iologi evaluation of polymer prodrug nanopartiles · master thesis performed at: universitÉ...

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


5.2.3. 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


5.5.2. 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


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.


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


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


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

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iologi evaluation of polymer prodrug nanopartiles · master thesis performed at: universitÉ...

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