GHENT UNIVERSITY
FACULTY OF PHARMACEUTICAL
SCIENCES
Department of Pharmaceutics
Laboratory of General Biochemistry and
Physical Pharmacy
Master thesis performed at:
UNIVERSITÉ PARIS-SUD
FACULTÉ DE PHARMACIE
Department of Pharmaceutics
Laboratory of Organic Chemistry
Academic year 2015 – 2016
BIOLOGIC EVALUATION OF POLYMER PRODRUG NANOPARTICLES
Kyra LEMEIRE
First Master of Pharmaceutical Care
Promotor
Prof. dr. K. Remaut
Co-promotor
Dr. Julien Nicolas
Commissioners
Dr. Sangram Samal
Prof. B. De Geest
GHENT UNIVERSITY
FACULTY OF PHARMACEUTICAL
SCIENCES
Department of Pharmaceutics
Laboratory of General Biochemistry and
Physical Pharmacy
Master thesis performed at:
UNIVERSITÉ PARIS-SUD
FACULTÉ DE PHARMACIE
Department of Pharmaceutics
Laboratory of Organic Chemistry
Academic year 2015 – 2016
BIOLOGIC EVALUATION OF POLYMER PRODRUG NANOPARTICLES
Kyra LEMEIRE
First Master of Pharmaceutical Care
Promotor
Prof. dr. K. Remaut
Co-promotor
Dr. Julien Nicolas
Commissioners
Dr. Sangram Samal
Prof. B. De Geest
COPYRIGHT
“The author and the promotors give the authorization to consult and copy parts of this thesis
for personal use only. Any other use is limited by the laws of copyright, especially concerning
the obligation to refer to the source whenever results from this thesis are cited.”
May, 2016
Promotor
Prof. dr. K. Remaut
Author
Kyra Lemeire
SUMMARY
Cancer is often treated with therapies based on chemotherapeutic drugs. The
chemotherapeutic drug has several disadvantages. It is distributed all over the body which
may cause side effects and it has to be administered in a high dose to reach an adequate
concentration at the site of action. Also, drugs can be metabolized resulting in reduced
therapeutic efficacies. The aim of this research is to develop and characterise polymer prodrug
nanoparticles, made from Cladribine- or Paclitaxel-polymer conjugates obtained by “drug-
initiated” living/controlled radical polymerization, to prevent those disadvantages as well as
to improve the efficacy of chemotherapeutic drugs for anticancer therapy.
The nanoparticles were prepared by the nanoprecipitation technique and characterised
based on diameter, zeta potential, size stability and in vitro anticancer activity. The results of
the size measurements showed that the size of the nanoparticles and the drug loading are
tuneable by changing the length of the polymer chains. The longer the polymer chains are, the
smaller is the size of the nanoparticles and the lower the drug loading.
The results of the stability test showed that all of the polymer prodrug nanoparticles
possessed high stability in water for a time as long as 2-3 months. They even exhibited
satisfying stability in a PBS solution for a short period without any stabilizer. This is a significant
advantage compared to other reports about polymer prodrug nanoparticles in literature,
especially for the Paclitaxel-polyisoprene conjugate, which is all-hydrophobic but showed
good stability in aqueous solution.
The cytotoxicity of the polymer prodrug nanoparticles was tested in vitro on a leukemia
mouse cell line (L1210), a human lung cancer cell line (A549) and a human breast cancer cell
line (MCF-7). The results showed that the target polymer prodrug nanoparticles exhibited
significant in vitro anticancer activity with tuneable IC50 values by changing the polymer chain
length. On the other hand, the polymer nanoparticles made from a fluorescent and nontoxic
initiator did not show any cytotoxicity, which is ideal for further imaging and diagnostic
applications.
SAMENVATTING
Kanker wordt vaak behandeld met therapieën gebaseerd op chemotherapie. Een
chemotherapeuticum heeft echter een aantal nadelen. Het chemotherapeuticum wordt
verdeeld over het hele lichaam, wat bijwerkingen kan veroorzaken, en het moet in hoge dosis
toegediend worden om in voldoende concentratie de plaats van actie te bereiken. Ook kan
het geneesmiddel gemetaboliseerd worden, wat resulteert in een gedaald therapeutisch
effect. Het doel van dit onderzoek is het ontwikkelen en het testen van polymere prodrug
nanopartikels, gemaakt van polymeer-conjugaten van Cladribine of Paclitaxel via
geneesmiddel-geïnitieerde levende/gecontroleerde radicaal polymerisatie, om de nadelen
van chemotherapeutica te voorkomen en om de werkzaamheid van antitumorale
toepassingen te verbeteren.
De nanopartikels werden bereid via de nanoprecipitatie techniek en gekarakteriseerd
op basis van diameter, zeta potentiaal, stabiliteit van de grootte van de nanopartikels en de
in vitro antikanker activiteit. De resultaten van de metingen van de grootte van de
nanopartikels toonden aan dat de grootte en de drug loading van de nanopartikels aangepast
kunnen worden door de lengte van de polymeerketens aan te passen. Hoe langer de
polymeerketen is, hoe kleiner de nanopartikels zijn en hoe lager de drug loading.
De resultaten van de stabiliteitstest toonden aan dat de polymere prodrug nanopartikels
gekenmerkt worden door een hoge stabiliteit in water voor een lange periode van 2 tot 3
maanden. Het werd zelfs duidelijk dat de nanopartikels stabiel zijn in een PBS oplossing voor
een korte periode zonder stabilisator. Dit is een significant voordeel in vergelijking met andere
verslagen over polymere prodrug nanopartikels in de literatuur, voornamelijk voor het
Paclitaxel-polyisopreen conjugaat, dat volledig hydrofoob is en toch een goede stabiliteit in
waterige oplossing vertoont.
De cytotoxiciteit van de polymere prodrug nanopartikels werd in vitro getest op een
leukemie cellijn afkomstig van een muis (L1210), een humane longkanker cellijn (A549) en
een humane borstkanker cellijn (MCF-7). De resultaten tonen een significante in vitro
antikanker activiteit met een IC50 waarde die aangepast kan worden via de ketenlengte van
het polymeer. De polymeren gemaakt van een fluorescente en niet-toxische initiator toonden
geen enkele cytotoxiciteit, wat ideaal is voor beeldvormende en diagnostische toepassingen.
ACKNOWLEDGEMENTS
First and foremost, I would like to thank the University of Ghent and Université Paris-Sud, in
particular my promotors Prof. Dr. Katrien Remaut and Dr. Julien Nicolas for making it
possible for me to be a part of the UMR CNRS 8612 and to work in the team of Prof. Dr. Elias
Fattal and Prof. Dr. Patrick Couvreur.
Secondly, I want to express my gratitude to Dr. Yinyin Bao for his invaluable guidance while
setting up experiments, analysing the results and during the writing process of my master
thesis. It would not have been possible to complete this master thesis without him.
Thirdly, I wish to extend a special thank you to everyone of équipe 7, they have helped me in the
lab, created a fun atmosphere and made my time there very pleasant.
Furthermore I am very grateful to have met amazing friends and want to thank them for their
support for my work inside the laboratory while discovering the Paris outside of the
laboratory.
Finally, I would like to thank my parents and family, who have always supported and encouraged
me in everything I do.
CONTENT
1. INTRODUCTION ........................................................................................... 1
1.1. CANCER ................................................................................................... 1
1.1.1. Pathology .............................................................................................................. 1
1.1.2. Cancer therapy: conventional methods ............................................................... 2
1.2. NANOPARTICLES FOR DRUG DELIVERY ..................................................... 2
1.2.1. Liposomes ............................................................................................................. 4
1.2.2. Polymeric nanoparticles ....................................................................................... 5
1.2.1. Dendrimers ........................................................................................................... 7
1.2.2. Nanoscale metal organic frameworks.................................................................. 7
1.2.3. Inorganic nanoparticles ........................................................................................ 8
1.2.4. Carbon nanostructures......................................................................................... 9
1.3. SYNTHESIS OF POLYMER PRODRUGS ....................................................... 9
1.3.1. “Drug-initiated” ring-opening polymerisation ................................................... 10
1.3.2. “Drug-initiated” controlled/living radical polymerization (CLRP) ...................... 11
1.4. POLYMERS .............................................................................................. 13
1.4.1. Polyisoprene ....................................................................................................... 13
1.4.2. Poly(squalenyl-methacrylate) ............................................................................ 13
1.4.3. Poly[oligo (ethylene glycol) methacrylate] (POEGMA) ...................................... 14
1.5. DRUGS .................................................................................................... 14
1.5.1. Cladribine ........................................................................................................... 14
1.5.2. Paclitaxel ............................................................................................................ 15
2. OBJECTIVES ................................................................................................ 16
3. MATERIALS AND METHODS ....................................................................... 17
3.1. MATERIALS ............................................................................................. 17
3.2. NANOPARTICLE FORMATION ................................................................. 20
3.3. DYNAMIC LIGHT SCATTERING (DLS) AND ZETA POTENTIAL .................... 21
3.4. CELL CULTURE ........................................................................................ 23
3.4.1. Cell line L1210 .................................................................................................... 23
3.4.2. Cell line A549 ...................................................................................................... 23
3.4.3. Cell line MCF-7 .................................................................................................... 24
3.5. IN VITRO ANTICANCER ACTIVITY ............................................................ 24
4. RESULTS ..................................................................................................... 26
4.1. NANOPARTICLE FORMATION ................................................................. 26
4.2. ANALYSIS OF SIZE, PDI AND ZETA POTENTIAL ......................................... 26
4.2.1. Cla-PI and Cla-d-PI .............................................................................................. 26
4.2.2. Cla-PSqMA and Cla-d-PSqMA ............................................................................. 27
4.2.3. Ptx-d-PI ............................................................................................................... 27
4.2.4. Ptx-d-POEGMA ................................................................................................... 28
4.2.5. Napht-PI.............................................................................................................. 28
4.3. SIZE STABILITY ........................................................................................ 28
4.3.1. Cla-d-PI and Cla-PI .............................................................................................. 28
4.3.2. Cla-d-PSqMA ....................................................................................................... 30
4.3.3. Ptx-d-PI ............................................................................................................... 30
4.4. MTT ........................................................................................................ 31
4.4.1. Cla-d-PSqMA and Cla-PSqMA ............................................................................. 31
4.4.2. Ptx-d-PI ............................................................................................................... 32
4.4.3. Ptx-d-POEGMA ................................................................................................... 32
4.4.4. Napht-PI.............................................................................................................. 34
4.4.5. IC50 values ........................................................................................................... 34
5. DISCUSSION ............................................................................................... 35
5.1. MEDICINE ............................................................................................... 35
5.2. SIZE AND ZETA POTENTIAL ..................................................................... 35
5.2.1. Cla-PI and Cla-d-PI .............................................................................................. 36
5.2.2. Cla-PSqMA and Cla-d-PSqMA ............................................................................. 36
5.2.3. Ptx-d-PI ............................................................................................................... 36
5.2.4. Ptx-d-POEGMA ................................................................................................... 36
5.2.5. Napht-PI.............................................................................................................. 37
5.3. SIZE STABILITY ........................................................................................ 37
5.3.1. Cla-PI and Cla-d-PI .............................................................................................. 37
5.3.2. Cla-d-PSqMA ....................................................................................................... 37
5.3.3. Ptx-d-PI ............................................................................................................... 38
5.4. CELL CULTURE ........................................................................................ 38
5.5. MTT ........................................................................................................ 38
5.5.1. Cla-PSqMA and Cla-d-PSqMA ............................................................................. 38
5.5.2. Ptx-d-PI ............................................................................................................... 39
5.5.3. Ptx-d-POEGMA ................................................................................................... 39
5.5.4. Napht-PI.............................................................................................................. 39
6. CONCLUSION ............................................................................................. 40
7. REFERENCES ............................................................................................... 41
ABBREVIATIONS
AIBN: Azobisisobutyronitrile
AMA-SG1: 2-[N-tert-butyl-N-(1-diethoxyphosphoryl-2,2-dimethylpropyl)
aminooxy]propanoic acid
ATRP: Atom-transfer radical polymerization
CAC: Critical aggregation concentration
CDP: 4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid
CDs: Carbon dots
Cla-d-PI: Cladribine-diglycolic-polyisoprene
Cla-d-PSqMA: Cladribine-diglycolic-poly(squalenyl methacrylate)
Cla-PI: Cladribine-polyisoprene
Cla-PSqMA: Cladribine-poly(squalenyl methacrylate)
CLRP: Controlled/living radical polymerization
d-AMA-SG1: Diglycolate-AMA-SG1
DLS: Dynamic light scattering
DMEM: Dulbecco’s modified eagle’s medium
DMSO: Dimethyl sulfoxide
EPR effect: Enhanced permeation and retention effect
FBS: Fetal bovine serum
Gem: Gemcitabine
IC50: 50% inhibition concentration
LA : Lactide
Mn: Number average molecular weight
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Nano MOFs or NMOFs: Nanoscale metal-organic frameworks
Napht: Naphthalimide
NMP: Nitroxide-mediated polymerization
OEGMA: Oligo (ethylene glycol) methacrylate
PAA: Polyaspartate acid
PBS: Phosphate buffered saline
PCL: Polycaprolactone
PDI: Polydispersity index
PEG: Poly(ethylene glycol)
PEGMA: Poly(ethylene glycol) methacrylate
PGA: Polyglycolic and polyglutamic acid
Phe-OCA: Phenyl O-carboxyanhydride
PI: Polyisoprene
PLA: Poly-lactic acid
PLGA: Poly(lactide-co-glycolide acid)
PLGA-mPEG: Poly(glycolide-co-lactide )-b-methoxylated PEG
PMMA: Poly(methyl methacrylate)
POEGMA: Poly[oligo (ethylene glycol) methacrylate]
PS: Polystyrene
PSD: Particle size distribution
PSqMA: Poly(squalenyl methacrylate)
Ptx-d-PI: Paclitaxel-diglycolic-polyisoprene
Ptx-d-PSqMA: Paclitaxel-diglycolic-poly(squalenyl methacrylate)
Ptx-POEGMA: Paclitaxel-diglycolic-Poly[oligo (ethylene glycol) methacrylate]
QDs : Quantum dots
RAFT: Reversible addition-fragmentation chain transfer
ROP: Ring-opening polymerization
SEC: Size exclusion chromatography
SqMA: Squalenyl methacrylate
TBAF: Tetra-n-butylammonium fluoride
TBDMS: Tert-butyldimethylsilyl
THF: Tetrahydrofuran
ζ : Zeta potential
1
1. INTRODUCTION
1.1. CANCER
1.1.1. Pathology
Cancer can originate anywhere in the body. Healthy tissues produce cells and distribute
them as needed in the body. [1] The regulation of the cell cycle is a very fine-tuned process.
A balance is needed between the proliferation and apoptosis of cells. If this balance is
disturbed, cancer can occur. The development of cancer is a multistep process which consists
of genetic and epigenetic changes. These changes can originate from genetic inheritance,
exposure to carcinogenic substances or can occur at random. A genetic modification will
ensure a change in DNA sequence. An epigenetic modification can cause cancer without
changing the DNA sequence. An accumulation of these modifications can lead to cancer. [2],
[3]
The development of cancer follows the 6 hallmarks, described by Hanahan and
Weinberg (2011): “sustaining proliferative signalling, evading growth suppressors, activating
invasion and metastasis, enabling replicative immortality, inducing angiogenesis and resisting
cell death”. These hallmarks describe the actions through which cancer develops and
stimulates itself. [4], [5] The metastasis of malignant tissue causes the spread of malignant
cells in the bloodstream and to other tissues, in this way the tumor can invade healthy tissue
and cause destruction. This leads to the formation of secondary tumors. [6]
Rapidly growing tumors require the formation of new blood vessels in order to have a
sufficient supply of nutrients and oxygen. Since the formation of these blood vessels happens
fast, they contain several small abnormalities. In healthy tissue, cells are close to each other
with only tight junctions (< 2nm) in between. An abnormal feature of blood vessels that are
formed too fast consists of gaps between the endothelial cells. This makes it easier for a drug
to permeate into the tumor. Moreover, tumors often lack a good lymphatic system, which
ensures a longer retention time for the drug in the tumor tissue. The combination of the effect
of leaky blood vessels and the absence of sufficient lymphatic drainage is called the enhanced
permeation and retention effect (EPR effect). [7]–[10]
2
1.1.2. Cancer therapy: conventional methods
Nowadays the main treatments for cancer are surgery, radiotherapy and chemotherapy.
Surgery will be performed if the cancer concerns a solid primary tumor. When surgery is used
as a single therapy, the aim is to remove all cancer cells and kill the tumor. This means that
there is a zero-order kinetic, but it is difficult to ensure that every malignant cell is removed.
Radiotherapy and chemotherapy on the other hand will only kill a certain percentage of the
cancer cells. Radiotherapy reduces tumors through high-energy radiation such as X-ray,
gamma rays and charged particles. Radiotherapy is important because it works fast and is
effective for both local and metastatic tumors. Chemotherapy consists of cytotoxic drugs,
which cure the cancer by killing the malignant cells or inhibiting the growth of the tumor.
However, most chemotherapeutics do not differentiate between healthy or malignant tissue
and damage DNA. It is an adjuvant to the treatment with surgery and radiation. For primary
tumors the main treatment is surgery and/or radiotherapy. Chemotherapy will be added to
the treatment for metastatic tumors. In most cases a combination of radiotherapy, surgery
and chemotherapeutics will be used, in order for the different therapies to complement each
other in curing the cancer. [6], [11]–[13]
The main problem with chemotherapeutics is that they do not reach adequate
concentrations at the site of action. They are distributed all over the body and only a small
amount of the drug will actually reach the site of action, which makes them less effective. If
the amount of drug delivered at the site of action can be increased, chemotherapeutics could
have a larger chance to cure cancer. In order to do this, researchers have been trying to
develop specific drug delivery systems, to deliver a larger amount of the drug at the site of
action. Such drug delivery systems are obtained by encapsulating (or linking) drugs into
nanocarriers, creating a nanomedicine. [6], [14]
1.2. NANOPARTICLES FOR DRUG DELIVERY
To improve the efficacy of chemotherapeutics in vivo, nanomedicine was developed.
The use of nanoparticles in medicine is called nanomedicine, which exists in many different
forms such as drug delivery, imaging, diagnostics and tissue-engineering. Nanoparticles are
commonly used in drug delivery as nanocarriers and they have important advantages for drug
3
delivery when it comes to drug release, drug loading, particle size, particle surface properties
and targeted drug release. In this section the use of nanoparticles as drug delivery systems
will be explained. [6], [14], [15]
The drug can be loaded into a nanoparticle, which is usually composed of a hydrophobic
core and a hydrophilic corona, in two different ways. Firstly, the core can physically
encapsulate one or several drugs. The encapsulation of several drugs can be important for
drugs that enhance each other (synergistic effect) or drugs that are active on the same tissue
in different ways. However, physically encapsulated drug delivery has several limitations. For
example, burst release can occur, meaning that a big amount of the drug is released just after
administration, which can cause toxicity. Furthermore a physical encapsulation often leads to
a small amount of drugs (drug loading) and if the drug has a poor affinity for the nanocarrier,
it is very difficult to reach high drug loadings. [6], [16], [17]
Secondly, the drug can be covalently linked to a nanocarrier to form a prodrug
nanoparticle. Once the drugs are linked to the nanocarriers, the drug loading is generally
higher and the solubility of the drugs is greatly improved. This method avoids burst release
because the drug can only be released if the bond between the drug and the nanocarrier is
cleaved. Moreover, if the core of the nanoparticles is stimuli-responsive, the drug can be
released by a stimulus at the site of action (e.g., enzyme, redox status, magnetic field,
temperature, etc.), which cleaves the bond between the drug and the nanocarrier and
releases the drug. [6], [10], [16], [18], [19]
Generally the size of nanoparticles is in the range of 10-130 nm, which makes them of
similar size as viruses (20–400 nm). This means the body can react in the same way to
nanoparticles as to viruses. Viruses and nanoparticles are easily recognised by the body as
non-self-material because they often have a hydrophobic surface. This recognition occurs by
opsonin proteins through a process called opsonisation. Once a nanoparticle is recognised by
opsonin proteins, it will be degraded by phagocytosis. The larger a particle, the faster it will
be removed by phagocytosis or endocytosis, and the easier it can obstruct capillary vessels.
Therefore, nanoparticles should not be too large. To further avoid opsonisation, the surface
4
of the nanoparticle can be shielded or coated by biocompatible materials such as hydrophilic
polymers including poly(ethylene glycol) (PEG, the so-called PEGylation) or polysaccharides.
Coating the nanoparticle improves its stealth properties, making the nanoparticle invisible for
the immune system. [6], [10], [18], [19]
According to the literature, different kinds of nanoparticulate systems can be used for
drug delivery such as liposomes, micelles, polymer nanoparticles, dendrimers, nanoscaled
metal organic frameworks, etc. These nanoscale systems are able to carry the drug to a
specific site in the body. The precise delivery is important to avoid cytotoxic effects on healthy
tissue and to restrain several side effects. This method is especially used for serious diseases
such as cancer and infectious or neurodegenerative diseases. [6], [16]
1.2.1. Liposomes
Liposomes are usually made of
biocompatible lipids such as phospholipids or
cholesterol. These lipids contain a hydrophilic
head and a hydrophobic tail. They aggregate into
spherical vesicles, consisting of 1 or more bilayers
with a size varying between 50 and 1000 nm
(Figure 1.1). Liposomes can contain hydrophilic
drugs in the aqueous core and/or hydrophobic
drugs in the bilayer of the vesicle. The main
disadvantage of liposomes is the poor drug
loading: only a limited amount of drugs can be inserted in the core. In the membrane there is
little place for hydrophobic drugs because the drugs would destabilise the membrane.
Liposomes have several advantages due to the use of physiological lipids such as
biodegradability, biocompatibility and the property to avoid immunologic reaction. When PEG
is attached to the surface of the liposome, the charge of the liposome will be shielded, which
prevents phagocytosis. The steric hindrance will provide a longer retention time of the
liposomes in the blood circulation. These advantages are the reason why liposomes are widely
Figure 1.1: Schematic representation of liposomes formed by phospholipid [20]
5
used as carriers to deliver biologically active substances in research therapeutics and analytical
applications. [6], [13], [20]
1.2.2. Polymeric nanoparticles
Polymer nanoparticles are composed of polymers, which can either be amphiphilic
polymers that self-assemble into nanospheres or nanocapsules, or polymersomes from
diblock copolymers.
Polymeric nanoparticles can either be
nanospheres or nanocapsules, depending on
the formulation process. If the nanoparticles
are made out of a polymeric matrix where
the drug is dispersed in the matrix, they are
called nanospheres, such as polymeric
micelles (Figure 1.2). When the drug is in an
oily internal cavity, surrounded by a polymer
membrane/shell, it is known as a nanocapsule. The size of these polymeric nanoparticles is
below 1 µm. These formulations are very stable, even when they are diluted in the blood, they
will not disaggregate. Polymeric micelles are uniform in terms of size (20-80 nm), they
aggregate at a low concentration and dissociate very slowly. Micelles have a significantly high
stability in vitro and in vivo. Although, when the micelles are strongly diluted, in the blood for
example, they can disintegrate as governed by the critical aggregation concentration (CAC).
[6], [7], [13]
Polymeric nanoparticles share several advantages with other nanocarriers, such as the
possibility to deliver several drugs, stealth properties, a long retention time and a specific
release of the drug at the site of action. Polymer nanoparticles are better in comparison with
other nanocarriers (e.g., liposomes) in terms of stability, drug loading, size distribution and
control of the drug release. The problem with polymer nanoparticles is the biodegradability.
Most polymers are not biodegradable, which will cause an immune response and a rapid
elimination. Therefore it is ideal to use biodegradable polymers. Commonly used
Figure 1.2: A micelle [20]
6
biodegradable polymers are polylactic acid (PLA), poly(ethylene glycol) (PEG), polyaspartate
(PAA), polyglycolic and polyglutamic acid (PGA), poly(lactide-co-glycolide acid) (PLGA) and
polycaprolactone (PCL). [6], [7], [13]
Polymersomes are similar to liposomes. They consist of 1 hydrophobic region in the
middle and 2 hydrophilic regions on the sides (Figure 1.3). The size and the thickness of the
membrane can be adjusted by changing the molecular weight of the hydrophilic and
hydrophobic block of the diblock copolymers. By using such polymers, it is possible to create
a hydrophilic core and a hydrophilic surface,
while the liposome can still encapsulate a broad
variety of drugs. Due to their hydrophilic surface,
polymersomes will have good stealth properties,
there will be less recognition by the immune
system and a longer retention time in the body.
One of the biggest disadvantages of
polymersomes for drug delivery is the stability of
the membrane. The membrane of a
polymersome (8-21 nm) is thicker than the membrane of a liposome (3-5 nm). The minimum
CAC for polymersomes is smaller than for micelles, which means that polymersomes are more
stable than micelles. The thicker the membrane, the more stable the membrane and the more
difficult to release the drug from the polymersome. [7], [13], [20]
Up to now, polymer nanoparticles have exhibited great advantages due to the flexibility
offered by macromolecular synthesis methods and the great diversity of polymers in terms of
nature, properties, composition and functionalization, which makes polymer nanoparticles an
ideal platform for drug delivery. The polymers can be produced in various ways, such as
through ring opening polymerization and controlled/living radical polymerization. [14], [16]
Figure 1.3: A polymersome [20]
7
1.2.1. Dendrimers
Dendrimers form a special group of nanoparticulate systems. The synthetic process
which starts from a core and provides the possibility to add branches to the structure after
each step. This process is easy to control, which allows to make the required structure (10-
100 nm). The biocompatibility of the dendrimer can be
adjusted by adapting the synthesis. There are two ways to
connect drugs to a dendrimer. Firstly, a dendrimer can
contain drugs in the cavities between the branches (Figure
1.4). These drugs can be physically enclosed or chemically
linked to the branches during or after the synthesis of the
dendrimer. Secondly, the drug can be connected to the
dendrimer by conjugating the drug to surface groups. Finally,
a combination of those two methods is also possible. In this way a mixture of drugs in the
cavities and drugs conjugated to surface groups is obtained. If the drug is a small size organic
molecule, it will be more likely to be in the cavities of the dendrimer in contrast to a large
biomolecule, which will be more likely associated to the surface. One of the most important
advantages of dendrimers is their uniformity and the possibility to add several functional
groups to the surface. The main disadvantage is the cost and the synthesis time, which
increases by adding more steps to the synthesis. [6], [13]
1.2.2. Nanoscale metal organic frameworks
Nanoscale metal organic frameworks (NMOFs) have been recently used as inorganic
nanocarriers. These systems are rather new and are still being studied. A NMOF is a self-
assembled porous material mostly made out of metal ions (nodes) and carboxylates (organic
linkers) (Figure 1.5). Due to a wide range of combinations of metal ions and linkers, NMOFs
can be created with a specific size of pores, suitable for a particular drug. There are 2 ways to
get drugs in the NMOFs: the drug can be incorporated in the framework (covalent attachment)
Figure 1.4: Dendrimer containing three generations [6]
8
or the drug can be put in the pores of the
NMOFs (non-covalent encapsulation)
(Figure 1.5). In both cases there is an
unstable bound between the drug and the
framework, which facilitates the drug
release. A controlled release of the drug at
the site of action may be provided by
specific functional groups, such as organic
polymers or silica coatings, at the surface of
the NMOFs. Due to a relatively unstable
bound between the metal ion and the
linker, NMOFs can easily be degraded and
eliminated once the drug is released. In
comparison to other nanomedicines,
NMOFs have the advantages of high drug
loading capacity and wide diversity
(structural as well as chemical). [21], [22]
1.2.3. Inorganic nanoparticles
Nanoparticles made from inorganic nanocarriers are highly robust, stable and extremely
resistant to enzymatic degradation. These nanoparticles can be formulated in very small sizes
< 20 nm. They can easily be made multifunctional for imaging and drug delivery by controlling
the size, shape, crystal phase, composition and surface characteristics to adjust their
magnetic, electric and optical properties. However, a big disadvantage of these nanoparticles
is their toxicity because they contain heavy metals (e.g. Cd), which makes it necessary to cover
the surface with biocompatible materials. An example of inorganic nanoparticles is quantum
dots. Quantum dots are very small (3-10 nm), smaller than or similar to the length of the Bohr
radius of an exciton (electron-hole pair). These excitons produce energy in quantized levels
which causes a conduction band. The quantized levels of energy of the valence bands and the
conducting bands are dependent on the size of the quantum dot which means that the
emission is size-dependent and tunable by changing the wavelength of the exitation. The
Figure 1.5 Nanoscale metal organic framework. (a) self-assembly of NMOF (b1) non-covalent encapsulation of drug in NMOF (b2) covalent attachment of drug in NMOF. [21]
9
tunable wavelength and the small size make the quantum dots very convenient for the use in
imaging and other biomedical applications. [14]
1.2.4. Carbon nanostructures
Carbon nanostructures, such as carbon dots (CDs), are made out of carbon materials and
are especially usefull in imaging, sensing and drug delivery because of their unique tensile and
electrical characteristics. Carbon dots (CDs) form a class of fluorescent materials with a usual
size below 10 µm. It is easy to functionalize them to be dispersible in water. The size, surface
chemistry and cristallinity define their wavelength and their optical signals. CDs exhibit rapid
elimination from the body, low toxicity and high resistance to photobleaching. [14]
1.3. SYNTHESIS OF POLYMER PRODRUGS
As described before, the use of a prodrug strategy can avoid the burst effect and
improve the drug loading. Generally, a prodrug is made from a drug and a polymer. The drug
will be released if the bond between the polymer and the drug is cleaved. Benefiting from the
diversity of the macromolecular design and synthesis, there are several ways to prepare
polymer prodrugs. [16]
Firstly, the drug can be directly conjugated to preformed polymers through the side
chains or end groups by postfunctionalization, a method widely used to prepare polymeric
drug delivery systems. However, due to the high steric hindrance macromolecules, the drug
loading capacity is still modest. Secondly, the drug can be conjugated with the monomers
before polymerization. This results in well-defined polymer prodrugs and improves the drug
loading, but it is still quite complex to precisely control the number of drugs in the polymer
chain. [16]
Recently, the “drug-initiated” method has been developed to prepare well-defined
polymer prodrugs and their nanoaggregates. In this process, the drug is first conjugated to an
initiator, which further initiates the polymerization of different monomers. This makes it
possible to conveniently prepare well-defined polymer prodrugs with high drug loadings when
chain lengths are small. [16] The “drug-initiated” method was firstly developed by Cheng
10
group in 2008 [23]–[26] utilizing ring-opening polymerization, then “drug-initiated”
controlled/living radical polymerization was created by our group in 2013 [27][28] which will
be further explained in the next paragraphs.
1.3.1. “Drug-initiated” ring-opening polymerisation
Ring opening polymerisation (ROP) is a technique to produce polyesters which are
biodegradable and well-defined. ROP can produce polyesters in three different ways. A first
method is coordination-insertion polymerization. The monomer will coordinate with the
metal active centre, while the monomer cleaves the oxygen bond and opens the ring which
initiates the polymerization. [29], [16] The second technique is ionic polymerization, an
anionic or cationic active centre starts the polymerization. [16] The third technique is
nucleophilic polymerization, which uses a nucleophilic functionality as an active centre to
initiate the polymerization. [16]
Figure 1.6 Preparation of PEGylated Paclitaxel-polylactide nanoparticles through Ptx initiated lactide polymerization in the presence of [(BDI)MN(TMS)2] (M=Mg, Zn), followed by nanoprecipitation and non-covalent surface modification with poly(glycolide-co-lactide )-b-methoxylated PEG (PLGA-mPEG) [23]
11
Cheng et al [23] utilized Paclitaxel as the initiator for ROP of lactide to produce Paclitaxel-
conjugated polylactide nanoparticles (Figure 1.6). A metal alkoxide can be produced in situ by
adding an active metal complex (e.g. a metal-amido complex) to Paclitaxel containing a
hydroxyl-group. This will initiate the polymerization of lactide to generate the polylactide. The
in situ production of the metal-alkoxide is quantitative which will lead to an incorporation
efficiency of Paclitaxel into polylactide of 100%. The drug loading can be changed by adapting
the lactide/Paclitaxel ratio. After polymerization, the Paclitaxel molecules are covalently
linked to the polylactide and nanoparticles are formed. There will be a sustained release
through hydrolysis, avoiding a burst release.
After the use of Paclitaxel, Cheng et al [24] developed a technique to produce
doxorubicin-polylactide conjugate nanoparticles. The ROP of lactide with doxorubicin as
initiator happened in several hours and with almost 100% drug loading efficiency and 100%
monomer conversion. The regioselectivity in the initiating step can be influenced by the steric
hindrance of the chelating ligand and the metal catalyst. The nanoparticles obtained from
doxorubicin-polylactide are smaller than 150 nm, they have a narrow particle size distribution
and the drug release is sustained without burst release.
In following work, Cheng et al [25], [26] found out that it is possible to use docetaxel
instead of Paclitaxel and to use different biopolymers instead of polylactide to obtain a
controlled polymerization. These conjugates are supposed to be very useful in drug delivery,
coating and controlled release applications.[25] They developed a strategy to initiate
polymerization of phenyl O-carboxyanhydride (Phe-OCA) by the use of a hydroxyl-containing
drug, such as Paclitaxel, docetaxel, doxorubicin and camptothecin. The properties of
nanoparticles formed with the drug-poly(Phe-OCA) conjugates are easily controllable and the
sustained drug release is without burst release. [26]
1.3.2. “Drug-initiated” controlled/living radical polymerization (CLRP)
Controlled/living radical polymerization (CLRP) have emerged as efficient tools to
synthesize well-defined polymers with various applications, and also to prepare well-
dispersed nanoaggregates from amphiphilic polymers for drug delivery. CLRP gathers a series
12
of polymerization techniques which enable minimization of irreversible termination reactions
such as transfer and termination reactions, thus allowing well-defined architectures to be
obtained. The three typical techniques are nitroxide-mediated polymerization (NMP), atom-
transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer
(RAFT) polymerization. [16], [30], [31] Compared to ROP, it is possible to use milder reaction
conditions and obtain a wider range of functionalities, due to the radical mechanism.
The techniques used in the field of the drug-initiated method are NMP and RAFT
polymerization. NMP represents one of the most well-established techniques in CLRP. NMP is
based on a reversible termination reaction between growing (macro)radical and a free
nitroxide to form a (macro)alkoxyamine. This equilibrium is only governed by temperature.
The conventional initiator for NMP is a preformed alkoxyamine. It consists of 2 parts; a carbon-
centered radical and a nitroxide, which will be separated at elevated temperature to initiate
the polymerization. [16] Nicolas et al [27] synthesized an alkoxyamine conjugated with
gemcitabine (Gem), an anticancer drug active against various tumors, and utilized it as an
initiator for NMP of isoprene, resulted in gemcitabine-polyisoprene conjugates with
amiphiphilic properties. The controlled growth of polyisoprene led to a library of gemcitabine-
polyisoprene conjugates with different molar masses and where every polymer was linked to
a Gem molecule. These polymer prodrugs were formed into well-dispersed nanoparticles by
nanoprecipitation, which exhibited significant anticancer activity both in vitro and in vivo.
RAFT polymerization is a reversible reaction between a radical and a dormant RAFT
agent which creates the RAFT equilibrium. The RAFT agent is a thiocarbonylthio group such as
a trithiocarbonate. The reaction needs an initiator, which is a radical, formed by the
conversion of the growing radical to the dormant species. This will produce an intermediate
radical and its fragmentation will initiate the polymerization. [16] [30] Nicolas et al [28] used
a RAFT agent conjugated to gemcitabine for the polymerization of squalenyl methacrylate
(SqMA) to produce a series of polymer prodrugs with controlled molar mass. The resulting
polymer-drug conjugates were self-assembled into nanoparticles because of their
amphiphilicity. These nanoparticles are stable, they contain a narrow dispersity on the size
13
and they show an adequate cytotoxicity in vitro on several cancer cell lines. More importantly,
the polymer prodrug nanoparticles also show significant anticancer efficacy in vivo. [32]
1.4. POLYMERS
In this section the different polymers used to make the prodrug polymer nanoparticles
are described.
1.4.1. Polyisoprene
Polyisoprene (Figure 1.7) is one of the most well-known natural
polymers (natural rubber). The properties of polyisoprene make it
interesting for the use in nanoparticles for drug delivery, because it is
enzymatically and chemically degradable, biocompatible and its
structure is similar to naturally occurring polyisoprenoids. The basic
structure of naturally occurring terpenes is isoprene, for this reason
synthetic polyisoprene can be used in biomedical applications. [27]
1.4.2. Poly(squalenyl-methacrylate)
Before the use of poly(squalenyl methacrylate), squalene
was used to make self-assembled nanostructures in aqueous
solution. Squalene is a precursor of cholesterol biosynthesis
which was used for the synthesis of prodrugs with promising in
vivo results for several pathologies. [28], [33], [34] However,
there were important limitations for this approach such as the
rigidity of the synthetic pathway and the relative colloidal instability upon PEGylation. These
drawbacks are improved by the use of a methacrylate monomer based on squalene (SqMA)
(Figure 1.8), polymerized by reversible addition-fragmentation chain transfer (RAFT) into the
hydrophobic poly(squalenyl methacrylate). [28]
Figure 1.7 Structure of polyisoprene [43]
Figure 1.8 Structure of the monomer SqMA [28]
14
1.4.3. Poly[oligo (ethylene glycol) methacrylate] (POEGMA)
Poly[oligo (ethylene glycol) methacrylate] (POEGMA) is a new class of biocompatible,
not toxic and water-soluble polymers, polymerized by the monomer oligo (ethylene glycol)
methacrylate (OEGMA) (Figure 1.9). This class of polymers are produced by controlled/living
radical polymerization (CLRP) where the backbone is a
hydrophobic polymethacrylate and the amphiphilic side
chains consist of oligo(ethylene glycol) moieties.
Modification of the chain length can alter the hydrophilicity
of the polymer. [35], [36]
1.5. DRUGS
The drugs used in this research are anticancer drugs: Cladribine and Paclitaxel. In our
research the drug was directly linked to the polymer in some cases and in other cases, we used
a diglycolic linker to link the drug to the polymer.
1.5.1. Cladribine
Cladribine is a first-line anticancer drug to treat hairy cell leukemia. Hairy cell leukemia
is a rare form of leukemia which is associated with an enlargement of the spleen, recurring
infections and a reduction of red blood cells, white blood cells and platelets. The diagnosis of
hairy cell leukemia is confirmed by measuring the mineral bone density
through flow cytometrie. Cladribine or 2-chlorodeoxyadenosine
(Figure 1.10) is easy to administer, because it is a hydrophilic molecule,
which makes it easy to be absorbed in the blood. This molecule is a
chlorinated deoxyadenosine, which makes it resistant to degradation
by adenosine deaminase. This chlorine function makes sure the
Cladribine accumulates in the lymphocytes as Cladribine-ATP. The ATP
and Cladribine-ATP induce an accumulation of DNA strands, which
leads to apoptosis of the lymphocyte. [37]–[39]
Figure 1.10: Structure of Cladribine [45]
Figure 1.9 Structure of the monomer OEGMA [44]
15
1.5.2. Paclitaxel
Paclitaxel or taxol® (Figure 1.11) is a chemotherapeutic drug, derived from Taxus spp.
Paclitaxel causes stabilisation of the microtubules in cells by inhibiting the mitosis in the G2/M
phase, leading to apoptosis. This will happen without interfering with the synthesis (S) phase.
Microtubules are dynamic and unstable structures with
several cellular functions such as cell shape, cell division and
transport in the cell. Microtubules are heterodimers
consisting of an α and β subunit. The Paclitaxel will bind to
the β subunit to stabilize the microtubule and inhibit the
mitosis. This cytotoxicity is time- and concentration-
dependent.
Paclitaxel is used for a variety of cancers such as non-small cell lung cancer, AIDS related
Kaposi sarcoma, ovarian cancer and breast cancer. In some research there have been results
of a positive effect to use Paclitaxel in B-cell leukemia 2 (Bcl2). Paclitaxel, being a very
hydrophobic molecule, is difficult to administer and has several adverse effects. The
administration can be simplified by using nanocarriers. Paclitaxel may result in neutropenia,
cardiotoxicity and peripheral neuropathy. That is why it is advised to follow up on the quantity
of neutrophils. [40]–[42]
Figure 1.11: Structure of Paclitaxel [41]
16
2. OBJECTIVES
Chemotherapeutic drugs often provide severe side effects. These side effects can be
restricted by targeted drug delivery to limit the amount of drug that is distributed over the
body and leads to side effects. As described previously, polymer prodrug nanoparticles have
exhibited significant anticancer activity. They benefit from the diversity of the macromolecular
design and synthesis, and the biocompatibility of polymers. Especially for the polymer prodrug
nanoparticles prepared by the “drug-initiated” living/controlled radical polymerization, the
drug-loading can be controlled precisely and both in vitro and in vivo anticancer activity were
obtained.
The aim of this research is to further prove the versatility of this “drug-initiated”
prodrug-construction approach. There has already been done a lot of research with
Gemcitabine, but not with other drugs. We aim to develop new polymer-drug conjugates and
investigate the anticancer activity of the nanoparticles formed by polymer-drug conjugates.
These were made utilizing new drugs besides Gemcitabine, such as Cladribine and Paclitaxel,
linked with various polymer groups, such as polyisoprene, poly(squalenyl methacrylate), and
poly[oligo (ethylene glycol) methacrylate].
The polymers were developed and linked to the drug through drug-initiated
controlled/living radical polymerization, especially by nitroxide-mediated polymerization
(NMP) and reversible addition-fragmentation chain transfer (RAFT) polymerization. These
polymers were used to form nanoparticles by using the nanoprecipitation technique.
The nanoparticles were tested and characterized by dynamic light scattering (DLS) and
the zeta sizer. The diameter, polydispersity index (PDI) and zeta potential were measured and
the stability of the nanoparticles was observed over time. Furthermore the cytotoxicity was
measured by in vitro experiments on L1210, A549 and MCF-7 cell lines. The MTT test was used
to measure the in vitro anticancer activity and to calculate the IC50 values.
17
3. MATERIALS AND METHODS
3.1. MATERIALS
Cladribine and Paclitaxel were purchased from Sequoia Research Products Limited (UK).
Dulbecco’s modified eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased
from Dulbecco (Invitrogen, France). Penicillin was purchased from Lonza (Verviers, Belgium).
All other materials were purchased from Aldrich at the highest available purity and used as
received.
The polymer prodrugs were synthesized by Dr. Yinyin BAO in the laboratory (figure 3.1 -
3.7). Tert-butyldimethylsilyl (TBDMS) was used to selectively protect the primary hydroxyl
group in Cladribine prior the polymerization. After polymerization tetra-n-butylammonium
fluoride (TBAF) was used to deprotect the hydroxyl group. 2-[N-tert-butyl-N-(1-
diethoxyphosphoryl-2,2-dimethylpropyl) aminooxy] propanoic acid (AMA-SG1) and
diglycolate-AMA-SG1 (d-AMA-SG1) counterpart are alkoxyamines that are linked to the drug
to initiate the NMP to obtain the polyisoprene (PI) drug conjugates. POEGMA and PSqMA were
synthesized by RAFT polymerization, using 4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl]
pentanoic acid (CDP) linked to the drug as a RAFT agent and azobisisobutyronitrile (AIBN) as a
radical initiator. In all these synthesis reactions dioxane was used as a solvent. Due to the
amphiphilic nature of Cla-PI, Cla-d-PI, Cla-PSqMA and Cla-d-PSqMA, tetrahydrofuran (THF)
was used to solubilize the deprotected conjugates. Similar syntheses have been performed
from paxclitaxel (Ptx) as a drug and from Naphthalimide (Napth) as a fluorescent dye for
aggregation-induced emission (AIE) purposes.
Figure 3.1 synthesis scheme of Cla-PI
18
Figure 3.2 synthesis scheme of Cla-d-PI
Figure 3.3 Synthesis scheme of Cla-PSqMA
Figure 3.4 Synthesis scheme Cla-d-PSqMA
19
Figure 3.5 Synthesis scheme of Ptx-PI
Figure 3.6 Synthesis of Ptx-POEGMA
Figure 3.7 Synthesis of Napht-PI
20
3.2. NANOPARTICLE FORMATION
The nanoparticles were formed using the nanoprecipitation technique. 1,2 mg of
polymer was dissolved in 240 µl tetrahydrofuran (THF). The THF was added to 480µl water
(MilliQ water) drop by drop while stirring. The ratio of mixture solvent THF/water (v/v) was
1/2. To make sure the cytotoxic effect is due to the nanoparticles, the tetrahydrofuran needed
to be removed completely. The THF was evaporated at ambient temperature with a rotavapor.
After approximately 20 min, the THF was totally removed. This protocol was used for most of
the polymers, but a lower concentration of 0,1 mg/ml or 1 mg/ml was used for some polymers
due to the large size or high cytotoxicity (Table 3.1).
Table 3.1 Theoretical polymer concentrations
Polymer Concentration (mg/ml)
Polymer Concentration (mg/ml)
Cla-d-PI
Cla-d-PI 3980 2.5 Cla-d-PI 2960 2.5
Cla-PI
Cla-PI 1410 2.5 Cla-PI 2270 2.5
Cla-PI 1560 2.5 Cla-PI 2520 2.5
Cla-d-PSqMA
Cla-d-PSqMA 3590 2.5 Cla-d-PSqMA 4470 2.5
Cla-d-PSqMA 3090 2.5 Cla-d-PSqMA 2920 2.5
Cla-d-PSqMA 3180 2.5
Cla-PSqMA
Cla-PSqMA 2710 2.5 Cla-PSqMA 3420 2.5
Cla-PSqMA 3080 2.5 Cla-PSqMA 4040 2.5
Cla-PSqMA 3340 2.5
Ptx-d-POEGMA
Ptx-d-POEGMA 5270
1 Ptx-d-POEGMA 8020 2.5
Ptx-d-PI
Ptx-d-PI 3560 2.5 Ptx-d-PI 2790 1
Ptx-d-PI 2640 1
Napht-PI
Napht-PI 2070 2.5 Napht-PI 3710 2.5
Napht-PI 2070 0.1 Napht-PI 3710 0.1
Napht-PI 2070 1 Napht-PI 3710 1
21
To make nanoparticles with a concentration of 0,1 mg/ml, the weight of the polymer
was approximately 0,3 mg to avoid making a mistake by weighing the polymer. The 0,3 mg
was dissolved in 1500 µl THF and only 500 µl THF was added to 1000 µl H2O. The preparation
of nanoparticles with a concentration of 1 mg/ml proceeded similarly to the preparation of
2,5 mg/ml. For 1 mg/ml, approximately 1mg was weighed, dissolved in 500 µl THF and added
to 1000 µl H20.
3.3. DYNAMIC LIGHT SCATTERING (DLS) AND ZETA POTENTIAL
Nanoparticle diameters (Dz) and zeta potentials (ζ) were measured by dynamic light
scattering (DLS) with a zetasizer Nano ZS from Malvern (173° scattering angle) (Malvern
Instruments Ltd, Worcestershire, UK) at a temperature of 25 °C. The surface charge of the
nanoparticles was investigated by ζ-potential (mV) measurement at 25 °C after dilution with
1 mM NaCl, using the Henry equation with a Smoluchowski approximation.
The size measurements are based on the Brownian motion of the particles. A laser emits
light, which encounters particles in the solution that scatter the light. The zetasizer nano ZS
measures the fluctuations in the intensity of the scattered light allowing to detect the
Brownian motions of the particles. The size of the particles is calculated according to the
Stokes-Einstein equation which explains the relationship between the speed, caused by the
Brownian motion, and the size of a particle. Based on the fluctuations in intensity it is possible
to see a size distribution and to calculate the range of the particle-size distribution (PSD) or
polydispersity index (PDI).
In order to measure the size of the nanoparticles, the prepared nanoparticle solutions
were filtered before doing the measurement to prevent the interference of dust. The solution
was transferred with a glass pipette to a syringe and filtrated with a 0,22 µm filter. For the
measurement, 100 µl of the filtered nanoparticle solution was diluted in 2 ml phosphate
buffered saline (PBS) or in MilliQ water in a disposable cuvette. The polydispersity index (PDI)
was also calculated by the zetasizer during the same measurement. Each measurement was
performed 3 times at 25°C. It was important to know the refraction index of the medium, PBS
or water in this case, to be able to measure the size correctly. These values were saved in the
22
software of the zetasizer, they can also be entered in the software by checking the values in
literature.
A combination of techniques is used to measure the zeta potential, these are
electrophoresis and laser doppler velocimetry. These techniques measure the velocity of a
particle in a fluid located in an electric field. If two known constants, the viscosity and the
dielectric constant, are applied in the Henry equation (equation 3.1) with the velocity of the
particles and the electrical field, the zeta potential can be obtained. The Henry equation is
applied with a Smoluchowski approximation because the measurement occurs in an aqueous
medium and there is a electrolyte concentration of 10-3M.
UE=2ɛ 𝑧 𝑓(𝐾𝑎)
3ɳ (equation 3.1)
UE = electrophoretic mobility or the velocity of a particle
z = zeta potential
ɛ = dielectric constant
ɳ = viscosity
f(Ka) = Henrys function, which is 1,5 if the Smoluchowski approximation
applies
The zeta potential was also measured with the zetasizer. The medium for this
measurement was 1 mM NaCl and the sample solution was filtered. Firstly a dilution was made
by adding 100 µl of filtered nanoparticle solution into 2 ml NaCl solution. Secondly 0,75 ml of
the dilution was injected into the zeta-cuvette with a 1 ml syringe. It is very important to avoid
air bubbles while injecting the dilution to avoid interference with the measurement. Each
measurement was performed 3 times at 25°C.
23
3.4. CELL CULTURE
The anticancer activity of the prodrug nanoparticles was tested in vitro on different cell
lines.
3.4.1. Cell line L1210
The L1210 cell line originated from a lymphocytic leukemia in an 8 years old female
mouse. These cells were grown in suspension in dulbecco’s modified eagle’s medium with an
added 50 ml FBS and 2,5 ml of a mixture of penicillin and streptomycin. The FBS contains a lot
of growth factors and nutrients and the antibiotics are added to prevent the grow of bacteria.
The cell line was renewed every 3 to 4 days. For the renewing of the cell line, 50 000 cells per
ml in a total of 7 ml were used. The cells were transferred from the cell flask to a 50 ml tube
and the amount of cells in 1 µl was counted and calculated using a glasstic slide 10 with grids
(Kova International Inc., Garden Grove, California, USA). The volume of cells necessary to
obtain 350 000 cells was calculated and was added to 7 ml of fresh medium in a new cell flask.
Two flasks were always prepared by renewing the cell line .
3.4.2. Cell line A549
The cell line A549 originated from a lung carcinoma of a 58 year old Caucasian male
human. The cells were adherent and were cultured in dulbecco’s modified eagle’s medium
with high glucose extended with 50 ml FBS and 2,5 ml of a mixture of penicillin and
streptomycin. This cell line was renewed every 3 to 4 days. To renew the cell line, the medium
was removed with a vacuum pump and the cells were washed with 10 ml PBS, which was also
removed with the vacuum pump. Afterwards, 3 ml trypsin was added, the flask was shaken
and incubated. After 5 minutes, 7 ml of medium was added and the suspended cells in 1 µl
were counted and calculated using a glasstic slide 10 with grids (Kova International Inc.,
Garden Grove, California, USA). 50 000 cells per ml were prepared in a volume of 15 ml
medium. The volume containing 750 000 cells was transferred into a Falcon tube and
centrifuged (5 minutes, 2000 rpm). The medium above the cells was removed with the vacuum
pump and the cells were redispersed in 15 ml fresh medium. The renewing of the cell line was
always done in duplicate.
24
3.4.3. Cell line MCF-7
The cell line MCF-7 originated from a adenocarcinoma in the mammary gland of a 69
year old Caucasian female human. These cells were adherent and the renewing of the cell line
happened in the same way as renewing cell line A549.
3.5. IN VITRO ANTICANCER ACTIVITY
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was used to test
cytotoxicity of polymer prodrug nanoparticles and cell viability on cell lines L1210, A549 and
MCF-7. First, two 96-well plates were seeded with cells (5 × 103/well). This happened by
counting the cells in the cell solutions and calculating the number of cells necessary to obtain
5000 cells in every well. The cells were added to the 60 inner wells. In the calculations we used
a number of 70 wells to make enough mixture. The volume of the cell solution needed to
obtain 350 000 cells was added to 7 ml of medium. After mixing the solution, 100 µl was
injected into the inner wells. In the outer 36 wells 100 µl of PBS (phosphate buffer saline) was
added to prevent dehydration of the cells.
After incubation of 1hour for cell line L1210 or 24 hours for cell line A549 and MCF-7,
the cells were then exposed to a series of concentrations of polymer prodrug nanoparticles,
for 72 hours. Before adding them to the wells, several solutions with different concentrations
were made to have a concentration gradient. In each column of the plates, 100 µl of a prodrug
nanoparticle solution with a different concentration was added, starting with medium without
nanoparticles and ending with the highest concentration of nanoparticles.
After drug exposition, 20 μL of MTT solution was added to medium and cells for each
well. For the MTT solution, 50 mg of MTT product (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) was weighed, dissolved in 10 ml PBS and mixed by a vortex.
This solution (5 mg.mL-1 in PBS) was filtered with a 10 ml syringe and a 0,22 µm filter. In every
well 20 µl of this solution was added except for the first well (well 2B), which served as the
blank. After adding the MTT solution, the plates were incubated for an hour at 37°C.
25
For the L1210 cell line, the cells grow in suspension, which means that the plates had to
be centrifuged before the medium was removed by a vacuum pump. Afterwards, 200 µl
dimethyl sulfoxide (DMSO) was added to the remaining living cells to dissolve the precipitates
and the precipitate was scratched from the bottom with a little pipette. The plate was put on
the orbital shaker (150 rpm) covered with aluminium foil to protect it from light. After several
minutes the plate was put on the plate reader (Metertech Σ 960, Fisher Bioblock, Illkirch,
France) and the absorbance was measured. The wavelength for this measurement was
570nm. The A549 and MCF-7 cell lines are adherent, so there was no centrifugation necessary.
The medium was directly removed and DMSO added. The plate was first put on the orbital
shaker (on speed 600 rpm) before the cells were scratched from the bottom with a pipette.
The percentage of surviving cells was calculated as the absorbance ratio of treated to
untreated cells. The inhibitory concentration 50% (IC50) of the treatments was determined
from the dose-response curve. All experiments were set up in duplicate to determine means.
26
4. RESULTS
4.1. NANOPARTICLE FORMATION
The polymer prodrug nanoparticles were obtained by nanoprecipitation of a THF
solution of the different polymer prodrugs into water to target a final concentration of 2.5, 1
or 0.1 mg.mL-1. As shown in the tables below (tables 4.1-4.7), well-dispersed polymer
nanoparticles were formed with average diameters in the 70–240nm range (depending on the
polymer type) and narrow particle-size distributions (PSD) or polydispersity index (PDI = ca.
0.1). In addition, the surface zeta potentials of the polymer nanoparticles were also measured.
4.2. ANALYSIS OF SIZE, PDI AND ZETA POTENTIAL
Different molecular weights were tested for each group of polymers. Each measurement
of size and PDI was performed in water and the zeta potential measurements were performed
in NaCl. These measurements were all performed 1 time, the measurements were not
repeated in time. The results of these experiments are shown in tables 4.1 – 4.7.
4.2.1. Cla-PI and Cla-d-PI
Table 4.1 Analysis Cla-PI nanoparticles
Cla-PI Mn a (g/mol)
Sizeb (nm)
PDIb ζc (mV)
%Clad (wt.%)
P1 1410 182 0.07 -69 20.2
P2 1560 201 0.12 -70 18.3
P3 2270 152 0.08 -64 12.6
P4 2520 137 0.10 -71 11.3
[a] Determined by SEC, calibrated with polystyrene (PS) standards and converted into polyisoprene (PI) by using Mark-Houwink-Sakurada parameters. [b] Determined by DLS. [c] Zeta potential. [d] %Cla=MWCla/Mn,PI
Table 4.2 Analysis Cla-d-PI nanoparticles
Cla-d-PI Mn a
(g/mol) Sizeb (nm)
PDIb ζc (mV)
%Clad (wt.%)
P1 1470 143 0.07 -66 19.4
P2 1710 128 0.10 -68 16.7
P3 2960 127 0.08 -70 9.6
P4 4980 111 0.11 -66 5.7
[a] Determined by SEC, calibrated with polystyrene (PS) standards and converted into polyisoprene (PI) by using Mark-Houwink-Sakurada parameters. [b] Determined by DLS. [c] Zeta potential. [d] %Cla=MWCla/Mn,PI
27
4.2.2. Cla-PSqMA and Cla-d-PSqMA
Table 4.3 Analysis Cla-PSqMA nanoparticles
Cla-PSqMA Mn a
(g/mol) Sizeb (nm)
PDIb ζc (mV)
%Clad (wt.%)
P1 2710 69 0.14 -51 10.5
P2 3080 82 0.11 -54 9.3
P3 3340 88 0.12 -57 8.5
P4 4040 94 0.09 -61 6.4
[a] Determined by SEC, calibrated with poly(methyl methacrylate) (PMMA) standards. [b] Determined by DLS. [c] Zeta potential. [d] %Cla=MWCla/Mn,PI.
Table 4.4 Analysis Cla-d-PSqMA nanoparticles
Cla-d-PSqMA Mn a
(g/mol) Sizeb (nm)
PDIb ζc (mV)
%Clad (wt.%)
P1 2920 112 0.12 -62 9.8
P2 3090 107 0.14 -60 9.2
P3 3180 96 0.12 -54 9.0
P4 4470 84 0.10 -53 6.4
[a] Determined by SEC, calibrated with poly(methyl methacrylate) (PMMA) standards. [b] Determined by DLS. [c] Zeta potential. [d] %Cla=MWCla/Mn,PI.
4.2.3. Ptx-d-PI
Table 4.5 Analysis Ptx-d-PI nanoparticles
Ptx-d-PI Mna
(g/mol)
Sizeb
(nm) PDIb Ζc
(mV) %Ptxd (wt.%)
P1 2640 236 0.10 -76 32.7
P2 2670 217 0.14 -75 32.3
P3 2790 145 0.12 -61 31.0
P4 3560 133 0.11 -63 24.3
P5 4310 110 0.07 -63 20.0
[a] Determined by SEC, calibrated with PS standards and converted into PI by using Mark-Houwink-Sakurada parameters. [b] Determined by DLS. [c] Zeta potential. [d] %Ptx=MWPtx/Mn,PI.
28
4.2.4. Ptx-d-POEGMA
Table 4.6 Analysis Ptx-d-POEGMA nanoparticles
Ptx-d-POEGMA
Mna
(g/mol) Sizeb (nm)
PDIb ζc (mV)
%Ptxd (wt.%)
P1 5270 133 0.06 -37 16.4
P2 8020 148 0.10 -33 10.8
[a] Determined by SEC, calibrated with PS standards and converted into PI by using Mark-Houwink-Sakurada parameters. [b] Determined by DLS. [c] Zeta potential. [d] %Ptx=MWPtx/Mn,PI
4.2.5. Napht-PI
Table 4.7 Analysis Napht-PI nanoparticles
Napht-PI Mna
(g/mol) Sizeb (nm)
PDIb ζc (mV)
P1 2070 197 0.19 -57
P2 3710 146 0.25 -38
[a] Determined by SEC, calibrated with PS standards and converted into PI by using Mark-Houwink-Sakurada parameters. [b] Determined by DLS. [c] Zeta potential.
4.3. SIZE STABILITY
To test the stability over time, the size and PDI of the polymer prodrugs nanoparticles in
different mediums were measured at different time points during a period of time.
4.3.1. Cla-d-PI and Cla-PI
The sizes of Cla-d-PI and Cla-PI were tested over a long period in water and Cla-d-PI was
also tested a short period in PBS. Different molecular weights were prepared and kept in the
fridge at 4°C during the experiment. The same samples were measured several times during
these days. The results of these experiments are shown in graphs 4.1-4.6. Each graph shows
the stability of the nanoparticles made from the polymer prodrugs with different molecular
weights (Mn) by showing the diameter and the polydispersity index of the nanoparticles.
29
Cla-d-PI: stability in water
Cla-PI: stability in water
Cla-d-PI: stability in PBS
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0 10 20 30 40 50 60 70 80 90
PD
I
Time (days)
Cla-d-PI
Mn 2640
Mn 2670
Mn 2790
Mn 3560
135
140
145
150
155
160
165
170
175
0 13 22 56 63
Dia
met
er (
nm
)
Time (days)
Cla-PI
Mn1410
Mn2520
0
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0,08
0,09
0,1
0 13 22 56 63
PD
I
Time (days)
Cla-PI
Mn1410Mn2520
Figure 4.1 Size Cla-d-PI nanoparticles in water Figure 4.2 PDI Cla-d-PI nanoparticles in water
Figure 4.3 Size Cla-PI nanoparticles in water Figure 4.4 PDI Cla-PI nanoparticles in water
Figure 4.6 PDI Cla-d-PI nanoparticles in PBS Figure 4.5 Size Cla-d-PI nanoparticles in PBS
80
90
100
110
120
130
140
150
160
0 10 20 30 40 50 60 70 80 90
Dia
met
er (
nm
)
Time (days)
Cla-d-PI
Mn 2640
Mn 2670
Mn 2790
Mn 3560
100
120
140
160
180
200
220
0 1 2 5 8
Dia
met
er (
nm
)
Time (days)
Cla-d-PI in PBS
Mn 3980
Mn 2960
0
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0 1 2 5 8
PD
I
Time (days)
Cla-d-PI in PBS
Mn 3980
Mn 2960
30
4.3.2. Cla-d-PSqMA
Four polymer samples of Cla-d-PSqMA with different Mn were tested in water once a
week, for a month. The different molecular weights of Cla-d-PSqMA were obtained by tuning
the polymerization time or the ratio of the monomer and the RAFT agent . In graph 4.7 and
4.8 the mean values of size and PDI at different time points are shown.
4.3.3. Ptx-d-PI
Four groups of nanoparticles prepared from Ptx-d-PI polymers with different molecular
weights were tested for several times over a period of 2 months. The stability of these
nanoparticles was tested in water and the results are shown in graph 4.9 and 4.10.
100
110
120
130
140
150
160
170
180
190
200
0 20 40 60
Dia
met
er (
nm
)
Time (days)
Ptx-d-PI
Mn 2640
Mn 2670
Mn 2790
Mn 3560
0
0,02
0,04
0,06
0,08
0,1
0,12
0 20 40 60
PD
I
Time (days)
Ptx-d-PI
Mn 2640
Mn 2670
Mn 2790
Mn 3560
Figure 4.7 Size measurements Cla-d-PSqMA nanoparticles
Figure 4.8 PDI measurements Cla-d-PSqMA nanoparticles
Figure 4.9 Size measurements Ptx-d-PI nanoparticles
Figure 4.10 PDI measurements Ptx-d-PI nanoparticles
70,00
80,00
90,00
100,00
110,00
120,00
130,00
0 7 14 21
Dia
met
er (
nm
)
Time (days)
Cla-d-PSqMA
Mn 3090
Mn 3180
Mn 4470
Mn 2920
0,000
0,050
0,100
0,150
0,200
0 7 14 21
PD
I
Time (days)
Cla-d-PSqMA
Mn 3090
Mn 3180
Mn 4470
Mn 2920
31
4.4. MTT
For each MTT test a concentration gradient of nanoparticles was prepared to add into
the wells. The exact concentrations added to the wells were calculated to make it possible to
obtain an IC50 value to indicate the cytotoxicity. The graphs show the results of the MTT tests,
through the relationship of the cell viability and the concentration of polymer prodrug
nanoparticles. For different groups of polymer prodrugs, we chose different cell lines for the
MTT test according to the anticancer activity of the corresponding drugs. The free drug and
the polymer nanoparticles without drug were also tested to be able to discuss the results.
4.4.1. Cla-d-PSqMA and Cla-PSqMA
Cla-d-PSqMA on L1210
Figure 4.11 Cell viability in function of the concentration of Cla-d-PSqMA nanoparticles
Cla-PSqMA on L1210
Figure 4.12 Cell viability in function of the concentration of Cla-PSqMA nanoparticles
-0,2
0
0,2
0,4
0,6
0,8
1
1,2
Cel
l via
bili
ty (
%)
Concentration (µM)
Cla-d-PSqMA
Free Cla
Mn 2920
Mn 3180
Mn 4470
0
0,2
0,4
0,6
0,8
1
1,2
Cel
l via
bili
ty (
%)
Concentration (µM)
MTT Cla-PSqMA
Free Cla
Mn 2710
Mn 3340
Mn 4040
32
4.4.2. Ptx-d-PI
Figure 4.13 Cell viability in function of the concentration of Ptx-d-PI nanoparticles tested on the A549 cell line
4.4.3. Ptx-d-POEGMA
Figure 4.14 Cell viability in function of the concentration of Ptx-d-POEGMA nanoparticles on the L1210 cell line
0
0,2
0,4
0,6
0,8
1
1,2
0 0,0005 0,002 0,0125 0,05 0,2 0,1 1 2 5 10
Cel
l via
bili
ty
Concentration (µM)
MTT Ptx-d-PI
Mn 2790
Mn 3560
AMA-d-PI
Free Ptx
0
0,2
0,4
0,6
0,8
1
1,2
0 5,00E-04 0,002 0,0125 0,05 0,2 1 2 5 10
Cel
l via
bili
ty
Concentration (µM)
Ptx-d-POEGMA on L1210
Mn 5270
Mn 8020
Free Ptx
33
Figure 4.15 Cell viability in function of the concentration of Ptx-d-POEGMA nanoparticles on the A549 cell line
Figure 4.16 Cell viability in function of the concentration Ptx-d-POEGMA nanoparticles on the MCF-7 cell line
0
0,2
0,4
0,6
0,8
1
1,2
Cel
l via
bili
ty
Concentration (µM)
Ptx-d-POEGMA (A549)
Mn 5270
Mn 8020
CDP-d-POEGMA
Free Ptx
0
0,2
0,4
0,6
0,8
1
1,2
0 5,00E-04 0,002 0,0125 0,05 0,2 1 5
Cel
l via
bili
ty
Concentration (µM)
Ptx-d-POEGMA (MCF-7)
Mn 5270
34
4.4.4. Napht-PI
Figure 4.17 Cell viability in function of the concentration of Napht-PI nanoparticles tested on the L1210 cell line
4.4.5. IC50 values
Table 4.8 IC50 values of the MTT experiments
IC50 values (µM)
Free Cla (L1210) 0.13 Free Ptx (L1210) 0.023 Free Ptx (A549) 0.0084 Cla-d-PSqMA Mn 2920 2.0 Mn 3180 2.1 Mn 4470 1.4 Cla-PSqMA Mn 2710 6.4 Mn 3340 8.0 Mn 4040 7.1 Ptx-d-PI Mn 2790 0.12 Mn 3560 0.033 Ptx-d-POEGMA Mn 5270 (L1210) 0.045 Mn 5270 (A549) 0.0059
Mn 5270 (MCF-7) 0.0091 Mn 8020 (L1210) 0.064 Mn 8020 (A549) 0.010
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
0 12,5 25 62,5 125 200 250
Cel
l via
bili
ty
Concentration (µg/ml)
Napht-PI
Mn 2070
Mn 3710
35
5. DISCUSSION
5.1. MEDICINE
The aim of this research is to further prove the diversity of this “drug-initiated” prodrug-
construction approach and to improve the important issues of anticancer drugs and the
anticancer behaviour. For this reason two anticancer drugs Cladribine and Paclitaxel were
chosen to prepare polymer prodrugs nanoparticles by modulating the drug/polymer
conjugate, amphiphilic properties, and polymer chain length. Considering that Cladribine is
not subjected to deamination in vivo, conversely to other nucleoside analogues such as
gemcitabine, it is more convenient to design and synthesize new initiators or RAFT agents
conjugated with Cladribine. Paclitaxel, on the other hand, is a highly efficient anticancer drug
with hydrophobic properties and limited water solubility. It would be interesting to investigate
the water stability and anticancer efficacy of the formed nanoparticles after conjugation with
hydrophobic or hydrophilic polymers through a “drug-initiated” strategy.
5.2. SIZE AND ZETA POTENTIAL
It should be noted that the diameter of the nanoparticles decreased when the polymer
chains were extended, which means the size can be tuned by controlling the molar mass of
the polymers. According to the literature, longer chains contain more functional groups, which
ensures more interactions with other polymers and denser nanoparticles. [28] The size of the
nanoparticles is important for the uptake in the cells. If the nanoparticles are too big, there
will not be a good uptake by the cells. If they become too small, there will be a higher chance
of an immune reaction. The optimal size is around 100nm. To be sure the size of the
nanoparticles is homogeneous, the polydispersity index (PDI) should be below 0,2, which is
fulfilled for all the polymers (tables 4.1-4.7). The zeta potential should be around -60 mV for
nanoparticles made of hydrophobic polymers and it should be around -35mV for nanoparticles
made of hydrophilic polymers. This indicates an efficient electrostatic stabilization provided
by the strongly negative surface charges, due to the amphiphilic property of the polymers. In
tables 4.1-4.7 the drug loading of Cladribine and Paclitaxel on different polymers is shown and
we can conclude that the drug loading decreases by increasing the chain length.
36
5.2.1. Cla-PI and Cla-d-PI
For Cla-PI and Cla-d-PI nanoparticles (Table 4.11 and Table 4.22), the size is between 110
and 200 nm and the PDI is around 0.1, which means well-dispersed polymer nanoparticles
were formed. We can see the drug loading decreases when the molecular weight increases.
The zeta potential of both nanoparticles is similar and they are all around -60mV.
5.2.2. Cla-PSqMA and Cla-d-PSqMA
The results of the analysis of Cla-PSqMA and Cla-d-PSqMA nanoparticles (Table 4.33 and
Table 4.44) show a PDI around 0.1, which means there is a homogeneous dispersity of the size.
The size and drug loading decrease when the molecular weight increases. The zeta potential
is about -60mV, which makes it a electrostatically stable nanoparticle.
5.2.3. Ptx-d-PI
The measurements of Ptx-d-PI nanoparticles (Table 4.55) show a wide range in diameter
of the nanoparticles when the molecular weight increases. The PDI is around 0.1 and the zeta
potential is about -60 mV, indicating the electrostatical stability. It is possible the wide range
of the size changes is related to intense of the aggregated hydrophobic polyisoprene in
aqueous solution. The drug loading increases when the length of the polymer chains decreases
and the drug loading is higher compared to the drug loading of Cla-d-PI nanoparticles. This is
due to the difference in molecular weight of Paclitaxel (854 g/mol) and Cladribine (286 g/mol).
5.2.4. Ptx-d-POEGMA
For the measurements of Ptx-d-POEGMA nanoparticles (Table 4.66), the size increases
when the molecular weight increases, which is opposite to the phenomenon observed with
the other polymer prodrug nanoparticles. The reason for this difference is that POEGMA-
polymer is hydrophilic while the other polymers are hydrophobic. When the molecular weight
increases for hydrophilic polymers, it leads to weaker interactions between the polymer
chains, resulting in higher size nanoparticles. There are homogenous dispersity’s of size
because the PDI is below 0.2. The zeta potential for these nanoparticles is about -35 mV, which
is lower compared to the other nanoparticles because the POEGMA polymer contains
37
different functional groups than the polyisoprene and poly(squalenyl methacrylate) polymers.
The drug loading decreases when the molecular weight increases.
5.2.5. Napht-PI
For all the measurements of size and PDI a concentration of 2,5 mg/ml was used except
for the measurements of Napht-PI (Table 4.77). A lower concentration (0,1 mg/ml) was used
to perform these experiments because this is a very hydrophobic molecule and a fluorescent
polymer with a diagnostic purpose, which makes it unnecessary to test this polymer in such a
high concentration. The size of these nanoparticles decreases when the molecular weight
increases. There is a wide range in zeta potential and the PDI values are about 0.2, because
these nanoparticles are not very stable. The stability of the nanoparticles could be further
increased by modifying the chemical structures of Naphthalimide-based fluorescent dyes.
5.3. SIZE STABILITY
5.3.1. Cla-PI and Cla-d-PI
The size stability was tested for Cla-PI and Cla-d-PI in water (Figure 4.1 - 4.4). The size of
these nanoparticles stayed unchanged with the PDI below 0.2 for more than two months,
which means these nanoparticles are highly stable in water due to their amphiphilic nature.
For Cla-d-PI in PBS (see Figure 4.5 - 4.6), there is a slight increase of size with PDI below 0.1
within 5 days for both of the two polymers, which is much more stable than the reported
polymer nanoparticles prepared by “drug-initiated” method in literature, without the
presence of any stabilizer.[23] These nanoparticles were tested in water in order to verify the
stability of the nanoparticles stored in water. The experiment in PBS was shorter, because the
purpose was to verify the stability of the nanoparticles for injection or circulation.
5.3.2. Cla-d-PSqMA
The size stability experiments were also conducted in water for Cla-PSqMA (Figure 4.7 –
4.8), the size of which stayed in the same level for 3 weeks with PDI below 0.2, indicated these
nanoparticles are highly stable in water due to their amphiphilic structure.
38
5.3.3. Ptx-d-PI
The results of the experiments to test the stability of polymer nanoparticles based on
Ptx-d-PI are shown as Figure 4.9 and Figure 4.10 Different from the polymers conjugated with
Cladribine, Ptx-d-PI is all hydrophobic, however, all of the polymer nanoparticles exhibited
high stability in water for more than two months, without the presence of any stabilizer. It is
a great advantage compared with the reported polymer nanoparticles prepared from “drug-
initiated” ring-opening polymerization, which always needs PEGylation to stabilize the
nanoparticles.
5.4. CELL CULTURE
In cell culture there are two different methods to renew the cell line (see section 3.4),
depending on the way the cells grow. In the L1210 cell line the cells grow in suspension and
the A549 cell line is adherent. The A549 cells need to be unattached from the bottom to work
with them by using trypsin, which is a protease that cleaves the proteins that attach the cell
to the bottom of the flask. To optimize the activity of trypsin, the cells are washed with PBS
before the trypsin is added. This washing step removed the remaining nutrients of the
medium, which makes it easier for trypsin to loosen the cells from the bottom. To make the
cell plates, trypsin is removed by centrifugation before adding fresh culture medium.
5.5. MTT
5.5.1. Cla-PSqMA and Cla-d-PSqMA
The cytotoxicity of Cla-PSqMA, Cla-d-PSqMA and free Cladribine were tested in vitro on
leukemia cells (L1210) by MTT, considering that Cladribine is used to treat leukemia. The MTT
of the polymer nanoparticle without the drug can be found in the literature [28] and shows
no significant cytotoxicity. The MTT test of Cla-d-PSqMA (Figure 4.11) shows the nanoparticles
prepared from Cla-d-PSqMA with exhibited obvious cytotoxicity with IC50 values of 1.4, 2.1,
and 2.0 µM for the polymers with Mn of 4470, 3180, and 2920 g/mol, respectively, which are
lower than the IC50 value of Cladribine free drug, due to the prodrug property. On the other
hand, Cla-PSqMA (Figure 4.12) showed much lower cytotoxicity for all of the polymers with
the molecular weights of 2710 , 3340 and 4040 g/mol, the IC50 values of which are higher than
39
5 µM. These results indicated the significant importance of the existence of the diglycolic
linker, which promotes more efficient drug delivery and anticancer activity.
5.5.2. Ptx-d-PI
The in vitro cytotoxicity of the polymer nanoparticles based on Ptx-d-PI was tested by
MTT on lung cancer cells (A549). As shown in Figure 4.13, both of the two polymers with a
molecular weight of 2790 and 3560 g/mol exhibited significant cytotoxicty to the A549 cells,
and the polymer with higher Mn showed a lower IC50 value (0.033) compared with the polymer
with lower Mn (0.12). Thus the cytotoxicity of these polymer prodrugs can be tuned by
changing the polymer chain length.
5.5.3. Ptx-d-POEGMA
The in vitro cytotoxicity of the polymer nanoparticles based on Ptx-d-POEGMA was
tested by MTT on the L1210, A549 and MCF-7 cell lines. As shown in Figure 4.15 both of the
two polymers with molecular weight of 5270 and 8020 g/mol exhibited significant cytotoxicty
to the A549 cells. The IC50 value of the polymer nanoparticles (Mn 5270) is even at the same
level with the Paclitaxel free drug, indicated the great potential for further in vivo anticancer
application. This is confirmed by the result on the MCF-7 cells for the polymer of 5270 g/mol
(Figure 4.16). The cytotoxicity of the polymers with molecular weight of 5270 and 8020 g/mol
is lower when they are tested on the L1210 cells (Figure 4.14). This is probably because
Paclitaxel is more active on lung cancer (A549) and breast cancer cells (MCF-7) compared to
leukemia cells (L1210).
5.5.4. Napht-PI
As shown in Figure 4.17, the MTT of Napht-PI-based polymer nanoparticles were
conducted on L1210 cell line. These polymers made from fluorescent nontoxic initiators didn’t
show any cytotoxicity even at very high concentration up to 250 µg/mL, which are ideal tools
for further imaging and diagnostic application.
40
6. CONCLUSION
The aim of this research was to develop and characterize new polymer prodrug
nanoparticles prepared from “drug-initiated” living/controlled radical polymerization utilizing
anticancer drugs Cladribine and Paclitaxel. The size, zeta potential and stability over time of
different groups of polymer nanoparticles was measured and the in vitro cytotoxicity was
investigated. The size of the nanoparticles and the drug loading are tuneable by changing the
length of the polymer chains, the longer the polymer chains, the smaller the size of the
nanoparticles and the lower the drug loading. The nanoparticles possessed highly negative
zeta potential up to -70 mV, indicated the good stability, which was further confirmed by the
stability test experiments.
Both Cladribine-based and Paclitaxel-based polymer prodrug nanoparticles exhibited
significant in vitro anticancer activity on L1210, A549, and/or MCF-7 cell lines. The cytotoxicity
of the polymers can be tuned by changing the polymer chain length, which provides an
efficient tool to optimise the anticancer efficacy of this type of polymer prodrugs. Not only the
amphiphilic polymers (Cla-d-PI or Cla-d-PSqMA) based on Cladribine, but more interestingly,
also the all-hydrophobic polymers based on Paclitaxel (Ptx-d-PI) showed highly stable colloid
property, which afford the new polymer prodrugs great advantage compared with the
reported examples in literature.
It should be pointed out that the polymer nanoparticles made from Ptx-d-POEGMA
exhibited extremely high in vitro anticancer activity, the IC50 value of which is at the same level
with free Paclitaxel drug, indicating the great potential for in vivo anticancer application.
The next step in this research is to conduct in vivo experiments to confirm the possible
use of these nanoparticles to treat cancer in humans. The aim of this research is to eventually
use nanoparticles with Cladribine or Paclitaxel to cure cancer with a limited amount of side
effects.
41
7. REFERENCES
[1] National institute for cancer, “What is cancer?,” 2015. [Online]. Available:
http://www.cancer.gov/about-cancer/what-is-cancer. [Accessed: 15-Feb-2016].
[2] B. Sadikovic, K. Al-Romaih, J. a Squire, and M. Zielenska, “Cause and consequences of
genetic and epigenetic alterations in human cancer.,” Curr. Genomics, vol. 9, no. 6, pp.
394–408, 2008.
[3] M. Macaluso, M. G. Paggi, and A. Giordano, “Genetic and epigenetic alterations as
hallmarks of the intricate road to cancer.,” Oncogene, vol. 22, no. 42, pp. 6472–6478,
2003.
[4] D. Hanahan and R. A. Weinberg, “Hallmarks of cancer: The next generation,” Cell, vol.
144, no. 5, pp. 646–674, 2011.
[5] D. Hanahan and R. A. Weinberg, “The Hallmarks of Cancer,” Cell, vol. 100, no. 1, pp. 57–
70, Jan. 2000.
[6] M. Estanqueiro, M. H. Amaral, J. Conceição, and J. M. Sousa Lobo, “Nanotechnological
carriers for cancer chemotherapy: The state of the art,” Colloids Surfaces B
Biointerfaces, vol. 126, pp. 631–648, 2015.
[7] R. H. Prabhu, V. B. Patravale, and M. D. Joshi, “Polymeric nanoparticles for targeted
treatment in oncology: current insights.,” Int. J. Nanomedicine, vol. 10, pp. 1001–18,
2015.
[8] H. Kobayashi, B. Turkbey, R. Watanabe, and P. L. Choyke, “Cancer drug delivery:
Considerations in the rational design of nanosized bioconjugates,” Bioconjug. Chem.,
vol. 25, no. 12, pp. 2093–2100, 2014.
[9] S. K. Sriraman, B. Aryasomayajula, and V. P. Torchilin, “Barriers to drug delivery in solid
tumors.,” Tissue Barriers, vol. 2, no. e29528, pp. 1–10, 2014.
[10] M. C. Garnett and P. Kallinteri, “Nanomedicines and nanotoxicology: Some
physiological principles,” Occup. Med. (Chic. Ill)., vol. 56, no. 5, pp. 307–311, 2006.
[11] a Urruticoechea, R. Alemany, J. Balart, a Villanueva, F. Viñals, and G. Capellá, “Recent
advances in cancer therapy: an overview.,” Curr. Pharm. Des., vol. 16, no. 1, pp. 3–10,
2010.
[12] National institute for cancer, “radiationtherapy for cancer,” 2010. [Online]. Available:
42
http://www.cancer.gov/about-cancer/treatment/types/radiation-therapy/radiation-
fact-sheet. [Accessed: 18-Feb-2016].
[13] E. Pérez-Herrero and A. Fernandez-Medarde, “Advanced targeted therapies in cancer:
Drug nanocarriers, the future of chemotherapy,” Eur. J. Pharm. Biopharm., vol. 93, pp.
52–79, 2015.
[14] C. Guanying, R. Indrajit, Y. Chunhui, and P. Paras N., “Nanochemistry and Nanomedicine
for Nanoparticle-based Diagnostics and Therapy,” Chem. Rev., no. 116, pp. 2826–2885,
2016.
[15] R. Singh and J. W. Lillard, “Nanoparticle-based targeted drug delivery,” Exp. Mol.
Pathol., vol. 86, no. 3, pp. 215–223, 2009.
[16] J. Nicolas, “Drug-initiated synthesis of polymer prodrugs: Combining simplicity and
efficacy in drug delivery,” Chem. Mater., vol. 28, pp. 1591–1606, 2016.
[17] S. C. Baetke, T. Lammers, and F. Kiessling, “Applications of nanoparticles for diagnosis
and therapy of cancer,” Br. J. Radiol., vol. 88, no. 1054, 2015.
[18] N. Kotagiri and J. W. Kim, “Stealth nanotubes: Strategies of shielding carbon nanotubes
to evade opsonization and improve biodistribution,” Int. J. Nanomedicine, vol. 9, no.
SUPPL.1, pp. 85–105, 2014.
[19] S. Salmaso and P. Caliceti, “Stealth Properties to Improve Therapeutic Efficacy of Drug
Nanocarriers,” J. Drug Deliv., vol. 2013, pp. 1–19, 2013.
[20] J. A. Pachioni-Vasconcelos, A. M. Lopes, A. C. Apolinario, J. K. Valenzuela-Oses, J. S.
Costa, L. O. Nascimento, A. Pessoa, L. R. Barbosa, and C. O. Rangel-Yagui,
“Nanostructures for protein drug delivery,” Biomater Sci, pp. 205–218, 2015.
[21] J. Della Rocca, D. Liu, and W. Lin, “Nanoscale Metal-Organic Frameworks for Biomedical
Imaging and Drug Delivery,” Acc. Chem. Res., vol. 44, no. 10, pp. 957–968, 2013.
[22] X.-G. Wang, Z.-Y. Dong, H. Cheng, S.-S. Wan, W.-H. Chen, M.-Z. Zou, J.-W. Huo, H.-X.
Deng, and X.-Z. Zhang, “A multifunctional metal–organic framework based tumor
targeting drug delivery system for cancer therapy,” Nanoscale, vol. 7, no. 38, pp.
16061–16070, 2015.
[23] R. Tong and J. Cheng, “Paclitaxel-initiated, controlled polymerization of lactide for the
formulation of polymeric nanoparticulate delivery vehicles,” Angew. Chemie - Int. Ed.,
vol. 47, no. 26, pp. 4830–4834, 2008.
43
[24] R. Tong and J. Cheng, “Ring-opening polymerization-mediated controlled formulation
of polylactide-drug nanoparticles,” J. Am. Chem. Soc., vol. 131, no. 13, pp. 4744–4754,
2009.
[25] R. Tong and J. Cheng, “Drug-initiated, controlled ring-opening polymerization for the
synthesis of polymer-drug conjugates,” Macromolecules, vol. 45, no. 5, pp. 2225–2232,
2012.
[26] Q. Yin, R. Tong, Y. Xu, K. Baek, L. W. Dobrucki, T. M. Fan, and J. Cheng, “Drug-Initiated
Ring-Opening Polymerization of O -Carboxyanhydrides for the Preparation of
Anticancer Drug–Poly( O -carboxyanhydride) Nanoconjugates,” Biomacromolecules,
vol. 14, no. 3, pp. 920–929, 2013.
[27] S. Harrisson, J. Nicolas, A. Maksimenko, D. T. Bui, J. Mougin, and P. Couvreur,
“Nanoparticles with in vivo anticancer activity from polymer prodrug amphiphiles
prepared by living radical polymerization,” Angew. Chemie - Int. Ed., vol. 52, no. 6, pp.
1678–1682, 2013.
[28] D. Trung Bui, A. Maksimenko, D. Desmaële, S. Harrisson, C. Vauthier, P. Couvreur, and
J. Nicolas, “Polymer prodrug nanoparticles based on naturally occurring isoprenoid for
anticancer therapy,” Biomacromolecules, vol. 14, no. 8, pp. 2837–2847, 2013.
[29] D. Chakraborty, B. Rajashekhar, S. K. Roymuhury, and R. V, “Group 4 metal complexes
of Trost’s semi-crown ligand: Synthesis, structural characterization and studies on the
ring-opening polymerization of lactides and ε-caprolactone,” Dalt. Trans., vol. 4, no. 8,
pp. 1166–1169, 2015.
[30] H.-S. Wang, M. Song, and T.-J. Hang, “Functional Interfaces Constructed by
Controlled/Living Radical Polymerization for Analytical Chemistry,” ACS Appl. Mater.
Interfaces, p. acsami.5b10465, 2016.
[31] V. Delplace, P. Couvreur, and J. Nicolas, “Recent trends in the design of anticancer
polymer prodrug nanocarriers,” Polym. Chem., vol. 5, pp. 1529–1544, 2014.
[32] A. Maksimenko, D. T. Bui, P. Couvreur, and J. Nicolas, “Signi fi cant Tumor Growth
Inhibition from Naturally Occurring Lipid- Containing Polymer Prodrug Nanoparticles
Obtained by the Drug- Initiated Method,” Chem. Mater., 2014.
[33] L. H. Reddy, P.-E. Marque, C. Dubernet, S.-L. Mouelhi, D. Desmaële, and P. Couvreur,
“Preclinical toxicology (subacute and acute) and efficacy of a new squalenoyl
44
gemcitabine anticancer nanomedicine.,” J. Pharmacol. Exp. Ther., vol. 325, no. 2, pp.
484–490, 2008.
[34] J. L. Arias, L. H. Reddy, M. Othman, B. Gillet, D. Desmaële, F. Zouhiri, F. Dosio, R. Gref,
and P. Couvreur, “Squalene based nanocomposites: A new platform for the design of
multifunctional pharmaceutical theragnostics,” ACS Nano, vol. 5, no. 2, pp. 1513–1521,
2011.
[35] N. Grishkewich, S. Parinaz, Z. Yao, R. Berry, and K. C. Tam, “Cellulose nanocrystal-poly (
oligo ( ethylene glycol ) methacrylate ) brushes with tunable LCSTs,” Carbohydr. Polym.,
vol. 144, pp. 215–222, 2016.
[36] M. Liu, J. C. Leroux, and M. A. Gauthier, “Conformation-function relationships for the
comb-shaped polymer pOEGMA,” Prog. Polym. Sci., vol. 48, pp. 111–121, 2014.
[37] J. B. Johnston, “Mechanism of action of pentostatin and cladribine in hairy cell
leukemia.,” Leuk. Lymphoma, vol. 52 Suppl 2, no. June, pp. 43–45, 2011.
[38] E. Huynh, D. Sigal, and A. Saven, “Cladribine in the treatment of hairy cell leukemia:
initial and subsequent results.,” Leuk. Lymphoma, vol. 50 Suppl 1, no. March, pp. 12–7,
2009.
[39] O. Khorshid, A. E. Namour, M. M. El-gammal, T. Y. Mahmoud, and C. Fortpied, “Efficacy
and Safety of Cladribine : Subcutaneous versus Intravenous Administration in Hairy Cell
Leukemia Patients,” pp. 1–8, 2015.
[40] C. Ferlini, G. Raspaglio, S. Mozzetti, M. Distefano, F. Filippetti, E. Martinelli, G.
Ferrandina, D. Gallo, F. O. Ranelletti, and G. Scambia, “Bcl-2 down-regulation is a novel
mechanism of paclitaxel resistance.,” Mol. Pharmacol., vol. 64, no. 1, pp. 51–58, 2003.
[41] A. Barbuti and Z.-S. Chen, “Paclitaxel Through the Ages of Anticancer Therapy: Exploring
Its Role in Chemoresistance and Radiation Therapy,” Cancers (Basel)., vol. 7, no. 4, pp.
2360–2371, 2015.
[42] J. Gligorov and J. P. Lotz, “Preclinical Pharmacology of the Taxanes : Implications of the
Differences,” Oncologist, vol. 9, no. suppl 2, pp. 3–8, 2004.
[43] Sigma-Aldrich, “Polyisoprene,” 2016. [Online]. Available:
http://www.sigmaaldrich.com/catalog/product/aldrich/431265?lang=fr®ion=FR.
[Accessed: 27-May-2016].
[44] Sigma-Aldrich, “Poly(ethylene glycol) methacrylate.” [Online]. Available:
45
http://www.sigmaaldrich.com/catalog/product/aldrich/409537?lang=fr®ion=FR.
[Accessed: 27-May-2016].
[45] Sigma-Aldrich, “Cladribine.” [Online]. Available:
http://www.sigmaaldrich.com/catalog/product/usp/1134200?lang=fr®ion=FR.