature with TMB
solution, followed by the addition of stop solution (0.2 N sulfuric acid), the presence
of bound specific antibody was detected at 450 nm. Anti-PA antibody was determined
similarly, except that the 5% horse serum in PBS/Tween 20 was replaced by 4% BSA
in PBS/Tween 20. Specific antibody responses were reported as the OD450 values or
as antibody titer. Antibody titers were determined by considering any absorbance
value higher than the 2-fold of the mean (2 × mean) of the negative control group as
positive. Total IgG was measured in both BALB/c and C57BL/6 mice. IgG subtypes
were measured in BALB/c mice only.
4.4.6 Anthrax lethal toxin neutralization assay
Lethal toxin neutralization activity was determined as previously described
(Sloat and Cui, 2006). Briefly, confluent J774A.1 cells were seeded (1 × 104
90
cells/well) in sterile, 96-well, clear-bottom plates and incubated overnight at 37 °C,
5% CO2. Fresh medium containing PA (800 ng/mL) and lethal factor (200 ng/mL) was
mixed at an equal volume with diluted serum sample and incubated for 2 h at 37 °C.
The cell culture medium was removed, and the serum/lethal toxin mixture was added
into each well at the final concentration of 400 ng/mL of PA and 100 ng/mL of lethal
factor. The cells were then incubated for 2 h at 37 °C, 5% CO2. Cell viability was
determined using the MTT assay with untreated and lethal toxin alone treated cells as
controls.
4.4.7 In vivo CTL assay and mouse tumor prevention study
In vivo CTL assay was carried out as previously described.(Qiu and Cui, 2007)
Female C57BL/6 mice were immunized with OVA-DOPA liposome complexes (20
µg OVA/mouse) on days 0, 7, and 14. OVA adjuvanted with IFA (Sigma-Aldrich) (25
µL/mouse) or sterile PBS were used as positive and negative controls, respectively.
On day 21, the splenocytes from naive mice were harvested and labeled with 0.2 µM
of SIINFEKL followed by labeling with a high concentration of CFSE (10 µM,
CFSEHigh). Same splenocytes without SIINFEKL labeling were labeled with a low
concentration of CFSE (1 µM, CFSELow). Ten million cells from each population were
mixed and injected intravenously via the tail vein into the immunized mice. Three
hours after the injection, mice were euthanized, and the relative abundance of
CFSEHigh and CFSELow cells in the splenocyte preparation was determined using a
91
flow cytometer (FC500 Beckman Coulter EPICS V Dual Laser Flow Cytometer,
Fullerton, CA).
For the tumor prevention study, 28 days after the first immunization, B16-OVA
cells (1 × 105) were subcutaneously injected into the flank of the mice (n = 5). The
tumor size was measured using a caliper and reported based on the following equation:
tumor size (mm) = ½ (width + length). Mice were euthanized 25 days after the tumor
cell injection, and their blood was collected.
4.4.8 In vitro uptake of OVA admixed with liposomes by DC2.4 cells
Liposomes were mixed with FITC-labeled OVA (FITC-OVA), which was
prepared following the manufacturer‟s instruction (Sigma-Aldrich). DC2.4 cells (1.0 ×
105 cells/well) were seeded into 48-well plates and allowed to grow overnight. The
cells were then incubated with 200 µL of the FITC-OVA-liposome mixtures for 3 h at
37 °C, 5% CO2. After incubation, the cells were washed three times with cold PBS,
lysed with a lysis buffer (0.5% Triton X-100), and centrifuged at 14000g to collect the
supernatant. The fluorescence intensity in a supernatant was measured using a BioTek
Synergy HT Multi-Mode Microplate Reader. As controls, cells were incubated with
FITC-OVA alone or fresh medium alone.
4.4.9 CD80 and MHC II expression on DC2.4 cells after in vitro stimulation
DC2.4 cells (5.0 × 105 cells/well) were seeded into six-well plates. After
overnight incubation, the cells were then incubated with 25 µL of DOTAP liposomes,
92
DOPA liposomes, or OVA solution (5 µg OVA) at 37 °C, 5% CO2. Cells were also
treated with sterile PBS as a negative control or LPS from Escherichia coli (200 ng,
Sigma) as a positive control. After 16 h, cells were washed twice with staining buffer
(1% FBS and 0.1% NaN3 in PBS), stained with FITC-labeled anti-CD80 antibody or
PE-labeled anti-I-A[b] MHC II for 20 min at 4°C, washed twice, and analyzed with a
FACSCalibur flow cytometer (BD Biosciences).
4.4.10 Mouse dendritic cell and antigen-presenting cell PCR array
DC2.4 cells (5.0 × 105 cells/well) were seeded into six-well plates. After
overnight incubation, the cells were incubated with 25 µL of DOPA liposomes or
sterile PBS for 24 h at 37 °C, 5% CO2. Total RNA was extracted using an RNeasy
Mini Kit from Qiagen (Valencia, CA) according to the manufacturer‟s instruction.
One microgram of total RNA was used to synthesize cDNA using an RT2 First Strand
Kit. Diluted first strand cDNA was used to prepare an experimental cocktail (RT2
SYBR Green/ROX qPCR Master Mix, diluted cDNA, nuclease-free water). Twentyfive microliters of the experimental cocktail was added to each well in the PCR array.
Real-time PCR was performed using an ABI 7900HT from Applied Biosystems, Inc.
(Foster City, CA). The plate was incubated at 95 ºC for 10 min and cycled 40 times at
95 ºC for 15 s, 60 ºC for 1 min. The Ct values were obtained from SDS Software 2.3
(Applied Biosystems) and used to calculate the fold change in gene expression by the
Web-Based PCR Array Data Analysis software from SABioscience.
93
4.4.11 Reverse transcription PCR
Total RNA was isolated from DC2.4 cells treated with 25 µL of DOPA
liposomes or PBS for 24 h as described above. Two-step RT-PCR was performed. One
microgram of total RNA was used to synthesize cDNA using SuperScript III FirstStrand Synthesis SuperMix for qRT-PCR (Invitrogen). Primer pairs for mouse B2m
(forward,
5‟-ACCGGCCTGTATGCTATCCAGAAA-3‟,
IL-6
AAGCATTGGGCACAGTGACAGACT-3‟),
(forward,
reverse,
ATCCAGTTGCCTTCTTGGGACTGA-3‟,
AACGCACTAGGTTTGCCGAGTAGA-3‟)
reverse,
and
TGTGATGGTGGGAATGGGTCAGAA-3‟,
β-actin
5‟5‟5‟-
(forward,
reverse,
5‟5‟-
TGCCACAGGATTCCATACCCAAGA-3‟) were synthesized by IDT technology
(Coralville, IA). The cDNA was used for PCR reaction mixture that included the
template DNA, primers, and Taq DNA polymerase. The mixture was incubated at 95
ºC for 5 min and cycled 20 times for B2m (33 times for IL-6) at 95 ºC for 30 s, 60 ºC
for 1 min, and 68 ºC for 1 min using an Eppendorf Mastercycler (Hauppauge, NY).
PCR products were analyzed using agrose gel electrophoresis, and the band intensity
was measured using the Syngene G-box GeneSnap software (Syngene, IL).
4.4.12 Determination of cytokine concentration
DC2.4 cells (1 × 106 cells/well) were seeded into six-well plates. After overnight
incubation, the cells were incubated with 25 µL of DOPA liposomes or DOPS
94
liposomes at 37 °C, 5% CO2. As controls, cells were also treated with sterile PBS,
LPS (200 ng), DOTAP liposomes, or DOPC liposomes. After 24 h incubation, the
supernatant was collected and analyzed for CCL-17 and IL-6 using a mouse CCL-17
ELISA kit from R&D Systems (Minneapolis, MN) and a mouse IL-6 ELISA kit (BD
Biosciences), respectively.
BMDC (3 × 105 cells/well) were seeded in twelve-well plates and incubated
overnight. Cells were stimulated with 12.5 µL of DOPA liposomes or DOPS
liposomes for 24 h. PBS and LPS (100 ng) were used as controls. The CCL-17 and IL6 production in supernatant were determined.
4.4.13 Statistics
Statistical analyses were completed using ANOVA followed by Fisher‟s
protected least significant difference procedure. A p-value of ≤ 0.05 (two-tail) was
considered statistically significant.
4.5 Results
4.5.1 Preparation and characterization of OVA-liposome mixtures
Net neutral, positively charged, and negatively charged liposomes were prepared
using DOPC, DOTAP, and DOPA, respectively. The mean size for the neutral,
positively charged, and negatively charged liposomes was 140 ± 4, 159 ± 3, and 163 ±
3 nm, respectively. OVA-liposome mixtures were prepared by simply mixing an equal
volume of liposomes and different concentrations of OVA in solution (e.g., 2.5, 5, 10,
95
25, or 50 µg in 25 µL). As shown in Figure 4.1A, significant aggregations were
formed when the positively charged liposomes (DOTAP) were mixed with high
concentrations of OVA, as indicated by the increased turbidity of the mixtures (Figure
4.1A). At a concentration less than 5 µg of OVA/50 µL, the turbidity of the mixture
was only slightly different from that of the liposomes alone. Therefore, OVAliposome mixtures containing 100 µg/mL of OVA were used in the immunization
studies. The particle sizes of liposomes and OVA-liposome mixtures (final OVA
concentration, 100 µg/mL) were not different for the neutral (DOPC) and net
negatively (DOPA) charged liposomes, whereas the size of OVA-DOTAP liposome
mixture was significantly larger than that of the DOTAP liposomes alone (Figure
4.1B). Finally, the percentage of OVA associated with the net neutral, positively, and
negatively charged liposomes was estimated to be 53 ± 1%, 91 ± 3%, and 46 ± 1%,
respectively.
4.5.2 OVA admixed with net negatively charged liposomes was as immunogenic
as OVA admixed with net positively charged liposomes
To evaluate and compare the adjuvant activities of liposomes with different net
charges, mice were immunized with OVA admixed with DOPC, DOTAP, or DOPA
liposomes. As expected, the OVA-DOTAP liposome mixture induced a strong antiOVA IgG response (Figure 4.2A), but the OVA-negatively charged DOPA liposome
mixture induced an anti-OVA IgG response that was as strong as that induced by the
OVA-DOTAP liposome mixture (Figure 4.2A).
96
Figure 4.1 Preparation of OVA-liposome mixtures. (A) Twenty-five microliters of
liposomes with different net charges (neutral, DOPC; positive, DOTAP; negative,
DOPA) were mixed with an equal volume (25 µL) of OVA solution containing
various amount of OVA. After 15 min of incubation at room temperature, the turbidity
was measured at 655 nm. (B) The size of liposomes alone or the OVA-liposome
mixtures. The final concentration of the OVA was 5 µg in a final volume of 50 µL.
Data shown are mean ± SD (n = 3).
97
(A)
0.6
DOTAP
DOPA
0.5
Tubidity (OD 655)
DOPC
0.4
0.3
0.2
0.1
0
0
2.5
5
10
25
50
Amount of OVA (µg)
(B)
250
w/o OVA
p < 0.001
w/OVA
Size (nm)
200
150
100
50
0
DOTAP
Figure 4.1
DOPA
DOPC
98
The OVA-DOPC liposome mixture was only weakly immunogenic (Figure
4.2A). The anti-OVA antibody response was IgG1 biased because both OVA-DOTAP
liposome mixture and OVA-DOPA liposome mixture induced a strong anti-OVA
IgG1 response (Figure 4.2B), whereas no significant level of anti-OVA IgG2a was
detected in the serum of all immunized mice (Figure 4.2C). In Figure 4.2D, the antiOVA IgG responses induced by OVA in PBS or OVA admixed with DOPA liposomes
were compared, confirming that the strong OVA-specific antibody response observed
above was not simply due to the OVA protein alone.
4.5.3 Net negatively charged DOPA liposomes admixed with anthrax protective
antigen protein induced a functional anti-PA antibody response
When the PA protein was used as an antigen, the PA-DOPA liposome mixture
induced an anti-PA IgG response significantly stronger than that induced by PA
adjuvanted with Alum (Figure 4.3A). The PA-neutral DOPC liposome mixtures
induced an anti-PA IgG response similar to that induced by the PA adjuvanted with
Alum, and the anti-PA IgG levels in mice immunized with the PA-DOPC liposome
mixture or PA adjuvanted with Alum were not different from that in mice that were
injected with sterile PBS at the 10000-fold dilution (Figure 4.3A). The anti-PA
antibodies induced by the PA-liposome mixtures were functional because the anti-sera
were able to neutralize anthrax lethal toxin and protect mouse macrophages (J774A.1)
from the lethal toxin challenge (Figure 4.3B).
99
Figure 4.2 Immunization of mice with OVA admixed with net positively or net
negatively charged liposomes induced a strong OVA-specific serum IgG response.
BALB/c mice (n = 5) were dosed with OVA admixed with neutral (DOPC), net
positively charged (DOTAP), or net negatively charged (DOPA) liposomes. As
controls, mice (n = 4) were injected (sc) with OVA adjuvant with Alum or PBS alone.
The IgG (A), IgG1 (B), and IgG2a (C) levels in the serum samples were determined
on day 41 after the serum samples were diluted 100-, 1000-, and 10000-fold. (D) A
comparison of the anti-OVA IgG levels in mice immunized with OVA in PBS or
OVA admixed with DOPA liposomes (OVA/DOPA). * For the anti-OVA IgG level,
DOPA vs. Alum, p = 0.004 after 1000-fold dilution, p = 0.02 after 10000-fold dilution.
DOTAP vs. DOPA, p = 0.422 after 1000-fold, p = 0.205 after 10000-fold dilution. **
p < 0.05, OVA/DOPA vs. OVA. Data shown are mean ± SD.
100
(A)
4
PBS
Anti-OVA IgG (OD450)
Alum
DOTAP
3
DOPA
DOPC
*
2
*
1
0
100
1,000
Dilution factor
10,000
(B)
5
PBS
Anti-OVA IgG1 (OD 450)
Alum
DOTAP
4
DOPA
DOPC
3
2
1
0
100
Figure 4.2
1,000
Dilution factor
10,000
101
(C)
Anti-OVA IgG2a (OD 450)
0.15
PBS
Alum
DOTAP
DOPA
0.1
DOPC
0.05
0
100
1,000
10,000
Dilution factor
(D)
3
PBS
Anti-OVA IgG (OD 450)
OVA
**
DOPA
2
**
1
**
0
100
1,000
Dilution factor
Figure 4.2 (Continued)
10,000
102
Again, all three liposomal formulations and the PA admixed with Alum induced
a strong anti-PA IgG1 response (Figure 4.3C). Anti-PA IgG2a was only detected in
the serum samples in mice that were treated with PA admixed with the DOTAP
liposomes or the DOPA liposomes, not in mice treated with PA admixed with the
DOPC liposomes or Alum (Figure 4.3D). In Figure 4.3E, the anti-PA IgG response
induced by PA in PBS was compared to that induced by PA admixed with Alum (50
µg/mouse/injection).
4.5.4 The specific antibody responses induced by liposomes with different charges
were not significantly different when cationized OVA was used as the
antigen
The pI of the OVA is 4.7. After cationization, the OVA and the cOVA migrated
in opposite directions when applied on an agrose gel with Tris-boric acid buffer (pH
8.5) (data not shown), demonstrating the successful cationization of the OVA. As
expected, when the negatively charged DOPA liposomes were mixed with high
concentrations of cOVA, significant aggregations were formed as shown by the
increase in turbidity (Figure 4.4A), whereas no aggregation was observed when the
cOVA was mixed with the positively charged DOTAP liposomes or the neutral DOPC
liposomes (Figure 4.4A). When 5 µg of cOVA in 25 µL of PBS was mixed with 25
µL of liposomes, the particle sizes of liposomes and cOVA-liposome mixtures were
not significantly different (Figure 4.4B). Therefore, the formulations containing 100
µg/mL of cOVA were used to immunize mice.
103
Figure 4.3 Immunization of mice with PA admixed with liposomes with different net
charges induced strong and functional PA-specific antibody responses. BALB/c mice
(n = 5) were dosed with PA admixed with liposomes (DOPC, DOTAP, or DOPA) on
days 0 and 14. Control mice were injected with PA admixed with Alum or PBS alone.
Mice were bled on day 26. (A) Anti-PA IgG levels in mouse serum samples. (B) The
anti-PA antiserum protected mouse macrophages from anthrax lethal toxin challenge.
Mouse serum samples were diluted 10-fold serially, and incubated with J774A.1 cells
in the presence of anthrax lethal toxin. The neutralization activity was determined with
3 replicates. (C, D) Serum anti-PA IgG1 and IgG2a levels. (E) Serum anti-PA IgG
level in mice immunized with PA in PBS or PA admixed with Alum (PA/Alum, 5
µg/50 µg). Data reported are mean ± SD (n = 5 except in E where n = 4 or 5). In (A), *
p < 0.003, PA/Alum vs. PA/DOPA. For PA/DOTAP vs. PA/DOPA, p = 0.18, 0.07,
0.05 at 100-, 1000-, and 10000-fold dilutions, respectively. At 10000-fold dilution,
PA/Alum is not different from PBS (p = 0.10). In (E), the values of the PA/Alum and
PBS were different (p < 0.05), except at 640000-fold dilution.
104
(A)
3.5
PBS
Anti-PA IgG (OD450)
3
Alum
*
DOTAP
2.5
DOPA
DOPC
2
1.5
*
1
0.5
*
0
100
1,000
Dilution factor
10,000
(B)
90
PBS
% Viability J774A.1 cells
DOTAP
DOPA
DOPC
60
Alum
30
0
10
100
1,000
Dilution factor
Figure 4.3
10,000
105
Anti-PA IgG1 (OD450)
(C)
4
PBS
3.5
Alum
DOTAP
3
DOPA
2.5
DOPC
2
1.5
1
0.5
0
100
1,000
Dilution factor
10,000
(D)
2.5
PBS
Anti-PA IgG2a (OD450)
Alum
2
DOTAP
DOPA
DOPC
1.5
1
0.5
0
100
1,000
Dilution factor
Figure 4.3 (Continued)
10,000
106
(E)
3
PBS
Anti-PA IgG (OD 450)
PA/Alum
PA
2
1
0
1,000
10,000
100,000
1,000,000
Dilution factor
Figure 4.3 (Continued)
Interestingly, cOVA admixed with three different liposomes induced comparable
levels of anti-cOVA IgG responses (Figure 4.4C). The cOVA itself was strongly
immunogenic (Figure 4.4D), although inclusion of Alum as an adjuvant still helped
significantly.
4.5.5 Net negatively charged liposomes prepared with different lipids had
different adjuvant activities
To evaluate the adjuvant activities of different negatively charged liposomes,
OVA was admixed with liposomes prepared with 3 other different negatively charged
lipids (DOPS, DOPG, and DCP) and used to immunize mice.
107
Figure 4.4 When admixed with cationized OVA, liposomes with different net charges
showed comparable adjuvant activities. (A) Twenty-five microliters of liposomes with
different net charges (neutral, DOPC; positive, DOTAP; negative, DOPA) were mixed
with an equal volume of cOVA in solution containing various amount of cOVA. After
15 min of incubation at room temperature, the turbidity was measured at 655 nm. (B)
The size of liposomes and the cOVA-liposome mixtures. The final concentration of
the cOVA was 5 µg in 50 µL. (C) Anti-cOVA IgG responses. BALB/c mice (n = 5)
were dosed with cOVA admixed with liposomes on days 0 and 14. The IgG levels
were determined on day 20 after the serum samples were diluted 100-, 1000-, and
10000-fold. ANOVA for Alum, DOTAP, DOPA, and DOPC revealed p values of
0.181, 0.138, and 0.096 at 100-, 1000-, and 10000-fold dilution, respectively. (D) The
immunogenicity of the cOVA alone. BALB/c mice (n = 5) were dosed with cOVA
alone or cOVA admixed with Alum on days 0 and 7. The IgG levels were determined
on day 20 after the serum samples were diluted 1000- or 10000-fold. Data shown are
mean ± SD (n = 3 in A, B).
108
(A)
0.5
DOTAP
DOPA
Tubidity (OD 655)
0.4
DOPC
0.3
0.2
0.1
0.0
0
2.5
5
10
25
50
Amount of cOVA (µg)
(B)
200
w/o cOVA
w/ cOVA
Size (nm)
150
100
50
0
DOTAP
Figure 4.4
DOPA
DOPC
109
(C)
5
PBS
Anti-cOVA IgG (OD 450)
Alum
DOTAP
4
DOPA
DOPC
3
2
p = 0.096
1
0
100
1,000
Dilution factor
10,000
(D)
1
PBS
Anti-cOVA IgG(OD 450)
cOVA
0.8
Alum
0.6
0.4
p = 0.003
0.2
0
1,000
10,000
Dilution factor
Figure 4.4 (Continued)
110
The sizes of liposomes prepared with different negatively charged lipids were
different (p < 0.001, ANOVA) (Figure 4.5A), but for all 4 liposomes, the sizes of the
liposomes and the corresponding OVA and liposome mixture were not significantly
different (Figure 4.5A). The anti-OVA IgG titers in the serum samples of mice
immunized with OVA admixed with DOPA liposomes, DOPS liposomes, and DCP
liposomes were strong, whereas the OVA-DOPG liposome mixture seemed to be only
weakly immunogenic (Figure 4.5B).
4.5.6 Immunization with OVA admixed with the negatively charged DOPA
liposomes significantly delayed the growth of B16-OVA tumors in mice
Mice immunized with OVA admixed with DOPA liposomes or with OVA
adjuvanted with IFA were subcutaneously injected with the OVA-expressing B16OVA tumor cells, and the growth of tumors was monitored. Tumors became visible 8
days after the injection and grew continuously in the unimmunized mice (Figure
4.6A). In contrast, tumors grew very slowly in mice immunized with OVA admixed
with DOPA liposomes (Figure 4.6A), and 4 of the 5 immunized mice were tumor-free
by the end of the study. Tumors did not grow in mice immunized with OVA
adjuvanted with IFA, but granulomas were visible at the injection site. The anti-OVA
IgG titers shown in Figure 4.6B were from the serum samples of the mice 25 days
after the tumor cell injection. The anti-OVA IgG titer in mice immunized with OVA
adjuvanted with IFA was significantly higher than that in mice immunized with OVA
admixed with DOPA liposomes.
111
Figure 4.5 Net negatively charged liposomes prepared with different lipids had
different adjuvant activities. (A) The size of liposomes prepared with different
negatively charged lipids (DOPA, DOPS, DOPG, or DCP) before (□) and after (■)
admixing with OVA (5 µg/50 µL). (B) Serum anti-OVA IgG responses induced by
OVA admixed with different net negatively charged liposomes. BALB/c mice (n = 45) were dosed with OVA admixed with liposomes (DOPA, DOPS, DOPG, or DCP) on
days 0, 14 and 28. Mice were bled on day 49. ANOVA comparison of DOPA, DOPS,
and DCP revealed p value of 0.737. Data reported are mean ± SD (n = 3 for A, 4 - 5
for B).
112
(A)
300
w/o OVA
Size (nm)
250
w/OVA
200
150
100
50
0
DOPA
DOPS
DOPG
DCP
(B)
p = 0.737
Anti-OVA IgG titer
16
p = 0.072
14
12
Alum
Figure 4.5
DOPG DOPA
DOPS
DCP
113
4.5.7 OVA admixed with the DOPA liposomes induced OVA-specific CTL
responses
To further characterize the immune responses induced by the OVA-DOPA
liposome mixture, the OVA-specific CTL response induced was measured using an in
vivo CTL assay. As shown in Figure 4.6C, CTL response (23.4% lytic activity) was
detected in mice immunized with OVA admixed with DOPA liposomes, and 35.1%
lytic activity was detected in mice immunized with OVA adjuvanted with IFA. In a
separate experiment, the OVA (SIINFKEL)-specific in vivo CTL activity in mice
immunized with the OVA-DOPA liposome mixture was compared to that in mice
immunized with OVA admixed with Alum or OVA adjuvanted with IFA. Again,
OVA adjuvanted with IFA induced the strongest CTL activity, followed by OVA
admixed with DOPA liposomes. On average, only a weak CTL activity was detected
in mice immunized with OVA admixed with Alum or OVA alone in sterile PBS (data
not shown).
4.5.8 The negatively charged DOPA liposomes only slightly enhanced the uptake
of the OVA by DC2.4 cells in culture
FITC-labeled OVA was admixed with liposomes with different net charges and
incubated with DC2.4 cells. As shown in Figure 4.7, the DOPA liposomes only
slightly facilitated the uptake of the OVA by DC2.4 cells, but far less than the DOTAP
liposomes. The neutral DOPC liposomes did not significantly affect the uptake of
OVA by DC2.4 cells (Figure 4.7).
114
Figure 4.6 Immunization of mice with OVA admixed with DOPA liposomes
significantly delayed the growth of B16-OVA tumors in mice and induced a specific
CTL response. (A) Tumor growth curve. C57BL/6 mice (n = 5) were immunized with
OVA admixed with DOPA liposomes, OVA adjuvanted with IFA, or PBS alone on
days 0, 7, and 14. On day 28, mice were sc injected with B16-OVA cells. The
numbers in parenthesis indicate tumor-free mice 25 days after the tumor cell injection.
(B) Titers of anti-OVA IgG in mice 25 days after the tumor injection. Data shown are
mean ± SD. (C) Immunization with OVA admixed with DOPA liposomes induced
OVA-specific CTL responses. C57BL/6 mice were dosed with PBS, OVA/IFA, or
OVA admixed with DOPA liposomes (OVA/DOPA) on days 0, 7, and 14. OVAspecific CTL responses were measured on day 21. Numbers shown are the % lytic
activity. Experiment was repeated in two mice with similar results.
115
(A)
12
PBS
OVA/DOPA
Tumor size (mm)
10
OVA/IFA
(0/5)
8
6
4
2
(4/5)
(5/5)
0
0
10
20
30
Time (day)
(B)
p < 0.001
20
Anti-OVA IgG titer
16
12
8
4
0
OVA/DOPA
Figure 4.6
OVA/IFA
116
(C)
PBS
OVA/DOPA
23.4%
Figure 4.6 (Continued)
OVA/IFA
35.1%
117
50
Fl (LP-OVA) / Fl (OVA)
p = 0.025
40
30
20
10
0
DOTAP
DOPA
DOPC
Figure 4.7 In vitro uptake of OVA admixed with liposomes with different charges by
DC2.4 cells. Cells (1.0 × 105) were incubated with FITC-OVA admixed with DOPC
(neutral), DOTAP (positively charged), or DOPA (negatively charged) liposomes for
3 h at 37 °C. Data reported are the ratio of fluorescence intensities of cells treated with
FITC-OVA admixed with liposomes over that treated with FITC-OVA alone. Data
shown are mean ± SD. (FI, fluorescence intensity; LP, liposomes)
4.5.9 The negatively charged DOPA liposomes up-regulated the expression of
genes related to DC activation and maturation
The DOPA liposomes significantly upregulated the expression of MHC II
molecules on the DC2.4 cells (Figure 4.8A), but did not have any detectable effect on
the expression of the CD80 (Figure 4.8B), even with increased concentration of
DOPA liposomes (data not shown). DOTAP liposomes, in contrast, slightly
upregulated both MHC II and CD80 on the DC2.4 cells (Figure 4.8).
118
(A) MHC II
(B) CD 80
OVA
DOPA
LPS
DOTAP
Figure 4.8 The expression of MHC II (A) and CD80 (B) on DC2.4 cells after in vitro
stimulation. Cells were incubated with DOTAP, DOPA, or OVA solution (5 µg of
OVA) for 16 h and stained with anti-I-A[b] MHC II or anti-CD80 antibody and
analyzed using a flow cytometer. The graphs of the treatment groups (dark line) were
overlaid on the graph of cells incubated with sterile PBS (gray area). Data shown were
one representative from three independent experiments with similar results.
Real-time PCR revealed the differential expression of 12 genes related to DC
and APC activation and maturation on the DC2.4 cells after stimulation with the
DOPA liposomes: (i) cytokines, chemokines and their receptors (Ccl17, Ccl19, Ccl5,
and Il6), (ii) antigen uptake (Cd44), (iii) antigen presentation (B2m, Cd1d1, Cd1d2,
119
Cd74, and Erap1), (iv) cell surface receptors (Tlr1), and (v) signal transduction (Relb)
(Figure 4.9A). The overexpression of B2m and IL-6 mRNA was confirmed by
semiquantitative RT-PCR (Figures. 4.9B, 4.9C). ELISA assay also confirmed the
upregulated expression of the CCL-17 and IL-6 by DC2.4 cells after stimulation with
the negatively charged DOPA or DOPS liposomes, although the CCL-17 level in
DC2.4 cells stimulated with the DOPA liposomes was not different from that when the
cells were treated with sterile PBS (Figures. 4.9D, 4.9E). Incubation with the
positively charged DOTAP liposomes and the net neutral DOPC liposomes did not
significantly upregulate the secretion of the CCL-17 and IL-6 by the DC2.4 cells
(Figures. 4.9D, 4.9E). Finally, the DOPA and DOPS liposomes up-regulated the
expression of CCL-17 and IL-6 in mouse BMDC as well (Figure 4.10), confirming the
findings in the DC2.4 cells.
4.6 Discussion
In the present study, it was shown that protein antigens admixed with certain net
negatively charged liposomes, such as those prepared with the negatively charged
DOPA lipid, induced a strong and functional antibody response when subcutaneously
injected into mice. The antibody response was IgG1 biased. Immunization with an
antigen admixed with negatively charged liposomes also induced antigen-specific
CTL responses and prevented the growth of antigen-expressing tumor cells in mice.
120
Figure 4.9 Effect of the negatively charged DOPA liposomes on DC2.4 cells. (A) The
upregulation (+) and downregulation (-) of genes related to DC activation and
maturation by DOPA liposomes detected using a PCR array. Genes with ≥ 2-fold
change in mRNA expression are shown. The B2m mRNA expressed was up-regulated
by 93,844-fold. (B) RT-PCR products of B2m, IL-6, and β-actin mRNA in DC2.4
cells after stimulation with DOPA liposomes (20 cycles for B2m, 33 cycles for IL-6).
(C) RT-PCR data revealed the up-regulation of B2m and IL-6 mRNA by DOPA
liposomes. Shown are ratios of the band intensity of the B2m or IL-6 gene mRNA
over that of the β-actin in B. (D, E) DOPA and DOPS liposomes upregulated the
expression of CCL-17 (D) and IL-6 (E) in DC2.4 cells. In panels C, D, and E, *, p <
0.001 vs. PBS. Data shown are mean ± S.D. (n = 3).
Relb
Tlr1
Erap1
Cd74
Cd1d2
Cd1d1
B2m
Cd44
Il6
Figure 4.9
Ccl5
(B)
Ccl19
-4
Ccl17
-2
93,844X
2
0
Fold change (DOPA/PBS)
121
(A)
4
122
(C)
Band intensity (vs. actin)
1.5
p = 0.03
PBS
DOPA
*
1
0.5
0
B2m
IL-6
(D)
CCL-17 (pg/mL)
80
60
*
40
20
Figure 4.9 (Continued)
DOPC
DOTAP
LPS
DOPS
DOPA
PBS
0
123
(E)
200
*
IL-6 (pg/mL)
150
100
*
50
DOPC
DOTAP
LPS
DOPS
DOPA
PBS
0
Figure 4.9 (Continued)
The adjuvanticity of the negatively charged liposomes was likely related to their
ability (i) to regulate the expression genes related to DC activation and maturation and
(ii) to slightly facilitate the uptake of the antigens by APC.
It is interesting to find that, when simply admixed with protein antigens, net
negatively charged liposomes prepared with certain negatively charged lipids such as
DOPA showed potent adjuvant activity. In cell culture, the negatively charged DOPA
liposomes only slightly enhanced the uptake of the OVA protein as a model antigen by
mouse DC2.4 cells, significantly less effective than the positively charged liposomes
prepared using DOTAP lipid (Figure 4.7), but the negatively charged DOPA
liposomes and the positively charged DOTAP liposomes both showed a strong
adjuvant activity (Figures. 4.2, 4.3).
124
(A)
10000
IL-6 (pg/mL)
1000
*
100
*
10
1
PBS
DOPA
DOPS
LPS
(B)
CCL-17 (pg/mL)
300
200
*
100
**
0
PBS
DOPA
DOPS
LPS
Figure 4.10 DOPA and DOPS liposomes up-regulated the expression of IL-6 (A) and
CCL-17 (B) in mouse BMDC as well. * p < 0.001 vs. PBS, ** p = 0.04 vs. PBS. Data
shown are mean ± S.D. (n = 3).
125
Therefore, mechanisms other than the ability to enhance antigen uptake have
likely contributed to the adjuvant activity of the negatively charged DOPA liposomes
when simply admixed with antigens. The DOPA liposomes also significantly
upregulated the expression of genes related to DC activation and maturation (Figures.
4.9-4.10). For example, the DOPA liposomes upregulated the expression of MHC II,
B2m (a component of MHC I molecule), Cd1d2, and Cd74, which are related to
antigen presentation by APC.(Granucci et al., 2001; Hashimoto et al., 2000) The
DOPA liposomes and the DOPS liposomes also significantly up-regulated the
expression of chemokines such as CCL-17 and CCL-19 (Figures. 4.9, 4.10). It is
known that immature DC can produce inflammatory chemokines including CCL-5. As
they mature, DC lose the ability to produce inflammatory chemokines and switch to
produce chemokines such as CCL-17 and CCL-19, which attract T and B
lymphocytes.(Sallusto et al., 1999; Yanagihara et al., 1998) It is speculated that the
ability for certain negatively charged liposomes to induce DC activation and
maturation may explain their adjuvant activity. Finally, the preliminary observation
that the positively charged DOTAP liposomes and the net neutral DOPC liposomes
failed to significantly upregulate the secretion of the CCL-17 and IL-6 by DC2.4 cells
suggested that the mechanisms of adjuvant activity of differently charged liposomes
are likely different.
Data in Figure 4.5 showed that not all negatively charged liposomes had a
strong adjuvant activity. For example, negatively charged liposomes prepared with
126
DOPA, DOPS, and DCP were shown to have comparable and strong adjuvant
activities when admixed with OVA, but the net negatively charged liposomes prepared
with the DOPG lipid showed only a weak adjuvant activity (Figure 4.5B). It was
unlikely that the size of the OVA-liposome mixtures was related to the different
adjuvant activities of the four different negatively charged liposomes because the sizes
of the DOPA, DOPS, and DOPG liposomes admixed with OVA were around 130-160
nm, whereas the size of the DCP liposome-OVA admixture was around 250 nm. More
experiments need to be completed to understand why certain negatively charged
liposomes have a strong adjuvant activity while others do not.
The OVA-DOPA liposome mixture induced a stronger OVA-specific antibody
response than OVA admixed with Alum (Figure 4.2A). Similarly, the PA-DOPA
liposome mixture also induced a stronger PA-specific antibody response than PA
admixed with Alum (Figure 4.3A). The purpose of using the PA as an antigen was 2fold. First, it allowed us to confirm that the adjuvant activity of the negatively charged
liposomes prepared with DOPA was not limited to the OVA as an antigen. Second, the
use of PA allowed us to evaluate the neutralizing activity of the specific antibodies
induced. Anthrax is a toxin-mediated disease. B. anthracis produces two toxins, lethal
toxin and edema toxin. Lethal toxin is composed of PA protein and anthrax lethal
factor; edema toxin is made of PA and edema factor. PA protein is required for the
entrance of lethal factor and edema factor into cells, and only when inside cells, the
lethal factor and edema factor are toxic.(Kenney et al., 2004; Scobie and Young, 2005)
127
Therefore, neutralizing anti-PA antibodies were shown to be able to effectively protect
against anthrax infection or against the anthrax toxins. As shown in Figure 4.3B, the
anti-PA antibodies induced by the PA-DOPA liposome mixture were functional.
Finally, it seemed that the negatively charged liposomes prepared with the DOPA can
help improve not only antibody responses, but also cellular responses against a protein
antigen. The OVA-DOPA liposome mixture induced OVA-specific CTL responses,
and immunization of mice with OVA admixed with the DOPA liposomes prevented
the growth of the OVA-expressing B16-OVA tumor cells in mice (Figure 4.6).
Three antigens were used in the present study. OVA is a 385 amino acid protein
with a molecule weight of 45 kDa and a pI value of 4.7. PA contains 735 amino acids;
its molecular weight is 83 kDa, and its pI is 5.6. Therefore, at pH 7.4, both OVA and
PA should be predominately net negatively charged. In order to understand the
adjuvant activity of the negatively charged DOPA liposomes when an antigen with a
pI value of larger than 7.4 is used, OVA was cationized by adding hexamethylene
diamine to it to generate the cOVA protein. Data in Figure 4.4C showed that the
cOVA-DOPA liposome mixture also induced a strong anti-cOVA IgG response. In
agreement with the previous finding that a catioinized protein is more immunogenic
than the original protein,(Muckerheide et al., 1987; Pardridge et al., 1994) the cOVA
itself was strongly immunogenic, which may explain why the cOVA-DOPA liposome
mixture, the cOVA-DOTAP liposome mixture, and the cOVA-DOPC liposome
mixture were all strongly immunogenic (Fig. 4.4D). At physiological pH (7.4), OVA
128
and PA are expected to be mainly negatively charged, whereas the cOVA is expected
to be mainly positively charged. That explains the finding that more than 90% of the
OVA was found binding to the positively charged DOTAP liposomes in PBS (pH
7.4). Although to a less extent, binding of OVA with the negatively charged DOPA
liposomes and the net neutral DOPC liposomes was also detected, possibly due to
interactions other than electrostatic interaction. It also explains the increase in
turbidity when a large amount of cOVA was admixed with the negatively charged
DOPA liposomes, but not the DOTAP liposomes (Figure 4.4A). As mentioned above,
the adjuvant activity of the negatively charged liposomes may be related to the
liposome‟s ability (i) to up-regulate the expression of molecules related to the
activation and maturation of APC and (ii) to facilitate the uptake of the antigens by
APC. However, when antigens with a pI value larger or smaller than 7.4 were used
(i.e., cOVA vs. OVA), the extent to which the antigens strongly bound to the
negatively charged liposomes was expected to be different. Therefore, the extent to
which the adjuvant activity of the negatively charged liposomes can be attributed to
their ability to facilitate antigen uptake by APC may vary depending on antigens used.
The same reasoning may be applied to the net positively charged liposomes. Finally,
when the net neutral DOPC liposomes were used, only antigens such as the PA and
cOVA were able to induce specific antibody responses (Figures. 4.3-4.4). Therefore,
the mechanisms of adjuvanticity of the liposomes with different net charges are likely
different.
129
As mentioned earlier, when liposomes were used as an antigen/vaccine delivery
system, often the antigen of interest was entrapped in the liposomes (Antimisiaris et
al., 1993; Bhowmick et al., 2010; Steers et al., 2009) or covalently conjugated onto the
surface of the liposomes.(Ernst et al., 2006; Ohno et al., 2009; Snyder and Vannier,
1984; Takagi et al., 2009) With the exception of the complexing of plasmid DNA with
positively charged liposomes, very rarely were immunizations carried out using
antigens simply admixed with preformed liposomes. The simple admixture of antigens
with preformed liposomes is convenient and commercially favorable because it will
avoid the procedures of entrapping antigens into the liposomes or chemically
conjugating antigens onto the surface of the liposomes. Based on the finding in the
present study that the negatively charged DOPA liposomes were more potent than
Alum in increasing the immunogenicity of OVA or PA (as an antigen), and that the
OVA admixed with the negatively charged DOPA liposomes also induced specific
CTL responses, it is concluded that negatively charged liposomes may have the
potential to become a suitable alternative to Alum to be admixed with certain antigens
in vaccine development. Other advantages of using the negatively charged liposomes,
instead of Alum, may include (i) the possibility of combining various antigen types
such as recombinant proteins and live attenuated viruses in a single delivery system
and (ii) the faster clearance of the negatively charged liposomes than Alum from the
injection site. However, it is premature to generalize that all protein antigens can be
simply admixed with negatively charged liposomes to induce a strong immune
130
response. Only three antigens (OVA, PA, and cOVA) were evaluated in the present
study, and the negatively charged liposomes prepared with DOPG did not show a
potent adjuvant activity when admixed with OVA (Figure 4.5). Moreover, data from
an early report showed that simply mixing BSA as a model antigen with negatively
charged liposomes prepared with egg lecithin, Chol, and phosphatidic acid failed to
induce any anti-BSA immune responses.(Shek and Sabiston, 1982a) Nonetheless, it is
interesting that all three antigens tested in the present study were able to induce strong
specific antibody responses when simply admixed with the net negatively charged
DOPA liposomes.
4.7 Conclusions
In conclusion, it was shown that liposomes with different net charges had
different adjuvant activities, depending on the antigens used. Certain net negatively
charged liposomes showed a potent adjuvant activity when simply admixed with the
antigen of interest, and the negatively charged liposomes promoted both antibody and
cellular immune responses. The strong adjuvant activity of the negatively charged
liposomes may be attributed to their ability to induce the activation and maturation of
APC. Simply admixing certain negatively charged liposomes with certain antigens of
interest may represent a novel and convenient strategy for vaccine development.
131
Chapter 5
GENERAL CONCLUSIONS
Lipid based nanocarriers are versatile and capable to deliver various kinds of
molecules. In the studies using lipid based nanocarriers for docetaxel delivery, solid
lipid docetaxel-nanoparticles were successfully engineered. The resultant docetaxelnanoparticles with the size around 100 – 200 nm were stable when store as an aqueous
dispersion. Comprised of biocompatible and biodegradable components, docetaxelnanoparticles are compatible with physiological condition and suitable for parenteral
administration. The docetaxel in nanoparticles was more effective in killing tumor
cells in culture. Data from the in vivo tumor uptake studies showed that intravenously
injected docetaxel-nanoparticles increased the accumulation of docetaxel in tumors.
When administered by intravenous injection or oral routes, docetaxel-nanoparticles
showed antitumor activity in tumor-bearing mice. The lecithin-based nanoparticles
have the potential to be a novel biocompatible and efficacious delivery system for
docetaxel.
In the study using lipid based carrier, liposomes, as a vaccine delivery system,
it was shown that liposomes with different net charges had different adjuvant
activities, depending on the antigens used. It was found that certain net negatively
charged liposomes showed a potent adjuvant activity when simply admixed with the
132
antigen of interest. The negatively charged liposomes promoted both antibody and
cellular immune responses. The strong adjuvant activity of the negatively charged
liposomes may be attributed to their ability to induce the activation and maturation of
APC. Simply admixing certain negatively charged liposomes with certain antigens of
interest may represent a novel and convenient strategy for vaccine development.
133
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