Licentiate Thesis_Heba Asem

Synthesis of Polymer Nanocomposites
for Drug Delivery and Bioimaging
Heba Asem
Licentiate Thesis in Physics
School of Information and Communication Technology
Royal Institute of Technology KTH
Stockholm, Sweden 2016
TRITA-ICT 2016:09
ISBN 978-91-7595-930-6
School of Information and
Communication Technology
SE-164 40 Kista
Sweden
Akademisk avhandling som med tillstånd av Kungliga Tekniska högskolan- KTH framlägges till
offentlig granskning för avläggande av teknologie licentiatexamen i fysik fredagen den 10 juni 2016
klockan 10:00 i Sal C, Electrum, KTH, Isafjordsgatan 26, Kista, Stockholm.
Cover image: schematic diagram showing the composition of multifunctional nanoparticle and its
multiple applications
© Heba Asem, June 2016
Universitetsservice US-AB, Stockholm 2016
This Thesis is Dedicated to my Family, my Lovely Husband
& In the Loving Memory of my Father
(1959-2013)
Abstract
Nanomaterials have gained great attention for biomedical applications due to their
extraordinary physico-chemical and biological properties. The current dissertation presents
the design and development of multifunctional nanoparticles for molecular imaging and
controlled drug delivery applications which include biodegradable polymeric nanoparticles,
superparamagnetic iron oxide nanoparticles (SPION)/polymeric nanocomposite for
magnetic resonance imaging (MRI) and drug delivery,
manganese-doped zinc sulfide
(Mn:ZnS) quantum dots (QDs)/ SPION/ polymeric nanocomposites for fluorescence
imaging, MRI and drug delivery.
Bioimaging is an important function of multifunctional nanoparticles in this thesis.
Imaging probes were made of SPION and Mn:ZnS QDs for in vitro and in vivo imaging. The
SPION have been prepared through a high temperature decomposition method to be used
as MRI contrast agent. SPION and Mn:ZnS were encapsulated into poly (lactic-co-glycolic)
acid (PLGA) nanoparticles during the particles formation. The hydrophobic model drug,
busulphan, was loaded in the PLGA vesicles in the composite particles. T2*-weighted MRI of
SPION-Mn:ZnS-PLGA phantoms exhibited enhanced negative contrast with r2* relaxivity of
523 mM-1 s-1 . SPION-Mn:ZnS-PLGA-NPs have been successfully applied to enhance the
contrast of liver in rat model.
The biodegradable and biocompatible poly (ethylene glycol)-co-poly (caprolactone)
(PEG-PCL) was used as matrix materials for polymeric nanoparticles -based drug delivery
system. The PEG-PCL nanoparticles have been constructed to encapsulate SPION and
therapeutic agent. The encapsulation efficiency of busulphan was found to be ~ 83 %. PEGPCL nanoparticles showed a sustained release of the loaded busulphan over a period of 10
h. The SPION-PEG-PCL phantoms showed contrast enhancement in T2*-weighted MRI.
Fluorescein-labeled PEG-PCL nanoparticles have been observed in the cytoplasm of the
murine macrophage cells (J774A) by fluorescence microscopy. Around 100 % cell viability
were noticed for PEG-PCL nanoparticles when incubated with HL60 cell line. The in vivo
biodistribution of fluorescent tagged PEG-PCL nanoparticles demonstrated accumulation of
PEG-PCL nanoparticles in different tissues including lungs, spleen, liver and kidneys after
intravenous administration.
Keywords: Biodegradable polymers, SPION, QDs, drug delivery, cytotoxicity, MRI, in
vivo fluorescence imaging, biodistribution
i
ii
Sammanfattning
Nanomaterial har fått stor uppmärksamhet för biomedicinska tillämpningar på grund av
deras extraordinära fysikalisk-kemiska och biologiska egenskaper. Denna avhandling
presenterar design och utveckling av multifunktionella nanopartiklar för molekylär
avbildning och kontrollerad drogleveransapplikationer som inkluderar bionedbrytbara
polymera nanopartiklar, superparamagnetiska järnoxidnanopartiklar (SPION)/ polymer
nanokomposit för magnetisk resonanstomografi (MRI) och drogleverans, mangan-dopade
zink sulfid (Mn:ZnS) kvantprickar (QDs) / SPION/polymera nanokompositer för
fluorescensavbildning, MRI och drogleverans.
Bioimaging är en viktig funktion av multifunktionella nanopartiklar i denna avhandling.
Avbildningssonder var gjorda av SPION och Mn: ZnS QDs för in vitro- och in vivoavbildning. SPION var tillverkad genom en hög temperatur sönderdelnings metod som skall
användas som kontrastmedel vid MRI. SPION och Mn:ZnS inkapslades i poly (mjölksyraCo-glykolsyra)
(PLGA)
nanopartiklar
under
partikelbildning.
Den
hydrofoba
modelläkemedel, busulphan, laddades i PLGA vesiklar i kompositpartiklar. T2* viktade MRI
av SPION-Mn:ZnS-PLGA fantomer uppvisade förstärkt negativ kontrast med r2*
relaxiviteten av 523 mM-1 s-1 . SPION-Mn: ZnS-PLGA NPs har tillämpats med framgång för
att förbättra kontrast av levern i råttmodell.
Den bionedbrytbara och biokompatibla poly (etylenglykol) -Co-poly (kaprolakton)
(PEG-PCL)
användes
som
matrismaterial
för
polymera
nanopartikelbaserade
drogleveranssystem. De PEG-PCL-nanopartiklarna har konstruerats för att inkapsla SPION
och terapeutiskt medel. Inkapslingseffektiviteten av busulphan var ~ 83 %. PEG-PCLnanopartiklar visade en oavbruten frisättning av den laddade busulphan under en period av
10 timme. De SPION-PEG-PCL fantomerna visade kontrastförbättring i T2*- viktade MRI.
Fluorescein-märkt PEG-PCL-nanopartiklar har observerats i cytoplasman hos de murina
makrofagceller (J774A) genom fluoreszenzmikroskopie. Omkring 100 % cellviabilitet
märktes för PEG-PCL nanopartiklar när de inkuberas med HL60-cellinje. In vivobiofördelningen av fluorescerande-märkta PEG-PCL-nanopartiklarna visade ackumulering
av PEG-PCL-nanopartiklar i olika vävnader inkluderande lungorna, mjälten, lever och
njurar efter intravenös administrering.
Nyckelord: Bionedbrytbara polymerer, SPION, kvantprickar, drogleverans, cytotoxicitet,
MRI, in vivo fluorescens avbildning, biofördelningen.
iii
Papers included in the Thesis
This thesis is based on the following publications:
I. Fei Ye, Åsa Barrefelt*, Heba Asem*, Manuchehr Abedi-Valugerdi, Ibrahim ElSerafi, Maryam Saghafian, Khalid Abu-Salah, Salman Alrokayan, Mamoun Muhammed,
Moustapha Hassan, “Biodegradable polymeric vesicles containing magnetic
nanoparticles, quantum dots and anticancer drugs for drug delivery and imaging”,
Biomaterials, 2014, 35, 3885–3894.
II. Heba Asem, Ahmed Abd El-Fattah, Noha Nafee, Ying Zhao, Labiba Khalil, Mamoun
Muhammed, Moustapha Hassan, Sherif Kandil, “Development and biodistribution of a
theranostic aluminum phthalocyanine nanophotosensitizer”, Photodiagnosis and
photodynamic therapy, 2016, 13, 48-57.
III. Heba Asem, Ying Zhao, Fei Ye, Åsa Barrefelt, Manuchehr Abedi-Valugerdi, Ramy ELSayed, Ibrahim El-Serafi, Svetlana Pavlova, Khalid Abu-Salah, Salman A. Alrokayan,
Jörg Hamm, Mamoun Muhammed, Moustapha Hassan,“ Biodistribution of busulphan
loaded biodegradable nano-carrier system designed for multimodal imaging”,
Manuscript submitted to Molecular Pharmaceutics.
Other work not included in the Thesis
I.
Åsa Barrefelt, Gaio Paradossi, Heba Asem, Silvia Margheritelli, Maryam Saghafian,
M., Letizia Oddo, Mamoun Muhammed, Peter Aspelin, Moustapha Hassan, Torkel
Brismar. “Dynamic MR imaging, biodistribution and pharmacokinetics of polymer
shelled micro bubbles containing SPION”, Nano, 2014, 9, 1450069.
II. Noha Nafee, Alaa Youssef, Hanan El-Gowelli, Heba Asem, Sherif Kandil, “Antibioticfree nanotherapeutics: Hypericin nanoparticles thereof for improved in vitro and in
vivo antimicrobial photodynamic therapy and wound healing”, International Journal
of pharmaceutics, 2013, 454, 249–258.
III. Ramy El-Sayed, Fei Ye, Heba Asem, Radwa Ashour, Wenyi Zheng, Mamoun
Muhammed, Moustapha Hassan. “Importance of the surface chemistry of nanoparticles
on peroxidase-like activity”, Manuscript.
*Equal contribution
iv
Conference presentations
I. Heba Asem, Noha Nafee, Ahmed Abd El-Fattah, Labiba El-Khordagui, Mamoun
Muhammed,
Moustapha
Hassan,
Sherif
Kandil,
“Biodegradable
Polymeric
Nanoparticles for Photodynamic Therapy” (poster), Photodynamic and Nanomedicine
for Cancer Diagnosis and Therapy Workshop, 2014, 13-15 February, GUC (German
University in Cairo), Berlin, Germany.
II. Heba Asem, Ahmed Abd El-Fattah, Noha Nafee, Labiba Khalil, Mamoun muhammed,
Sherif Kandil, Moustapha Hassan “synthesis and characterization of biodegradable
aliphatic polyester for drug delivery applications” (poster), EUPOC: Bio-based Polymer
and Related Biomaterials, poster prize, 2011, 29 May- 3 June, Gargnano, Italy.
Contribution of the author
Paper I. Planning some experiments, preparing the materials, coordinating collaboration,
performing some of materials characterization, analyzing the results and writing part of the
article.
Paper II. Initiating the idea, planning of experiments, preparing the materials, carrying out
materials characterization, parsing the results and writing the article.
Paper III. Initiating the idea, planning of experiments, preparing the materials, performing
materials characterization, analyzing the results and writing the manuscript.
v
List of abbreviations and symbols
APTMS
(3-aminopropyl) trimethoxysilane
CMC
critical micelle concentration
CT
computed X-ray tomography
DCM
dichloromethane
DDS
drug delivery systems
DI
deionized water
DMEM
dulbecco’s modified Eagle medium
DMF
dimethylformamide
DNA
deoxyribonucleic acid
DSC
differential scanning calorimetry
ε-CL
epsilon-caprolactone
EDC
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
FDA
food and drug administration
FBS
fetal bovine serum
FeCl3 · 6H2 O
iron chloride hexahydrate
FE-TEM
field emission transmission electron microscopy
FTIR
Fourier transform infrared spectroscopy
HPLC
high-performance liquid chromatography
IV
intravenous
IVIS
In vivo imaging system
Mn:ZnS
manganese-doped zinc sulfide
MnCl2
manganese chloride
MNPs
magnetic nanoparticles
MnSt2
manganese stearate
MPS
mononuclear phagocyte system
MRI
magnetic resonance imaging
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Mw
weight average molecular weight
N2
nitrogen gas
NHS
N- Hydroxysuccinimide
NIR
near-infrared
NMR
nuclear magnetic resonance
NPs
nanoparticles
ODE
octadecene
vi
OLA
oleylamine
O/W
oil-in-water emulsion
PBS
phosphate buffer saline
PEG-PCL
poly(ethylene glycol)-co- poly(caprolactone)
PEG
polyethylene glycol
PEO
polyethylene oxide
PCL
poly(caprolactone)
PGA
poly(glycolic acid)
PICM
polyion complex micelles
PLA
poly(lactic acid)
PLGA
poly(lactic-co-glycolic) acid
PS
photosensitizer
PVA
polyvinyl alcohol
QDs
quantum dots
RF
radio frequency
ROI
region of interest
ROP
ring opening polymerization
RPMI
Roswell Park Memorial Institute medium
SA
stearic acid
SEC
size exclusion chromatography
SPECT
single-photon emission computed tomography
SPION
superparamagnetic iron oxide nanoparticles
TE
echo time
TEM
transmission electron microscopy
TGA
thermogravimetic analysis
THF
tetrahydrofuran
TMAOH
tetramethylammonium hydroxide
UV/Vis
ultraviolet-visible
ZnAc2
zinc acetate dehydrate
σ
standard deviation
vii
viii
Table of contents
ABSTRACT.......................................................................................................... i
SAMMANFATTNING.......................................................................................... iii
Papers included in the Thesis .....................................................................................................iv
Other work not included in the Thesis..........................................................................................iv
Conference presentations .......................................................................................................... v
Contribution of the author ........................................................................................................... v
List of abbreviations and symbols ...............................................................................................vi
1 INTRODUCTION ............................................................................................. 1
1.1 Objectives ...................................................................................................................... 1
1.2 Outlines ......................................................................................................................... 2
1.3 Drug delivery systems (DDS)........................................................................................... 3
1.3.1 Polymeric matrices ........................................................................................................................ 4
1.3.2 Micellar nanostructures................................................................................................................. 5
1.3.3 Magnetic nanoparticles ................................................................................................................. 6
1.3.4 Quantum dots ................................................................................................................................. 6
1.4 Multifunctionality of drug carriers...................................................................................... 7
1.4.1 Drug loading and release ............................................................................................................. 7
1.4.2 Targeting ......................................................................................................................................... 8
1.4.3 Visualization ................................................................................................................................... 9
1.4.3.1 Magnetic resonance imaging ............................................................................................... 9
1.4.3.2 Fluorescence imaging ......................................................................................................... 11
1.5 Preparation methods of nanoparticles ............................................................................. 11
1.5.1 Preparation of polymeric nanoparticles.................................................................................... 11
1.5.2 Preparation of magnetic nanoparticles..................................................................................... 12
2 EXPERIMENTAL .......................................................................................... 13
2.1 Synthesis of PEG-PCL drug carrier ............................................................................... 13
2.2 Fabrication of nanoparticles for imaging ........................................................................ 13
2.2.1 Synthesis of SPION ................................................................................................................... 13
2.2.3 Preparation of SPION-polymeric NPs ..................................................................................... 14
2.2.4 Fluorescent PEG-PCL-NPs ...................................................................................................... 14
ix
2.3 Characterization ........................................................................................................... 14
2.4 Drug release ................................................................................................................ 15
2.5 Degradation of polymer matrix ....................................................................................... 16
2.6 Cell uptake and biocompatibility evaluation..................................................................... 17
2.6.1 Cell lines and culture conditions ................................................................................................ 17
2.6.2 Cellular uptake.............................................................................................................................. 17
2.6.3 Cell viability (MTT) assay............................................................................................................ 17
2.7 MRI and fluorescence imaging....................................................................................... 18
2.7.1 In vitro magnetic resonance imaging ........................................................................................ 18
2.7.2 In vivo fluorescence imaging/ computed tomography (CT) ................................................... 18
2.7.3 Necropsy and histology ............................................................................................................... 19
3 RESULTS AND DISCUSSION .......................................................................20
3.1 Synthesis and characterization of PEG-PCL drug carrier .................................................. 20
3.2 Inorganic imaging probes ............................................................................................. 20
3.2.1 Microscopic studies of SPION and QDs .................................................................... 20
3.2.2 Magnetic and optical properties................................................................................................. 22
3.3 Multifunctional DDS ...................................................................................................... 22
3.3.1 Microscopic studies and size control ........................................................................................ 22
3.3.1.1 SPION-Mn:ZnS-PLGA NPs ................................................................................................ 22
3.3.1.2 SPION-PEG-PCL-NPs ......................................................................................................... 23
3.3.2 Biocompatibility evaluation of polymeric NPs .......................................................................... 24
3.3.3 Cell uptake assessment of polymeric NPs .............................................................................. 24
3.4 Drug release................................................................................................................. 25
3.5 MRI and fluorescence imaging....................................................................................... 27
3.5.1 MRI phantoms of SPION-Mn:ZnS-PLGA and SPION-PEG-PCL-NPs ................................ 27
3.5.2 In vivo MRI .................................................................................................................................... 28
3.5.3 In vivo fluorescence imaging/ Computed Tomography (CT) ................................................ 29
3.5.4 Histological analysis .................................................................................................................... 29
4 CONCLUSIONS ............................................................................................32
FUTURE WORK ................................................................................................33
ACKNOWLEDGEMENTS ..................................................................................34
REFERENCES...................................................................................................35
x
1 Introduction
Nanotechnology refers to an interdisciplinary area of research such as chemistry, physics,
biology, and medicine that have been achieved tremendous progress in the past several
decades. Due to the size reduction to nanometer, the nanomaterials gained extraordinary
chemical, physical, optical and biological properties.1,2 These unique properties offer great
opportunities and potential benefits in a wide spectrum of applications involving energy
storage3, 4 , water remediation5, 6, food processing7, 8, health monitoring and diseases
therapy.9 The interaction between the nanotechnology and medicine will support the next
generation of nanomedicine and facilitate a personalized and efficient therapy with
minimized side effects.10 Nanotechnology has demonstrated a tremendous potential in
improving both biomedical research and clinical applications because the nanoobjects are
generally in the similar size range with biological entities, e.g. cells, organelles, DNA, and
proteins. Therefore, nanomaterials have potentials to tune the bioactivity in the molecular
level. Fabricating nanomaterials such as nanospheres, nanotubes, nanorods, porous
nanoparticles and nanocomposites have been designed for diverse biomedical applications
especially on cell separation11, 12, controlled drug delivery, gene delivery13-15, non-invasive
imaging16-21 , and cancer therapy.22-25 The advantages of nanostructures such as controlled
size and shape, design flexibility, surface modification with multivalent ligands are suitable
for multifunction applications including bioimaging and targeted drug delivery. This will
facilitate the improvement of drug pharmacokinetics and greatly increase the efficiency of
medical therapy at the level of single molecule or molecular assemblies.
1.1 Objectives
Two drug delivery systems are investigated in the present work: polymeric nanoparticles and
polymer/inorganic nanocomposites for imaging and controlled drug delivery:
A series of synthesized poly (ethylene glycol)-co-poly (caprolactone) (PEG-PCL)
copolymers are used to prepare nanophotosensitizer with different particles size. PEG-PCL
nanoparticles (NPs) are prepared to study the loading of a hydrophobic drug model
(photosensitizer) for photodynamic therapy with a sustained drug release. The
biodistribution of the free drug and drug-loaded PEG-PCL-NPs are studied in animal model
in different organs.
1
Multifunctional nanoparticles composed of PEG-PCL loaded with SPION are developed to
be used as a drug carrier to deliver anticancer drug such as busulphan. The drug release from
the NPs is studied over period of 10 h. The multifunctional drug carrier are examined as a
contrast agent for T2*-weighted MR imaging. The biocompatibility and cell uptake of PEGPCL-NPs are assessed in cell lines. For simultaneous biodistribution evaluation, the PEGPCL was modified with amino group and conjugated with VivoTag 680XL for in vivo
fluorescence imaging and monitoring the PEG-PCL-NPs in different organs.
Furthermore, nanocomposite system is designed for multimodal imaging and
controlled anticancer drug release. PLGA vesicles encapsulating two kind of inorganic
nanocrystals, SPION as contrast probe for MRI and Mn: ZnS QDs for fluorescence imaging.
SPION-Mn: ZnS-PLGA NPs are examined as drug delivery system for cytostatic drugs and
as T2 contrast agent for MRI. The SPION-Mn: ZnS-PLGA NPs are employed to study the cell
uptake by fluorescence microscopy. In addition, these nanocomposites are served to image
some organs in a rat model under MRI.
1.2 Outlines
This thesis addresses the development of multifunctional NPs such as drug carrier for
various biomedical applications including controlled drug release, MRI and photodynamic
therapy.
Chapter 1.3 briefly introduces different categories of drug delivery systems, e.g.
biodegradable polymers, liposomes, denderimers and smart polymers, contrast agents for
MRI and fluorescence imaging e.g. SPION and QDs. Chapter 1.4 introduces biomedical
application of multifunctional drug carriers such as drug loading of cytostatic drugs, drug
release, targeted drug delivery and bioimaging. Different imaging techniques such as MRI
and fluorescence imaging were mentioned in this chapter. A brief explanation of physical
background of MRI and superparamagnetism are discussed in this chapter as well. Chapter
1.5 briefly introduces different techniques for preparation of polymeric nanoparticles and
magnetic nanoparticles.
Chapter 2.1 starts with the synthetic methodology of the PEG-PCL copolymer and the
polymerization conditions. Chapter 2.2 presents the synthesis of SPION by thermal
decomposition method and Mn: ZnS QDs using nucleation-doping method. The loading of
different materials into the PEG-PCL and PLGA NPs such as SPION, QDs, busulphan and
aluminum phthalocyanine chloride (AlPc) drug are studied to form multifunctional
nanoparticles. Chapter 2.3 mentions various characterization tools to measure the physico-
2
chemical properties of the prepared polymers and nanoparticles. Chapter 2.4 and 2.5 study
the drug release from nanocarriers by dialysis method and the degradation of the polymer
matrix, respectively. The cell uptake of the nanoparticles in different cell lines is described
in chapter 2.6. Studies of in vitro and in vivo MRI and fluorescence imaging are summarized
in chapter 2.7.
Chapter 3 presents and discusses the results of synthesis and properties of
nanomaterials for biomedical applications. In chapter 3.1 analysis of PEG-PCL copolymer
are discussed using FTIR, 1 H-NMR, and TGA. Chapter 3.2 demonstrates the morphologies
of SPION and QDs as well as the magnetic and optical properties of the nanocrystals. In
chapter 3.3 the properties of multifunctional drug delivery system comprised of SPIONMn:ZnS-PLGA-NPs and SPION-PEG-PCL-NPs are studied. In this chapter the cytotoxicity
evaluation and cellular uptake of the PEG-PCL-NPs reveals that these particles are
biocompatible at the studied concentrations and can be taken by different cell lines. In
chapter 3.4, the drug release of busulphan from PLGA and PEG-PCL polymeric matrices
show sustain release profiles in period of 5 h and 10 h respectively. The imaging applications
of the nanoparticles as a contrast agent are investigated in chapter 3.5 in which the SPIONMn:ZnS-PLGA phantoms show high relaxivity in vitro and in vivo MRI. Furthermore the
VivoTag 680XL-PEG-PCL-NPs are presented in various organs such as lungs, spleen, liver
and kidneys.
1.3 Drug delivery systems (DDS)
DDS have received great attention and implementations in development of novel medical
devices. DDS enable the introduction of therapeutic agents to human body, improve their
efficacy and safety compared with using unmodified drugs by controlling the rate, time, and
place of release of drugs in the body.26-28 The ideal drug carrier should regulate and optimize
the drug release rate during the therapy in a controlled manner. Minimizing the drug
fluctuation in plasma level and extending its duration of action are the robust functions of
ideal drug carriers. Therefore the frequency of dosing of drug can be reduced and the patient
compliance increased.
Several biocompatible nanomaterials have been utilized to deliver different biological
molecules and therapeutic substances. Liposomes, polymeric nanoparticles, dendrimers,
carbon nanotubes, mesoporous silica and gold NPs (Figure 1.1) are usually used as drug
carriers.29 Liposomes and polymeric NPs are the most extensively investigated drug carrier.
Doxorubicin-loaded liposome (Doxil®) has been approved from FDA as first liposomal drug
delivery formulation for human cancer. 30 The hydrophilic drugs can be encapsulated in the
3
inner aqueous core of the liposome, while the lipid bilayer is used to deliver the lipophilic
drugs.31 The particulate drug carriers must be hidden from immune system especially from
the phagocytes uptake and glomerular filtration, as long as liver and kidney are not the target
organs.
Figure 1.1 Different types of nanocarrier systems for drug delivery 29, 32, 33
Phagocytic uptake of the drug carriers is influenced by particles size, surface charge and
hydrophobicity.34 The particles size from 200 nm to several microns showed higher
phagocytic uptake and adsorption of opsonins on the surface of the particle. However,
modification the particles surface with hydrophilic polymer (e.g. poly (ethylene glycol), PEG)
impedes interaction of the particle towards environmental proteins including opsonins.35-37
PEGylation of polymeric nanoparticles can be achieved by adhering surfactants like
poloxamers or polysorbates onto nanoparticles, or by formulating nanoparticles with
copolymers comprising PEG and a biodegradable moiety.
1.3.1 Polymeric matrices
The most attractive class of polymers for drug delivery system is biodegradable polymers.
Biodegradable polymers are one type of biomaterials that can decompose into nontoxic
products and normal metabolites.38 Biodegradable polymers are classified into two
categories: natural polymer, such as cellulose, chitosan, polypeptides and proteins, and
synthetic polymers including poly (glycolic-co-lactic) acid (PLGA), poly (lactic acid) (PLA),
poly (glycolic acid) (PGA), poly (caprolactone) (PCL), aliphatic polycarbonates and
polyphosphazenes.39 Biodegradable polymers such as poly esters are produced by two
general routs from a variety of monomer via ring opening polymerization (ROP) of lactones
and lactides (e.g. ROP of ε-caprolactone monomer (Figure 1.2)) or condensation polymer-
4
ization between diols and diacids.40-42 The active linkage (ester, amine, urethane and enolketone) along the main polymer chain has great influence on the biodegradability of
polymers, which can be hydrolyzed and/or oxidized to small compartments under hydrolysis
or enzymatic action.43 Lactic acid and glycolic acid are the main degradation products of PLA
and PGA, respectively which are biocompatible and are easily eliminated from the body.44
Additionally, the degradation rate of bulk polymer is influence by different factors including
the hydrophilicity, molecular weight, crystallinity, surface area, type of repeating units, and
polymer compositions.45
m
catalyst
+
ɛ-CL monomer
PEG
PCL-PEG
Figure 1.2 Scheme of ring opening polymerization of ε-caprolactone (ε -CL) monomer
Biodegradable polymers-based drug carriers have been developed for controlled release
of drug over prolonged period. Polymeric nanospheres protect drugs, particularly
hydrophobic drugs, from degradation by physiological environment. Further protection of
the drug is achieved by PEGylation of the polymeric nanoparticles.46 The drug can be
entrapped in the polymeric NPs in different ways; the payload disperses in the matrix space
of the nanoparticles forming nanospheres or the drug accumulates in the particle core
producing nanocapsule or core-shell nanostructure. Additionally, bioactive agents including
proteins, enzymes and antibodies can be chemically conjugated to polymers chain via
covalent bond.47 Another type of polymer-based drug carrier is environment responsive
polymers which demonstrate physicochemical changes in response to environmental
stimuli, such as change in pH, temperature, redox potential, ionic strength, biochemical
agents, and ultrasound48, 49. Hydrogels are three-dimensional, cross-linked networks of
water-soluble polymers which use as carriers for controlled drug delivery due to their
biocompatibility and inertness toward various drugs, especially proteins.50, 51
1.3.2 Micellar nanostructures
Micelles and core-shell structures are class of self-assembly materials in nanoscale which
enclose active constituents in their core. Block copolymers are well established building
blocks for preparation of micelles drug delivery. 52,
53
Block copolymer micelles can be
classified depending on the type of intermolecular force between the core and the aqueous
milieu54, 55 ; a) amphiphilic block micelles which formed by hydrophobic interaction between
5
the hydrophobic blocks producing core; b) polyion complex micelles (PICM) which resulting
from electrostatic interaction between polycation and polyanion such as plasmid DNA 56, 57;
c) micelles produced by metal complexation.58 The hydrophilic block such as polyethylene
oxide (PEO) can form hydrogen bond with the aqueous environment which creates tight
shell around the micelles core. The amphiphilic block copolymer can form stable micelles
at/or above the critical micelle concentration (CMC). The polymer chains association is
driven by entropy via the expulsion of water molecules into the bulk aqueous phase.59
Further stability of micelles can be achieved by crosslinking of the micelles core as well as
the micelles shell. The cross linking can be affected on the permeability of micelles corona
which in turn control the drug release rate from the core.60 Several methods of drug loading
into micelles have been developed include direct dissolution 61 , dialysis62, oil/water
emulsion63, chemical conjugation64 and various evaporation procedures.
1.3.3 Magnetic nanoparticles
Magnetic NPs (MNPs) have been applied in various clinical purposes such as targeted drug
delivery systems 65-67 , hyperthermia68 and contrast agents for MRI.69,
70
MNPs made of
superparamagnetic magnetite (Fe 3O4 ) and maghemite (γ-Fe2O3) are employed for
biomedical applications. One of the major advantages of MNPs is miniaturized particles size
which would strongly enhance their magnetic properties to be superparamagnetic. The ease
surface functionalization of MNPs would increase the chance of attaching various ligands
and cargos with particles surface. MNPs can be coated with polymers e.g. dextran71 ,
polysaccharides72 , PEG73, as well as mesoporous silica74 for delivering several drugs and
biomolecules. The coating layer protects and shields the particles from environment
conditions. The particles surfaces can be also attached to the function groups such as
carboxyl group, amino group, biotin and carbodi-imide. The drug carrier containing MNPs
inside are magnetically guided and delivers the active agent such as DNA, PDT-agents and
antineoplastic drugs to the site of action. Superparamagnetic iron oxide NPs and gadolinium
oxide are extensively used as MRI markers for tumor visualization in DDS.
1.3.4 Quantum dots
Early detection of tumor is an important prerequisite and crucial stage in cancer therapy.
Some inorganic NPs possess unique optical, electrical or magnetic properties which make it
possible to monitor the neoplastic tissues and the drug carrier with different imaging
modalities. Semiconductor nanocrystals, also called as QDs reveal high fluorescence
quantum yield for biological imaging75-78, higher brightness and photostability than conven-
6
tional organic dyes. The small size of QDs (≤10 nm) has great influence on their absorption
and emission in visible region due to quantum confinement effect. Biocompatible
semiconductors with strong and stable photoluminescence have been fabricated. Different
strategies of surface modification of QDs were used to achieve biocompatibility. Surface
functionalization of QDs with biomolecules such as antibodies, nucleic acids and peptides is
important to minimize their toxicity and greatly improve their use in biomedical
applications, particularly in vivo imaging. These biomolecules can be attached covalently or
non-covalently to the surface of QDs. 79, 80 QDs bearing surface functional groups such as
carboxylic, primary amine and thiol can be conjugated with antibodies and peptides by
exploiting cross-linking chemistry of carbodiimide, maleimide and succinimide. Recently,
doped semiconductor nanocrystals are developed presenting additional advantages than the
undoped nanocrystals including larger stockes shift, enhanced thermal and environmental
stabilities and reduced toxicity.81 By introducing transition metal dopant into semiconductor
host, a new class of materials are generated with unique optical, electronic and magnetic
properties.82, 83 ZnS and ZnSe nanocrystals have usually been doped with Mn2+ ion which act
as efficient luminescence center and interact strongly with the s-p electronic state of the host
nanocrystals.84, 85
1.4 Multifunctionality of drug carriers
Conventional drugs administration routes are mostly through intravenous injection causing
general systemic distribution resulting in exposure of drug to physiological degradation by
specific proteins in plasma and losing its therapeutic functions. Prolonged retention time of
drug in blood provides regular distribution to the desired tissue, leading to an increase in
duration of their pharmacological activities. 86 Successful combination of different
components of functional nanostructure materials in one entity will enable to integrate
therapeutic and diagnostic in synergistic way. Multifunctional drug carriers-based
nanoparticles will allow delivery of bioactive molecules to target tissues selectively via
passive targeting derived from their nanosize or adsorption of bioactive ligands on the
particles surface for active targeting. Additionally, real time non-invasive monitoring of
biological responses to the therapy can be achieved by these nanosystems which can provide
vital feedback in the treatment of the pathological diseases.
1.4.1 Drug loading and release
Enclosing the drugs and bioactive agents in delivery devices is vital to protect the drug from
Degradation and achieve the maximum therapeutic effect. The advantages of protected
7
devices encompass delaying the dissolution of drug molecules, inhibiting the diffusion of the
drug out of the device and controlling the flow of drug solutions.87 The drug can be loaded
into DDS by different means. In polymer-based DDS, drug can form covalent bond with
polymer chain generating polymer-drug conjugate systems. Another loading system that the
drug dissolves or disperses in the polymer matrix forming monolithic matrix system.88
Likewise the drug core can be enveloped by polymer coating and the drug release rate is
controlled by polymer composition, molecular weight and the thickness of the polymer layer.
Most of therapeutic drugs tend to be released rapidly after the administration which
may cause an increase of drug concentration leading to toxicity in the body. Controlled DDS
can prohibit the systemic toxicity of drug with relatively high drug concentration at the site
of treatment. Controlled drug release from polymer matrix can be explained generally
through two main mechanisms, diffusion and dissolution-based release systems. Drug
release can be defined as transfer of drug solutes from the initial position in drug carrier to
the outer of carrier surface and then to the release medium89. For instance, the drug is
uniformly mixed with a polymeric matrix and is present either in a dissolved or dispersed
structure. The release model of dissolved drug follows Fick’s laws of diffusion from devices
where the drug is enveloped. When the drug is dispersed in the polymeric matrix, the rate of
release follows square root of time kinetics until the concentration of the drug decreases
below the saturation value.
90
The desired releasing profiles of the drug in bulk degrading
systems can be manipulated by adjusting the molecular weight of the polymer, copolymer
composition, crystallinity, the loading amounts of the drug, interaction between polymer
and drug, etc. In addition, the physicochemical properties of the drug, such as solubility,
drug particle size and molecular weight can also affect the release rate.88, 91
1.4.2 Targeting
The main goal of DDS is to deliver the drug molecules to the desired pathological site with
sufficient amount and in selective manner. Two strategies of targeting can be applied, active
or passive targeting to the site of action. Active targeting requires the carrier system attach
covalently with the cell-specific ligand. While in passive targeting, the drug encapsulated in
carrier system reach passively the tumor via enhanced permeability and retention effect
(EPR). 92, 93 Varieties of target ligands can be attached to the drug carrier such as antibodies,
peptides, folate, sugar moieties and other ligands. Drug nanocarriers attached with
glucosamine saccharide moieties on the surface can target liver cells carcinoma.94 Folatemodified nanocarriers are widely used to target tumors because folate receptor is highly
overexpressed in the majority of human tumor cells. 95-97 many nanocarriers systems has
8
been developed with folate tagging on the surface including polymeric NPs 98, 99, iron oxide
NPs100, 101 , QDs102, mesoporous silica NPs.103
1.4.3 Visualization
Real-time imaging of body internal organs as well as biomolecules such as DNA, peptides
and proteins is of great interest in clinical trials. Various imaging modalities have been
involved including MRI, CT, SPECT and ultrasonography.104 Particularly, contrast probes
such as iron oxide NPs and QDs are used for MRI and fluorescence imaging respectively.
Among others, MRI is a potent imaging modality due to its high spatial resolution, excellent
soft tissue contrast, and non-invasive nature.105, 106 Recently, MRI markers-based drug
carriers have gained remarkable research interest. Polymeric nanoparticles encapsulating
SPION are synthesized as MRI-contrast agent.107 Mesoporous silica coated SPION are
fabricated for theranostic DDS incorporating chemotherapeutics and MRI. 21, 108
1.4.3.1 Magnetic resonance imaging
Magnetic resonance imaging has become one of the most important clinical imaging
modality which is widely used in cancer diagnosis. MRI is non-invasive techniques, it
produces high-definition images and detailed visualize the cellular structures and
functions.106, 107, 109 MRI is based on the nuclear magnetic resonance (NMR) which was
discovered in 1946 by two research groups Purcell et al110 and Bloch et al.111 NMR is an
interaction between magnetic field and proton. Under a static magnetic field (B₀), a torque
is produced which results in a precessional movement around the B₀ field. The frequency of
precession, also called the Larmor frequency 112, is proportional to B₀, and the constant called
gyromagnetic ratio (γ) as shown in Figure 1.3. All the protons in the magnetic field precess
at the same Larmor frequency, which is a resonance condition. Depending on the internal
energy state, proton will align along the B₀ field which corresponds to a lower energy state.
𝜔₀ = 𝛾𝐵₀
Figure 1.3 Larmor precession of a spinning nucleus about the axis of an applied magnetic field.
9
When a resonant radio frequency (RF) transverse pulse is perpendicularly applied to B₀, it
causes resonant excitation of the magnetic moment precession into the perpendicular plane.
Upon removal of the RF, the magnetic moment gradually relaxes to equilibrium by
realigning to B₀. Such relaxation processes involve two pathways, a) longitudinal or T1
relaxation associated with energy transfer from the excited state to its surroundings (lattice)
and recovery of decreased net magnetization to the initial state, and b) transverse or T2
relaxation from the disappearance of induced magnetization on the perpendicular plane by
the dephasing of the spins.113
An important requirement for useful T2 contrast agents is the large transverse and
longitudinal relaxivities (r2/r1 ) ratio, in combination with a high absolute value of r2.
Contrast agents that satisfy these conditions are SPION. Another important parameter used
to
characterize
ferri-
or
ferromagnetic
materials
are
superparamagnetism.
Superparamagnetic state has been shown in ferromagnetic material when the size is reduced
below critical size (Dc), dependent on the material, which is typically 10-20 nm.114, 115 The
magnetic nanoparticles contain a single magnetic domain with no domain boundary 116, upon
applied magnetic field, the magnetic moment is aligned along the direction of easy
magnetization. In the absence of an external magnetic field the magnetic nanoparticles
exhibit no net magnetization due to the rapid reverse of magnetic moment.117 Additionally,
the magnetic anisotropy energy (Ea) is proportional to the particle volume 118 which is
expressed as follows:
Ea = 𝐾𝑉 sin2 𝜃
(1.1)
Where V is the particle’s volume, K is anisotropy constant and θ is the angle between
magnetization and the easy axis. When the volume is small enough, the magnetic anisotropy
energy of the particle exceeds its thermal energy KBT, where KB is Boltzmann constant and
T is the absolute temperature, i.e. KBT < Ea, and the magnetization is easily flipped. The
average time to perform such a flip is given by the relaxation time (τ) which is determined
by Néel-Brown relaxation law119:
𝜏 = 𝜏0 (𝐸𝑎 ⁄𝐾𝐵 𝑇 )
(1.2)
Where τ0 is typically in the range of 10 -13-10-9 s. If the average time of flip is much smaller
than the measurement time (τm), τ << τm, the system is in supermagnetic state which implies
that the relaxation happens so fast that a time average of the magnetization orientation is
observed within the experimental time. SPION can cause remarkable shortening of T2
relaxation times with signal loss in the diseased tissue and generate contrast to other tissues.
10
1.4.3.2 Fluorescence imaging
Tissue observation with light is probably the most common imaging practice in medicine
and biomedical research ranging from the simple visual inspection of a patient to advance
in vitro and in vivo spectroscopic and microscopy techniques. Fluorescence imaging is
widely used to provide spatial information by measuring the illumination emitted from the
intended organ.120, 121 Traditional optical imaging approaches have been utilized for in vivo
surface and subsurface fluorescence imaging using confocal imaging, multiphoton imaging
microscopic and fluorescence microscopy. Recently, NIR range is used in fluorescence
imaging for in vivo visualization which can penetrate for several centimeters into tissues and
enables disease diagnosis and imaging of treatment response. Various fluorophores have
been developed such as organic dye 21 , QDs122 and lanthanide-doped upconversion
nanocrystals.123 Conventional organic dyes and fluorescent protein usually require specific
wavelength for excitation and have broad emission spectra, as well as affected by fast
photobleaching and subsequent short fluorescence lifetime.124 However, biocompatible QDs
arise with strong and stable photoluminescence distributed in the visible and NIR region.
Their unique optical properties confer the QDs great attractive for biological applications
due to broad absorption bands, size tunable light emission, large two-photon absorption
cross-section.79, 125
1.5 Preparation methods of nanoparticles
1.5.1 Preparation of polymeric nanoparticles
The properties of polymeric nanoparticles such as hydrodynamic diameter, morphology,
and zeta potential can be optimized depending on the intended application. The preparation
techniques of nanoparticles play a vital role to achieve the desired properties. Many methods
have been developed for preparing polymeric nanoparticles which can be broadly classified
into two main categories, a) the nanoparticles formed through a polymerization reaction or,
b) it is achieved directly from an already prepared polymer. Nanoparticles can be prepared
directly from the dispersion of the preformed synthetic or natural polymers by various
techniques including emulsification-solvent evaporation method, nanoprecipitation method
and salting-out method.126 Emulsification-solvent evaporation method is commonly used for
the preparation of polymeric nanoparticles. This method is characterized by two main steps,
emulsion formation and solvent evaporation. The polymers and drug are dissolved in
organic solvent forming oil phase. Subsequently, the emulsion formed by mixing the oil
phase with an aqueous phase with surfactant. Homogenization or sonication is used to
11
produce nanosized polymer droplets. The organic solvent is then evaporated and the
nanoparticles are usually collected by centrifugation and lyophilization. This method has
been used for encapsulating hydrophobic drugs, however, the drugs with hydrophilic nature
have poor incorporation into the nanoparticles by emulsion method.127, 128 The particle size
can be controlled by adjusting the stirring rate, type and amount of dispersing agent,
viscosity of organic and aqueous phases, and temperature.
The nanoprecipitation method was developed by Fessi et al.129 which based on the
interfacial deposition of a polymer following displacement of a semi-polar solvent miscible
with water from a lipophilic solution in the presence or absence of a surfactant. This method
has been applied to various polymeric materials such as PLA, PLGA, PCL, and poly (methyl
vinyl ether-co-maleic anhydride) (PVM/MA). High loading efficiencies are achieved for
lipophilic drugs when nanocapsules are prepared by nanoprecipitation method.130
1.5.2 Preparation of magnetic nanoparticles
Several chemical methods have been developed to describe the efficient synthesis
approaches of iron oxide NPs with controlled size and shape. Biocompatible and
monodisperse iron oxide NPs are prerequisites for biomedical applications not to induce the
immunogenicity. The most common synthetic approaches of SPION are coprecipitation,
thermal decomposition, hydrothermal synthesis, microemulsion, sonochemical synthesis
which are directed to the synthesis of high quality of iron oxide NPs. The chemical reaction
of Fe3O4 formation can be written as:
Fe 2  2 Fe 3  8OH   Fe3O4  4 H 2O
(1.3)
Coprecipitation method is the most conventional method for obtaining Fe 3O4 or αFe2O3. This method consists of mixing Fe +2/Fe+3 ions in highly basic solutions at room
temperature or at elevated temperature.131 The size and shape of the iron oxide NPs are
dependent on type of salts used (e.g. chlorides, sulfates, nitrates) and several other factors
such as Fe+2/Fe+3 ions ratio, reaction temperature, pH and ionic strength of the media. In
addition, Fe3O4 NPs are not very stable under ambient conditions and are easily oxidized to
Fe2O3 or dissolved in an acidic medium. In order to avoid the possible oxidation in the air,
the synthesis of Fe 3O4 NPs must be done in inert condition (oxygen free environment). The
reaction parameters such as stirring rate, dropping speed of basic solution are also effect on
the quality of the nanoparticles. The advantages of the coprecipitation method are high
reproducibility of NPs and large amount of magnetic NPs that can be produced per batch
however, difficulties in controlling the shape and size distribution of the NPs are well
12
identified. Efforts have been devoted to achieve monodisperse magnetic NPs.132 Surfactant,
temperature, pH and ions concentrations are reaction parameters which have been altered
to accomplish small size and morphology control.133-135
Thermal decomposition method has been widely used to synthesize monodisperse iron
oxide NPs.136, 137 Organometallic precursors are decomposed in organic solution at relatively
high temperature in presence of capping agent.138, 139 The organometallic precursors include
metal-oleate complex136, acetylacetonates137, or metal carbonyls.140 Fatty acids141 , oleic
acid142, and hexadecylamine 143 are often used as surfactants. Other methods, including
microemulsion144 , hydrothermal145, and sol-gel146, are also investigated for synthesis of
magnetic NPs.
2 Experimental
2.1 Synthesis of PEG-PCL drug carrier
Amphiphilic copolymer PEG-PCL was synthesized by ring opening polymerization of ε-CL
monomer using PEG as a macroinitiator. An appropriate amount of distilled ε-CL was added
into previously dried PEG in presence of tin (II) 2-ethylhexanoate as a catalyst. The mixture
was stirred under nitrogen atmosphere at 160 °C for 4h. The resulting copolymer was
precipitated in cold diethyl ether, filtered and dried under vacuum at 40 °C for 48 h.
2.2 Fabrication of nanoparticles for imaging
2.2.1 Synthesis of SPION
Monodisperse SPION was synthesized via thermal decomposition method according to
previously reported method.136 Iron-oleate complex was synthesized from sodium oleate and
FeCl3.6H2O followed by refluxing the complex in octyl ether and oleic acid at approximately
297 °C for 1.5 h.
2.2.2 Synthesis of Mn:ZnS QDs
Mn:ZnS QDs were synthesized by a nucleation-doping method. Briefly, manganese stearate
(MnSt2) was prepared by dropwise addition of MnCl2 /methanol solution into a mixture of
SA and TMAOH in methanol. A mixture of MnSt 2 and 1-dodecanethiol in ODE was then
degassed at 100 °C for 15 min, followed by the addition of sulfur and ZnAc 2 at 250 °C.
13
The Mn:ZnS nanoparticles obtained were washed by acetone and re-dispersed in
dichloromethane.
2.2.3 Preparation of SPION-polymeric NPs
SPION-PEG-PCL and SPION-PLGA nanoparticles were prepared by O/W emulsion solvent
evaporation technique. Briefly, an appropriate amount of polymer and SPION were
dissolved in organic solvent (DCM). The organic solution was mixed with aqueous PVA
solution (1:20 oil to water ratio) under probe type sonication for 15 min to form emulsion.
The resulting brownish emulsion was stirred overnight to evaporate organic solvent at room
temperature. The nanoparticles were washed three times against de-ionized (DI) water
(15 MΩ cm) to collect SPION-PEG-PCL and SPION-PLGA-NPs.
2.2.4 Fluorescent PEG-PCL-NPs
The fluorescent nanoparticles were prepared for further biological examination such as
cellar uptake. Fluorescein-labeled PEG-PCL-NPs have been prepared in one step
conjugation reaction. First, chain modification of hydroxyl terminated PEG-PCL copolymer
into amino group by adding 2mmol APTMS to 0.3mmol hydroxyl-PEG-PCL copolymer in
THF, and reflux under N2 overnight. The copolymer was collected by precipitation in excess
diethyl ether, filtered and dried. The carboxy fluorescein was then conjugated with amino
copolymer by adding proper amount of EDC and NHS. The nanoparticles were prepared
from fluorescein-PEG-PCL in DCM by emulsifying in PVA aqueous solution as described
above.
In order to study the biodistribution of the nanoparticles, the VivoTag 680XL labeledPEG-PCL were prepared as follows, a solution of VivoTag 680 XL in dimethyl sulfoxide (0.37
mg/ml) was mixed with amino terminated PEG-PCL copolymer solution under stirring for
1h. The labeled copolymer was collected by precipitation in cold diethyl ether and dried. The
non-conjugated VivoTag 680XL was removed by dialysis (MWCO 10kDa) against PBS at
room temperature. The VivoTag 680XL-labeled PEG-PCL-NPs were prepared by previously
described emulsion solvent evaporation method.
2.3 Characterization
The prepared PEG-PCL polymer was characterized using proton nuclear magnetic
resonance (1 H-NMR) with Bruker AM 400 at 400 MHz and deuterated chloroform (CDCl3)
as solvent. Fourier transform infrared (FTIR) spectroscopy was performed using a Thermo
14
Scientific Nicolet iS10 spectrometer in the attenuated total reflection (ATR) mode, with a
ZnSe focusing optic in ATR crystal plates. The molecular weight and polydispersity index
(PdI) of polymers were determined by size exclusion chromatography (SEC) with
dimethylformamide (DMF) as mobile phase. The analysis were performed on a TOSOH
EcoSEC HLC-8320GPC system equipped with an EcoSEC RI detector and three columns
(PSS PFG 5µm, Microguard, 100 Å, and 300 Å) (Mw resolving range: 100-300,000 g mol-1 )
from PSS GmbH, using DMF (0.2 ml min-1 ). Differential scanning calorimetry (DSC-Q2000
TA Instruments) was used to measure the thermal behavior of polymers. The samples
heating range was from 25 to 100°C with a constant rate (10°C/min). Thermogravimetic
analysis (TGA-Q500 TA Instruments.) was performed to study the changes of polymer
sample weight with regard to increase the temperature. The temperature was increased from
room temperature to 700°C with constant heating rate of 10°C/min under nitrogen gas.
The morphology of nanoparticles was investigated by JEM-2100 (JEOL Ltd., Tokyo,
Japan) field emission transmission electron microscopy (FE-TEM) at an accelerating voltage
of 200KV. The hydrodynamic diameter of nanoparticles in deionized water was measured
using Beckman Coulter DelsaTM Nano particle size analyzer. The concentration of elements
in suspension was measured by inductively coupled plasma-optical emission spectrometry
(ICP-OES) (Thermo Scientific iCAP 6000 series). The absorbance spectra of the particles
were evaluated by Perkin Elmer 750UV/Vis spectrophotometer. The fluorescence intensity
and images were examined using Perkin Elmer Ls55 Fluorescence spectrophotometer and
IVIS optical imaging system. The amount of lactic acid was measured by high-performance
liquid chromatography (HPLC). The HPLC system consisted of a Gilson autoinjector (100 μl
loop), an LKB HPLC pump 2150 (Pharmacia Inc., Sweden), an LDC analytical
spectromonitor 3200 UV detector (Riviera Beach, FL, USA) and a CSW 32 chromatography
station
integrator.
Separation
was
performed
on
a
Zorbax
SB-CN
column
(4.6 mm × 150 mm, 5 μm) from Agilent Technologies (Santa Clara, CA, USA), and the
column was maintained at room temperature during analysis. The mobile phase was
composed of NH4 H2PO4 (0.1 M, pH 2.5) and flow rate was set at 0.8 ml/min with a running
time of 6 min. Injection volume was 50 μl. Detection of lactic acid was performed at 210 nm
compared to a calibration curve for lactic and glycolic acids. Lactic acid eluted after 4 min.
2.4 Drug release
The release profile of busulphan (Figure 2.1) and/or AlPc from two nanocarrier systems,
SPION-PEG-PCL-NPs and SPION-Mn:ZnS-PLGA-NPs, was evaluated using dialysis
method. The drug loaded-NPs were redispersed in PBS and placed in dialysis tube (MWCO
15
10kDa). The dialysis tube was immersed in appropriate amount of PBS pH 7.4 at 37± 0.4°C
under regular shaking (Shaker InFors AGCH-4103 BOTTMNGEN) at 80 rpm. An aliquot of
the buffer was taken from the dissolution medium at different time points. The
concentration of busulphan was measured by gas chromatography (SCION 436-GC, Bruker)
with electron capture detector (ECD). The encapsulation efficacy of the drug was calculated
as amount of the drug released from the nanoparticles and the residual drug in the dialysis
membrane divided by the total amount of the drug. The concentration of the released AlPc
was measured by UV-VIS- spectrophotometer at 670 nm.
(a)
(b)
Figure 2.1 Chemical structure of: (a) busulphan drug and (b) Aluminum phthalocyanine
chloride (AlPc).
2.5 Degradation of polymer matrix
The PLGA polymer is degraded by hydrolytic cleavage of the ester linkage into lactic acid
and glycolic acid (Figure 2.2). The degradation of PLGA-NPs was determined by measuring
the concentration of lactic acid as a degradation product. The prepared PLGA-NPs were
dialyzed against 1 L PBS (pH 7.4) at 37 °C. At predetermined time intervals, a sample of PBS
solution was taken and fresh PBS was added into the dissolution medium. The
concentration of lactic acid released in PBS was measured by HPLC.
hydrolysis
+
PLGA
D,L Lactic acid
Figure 2.2 In vitro degradation of PLGA by hydrolysis.
16
Glycolic acid
2.6 Cell uptake and biocompatibility evaluation
2.6.1 Cell lines and culture conditions
The murine macrophage cell line (J774A, the European Type Tissue Culture Collection
(CAMR, Salisbury, UK) was a kind gift from Professor Carmen Fernandez (Department of
Immunology, Wenner-Gren Institute, Stockholm University, Stockholm, Sweden). J774A
cell line was cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with
10 % heat-inactivated FBS (Invitrogen), penicillin (100 µg /ml) (Invitrogen), and
streptomycin (100 µg/ml) (Invitrogen) in 50 cm2 tissue culture flasks (Costar, Corning, NY,
USA). The cultures were maintained at 37°C in a humidified atmosphere containing 5 % CO2.
J774A with electron capture detector (ECD). The encapsulation efficacy of the drug was
calculated as amount of the drug released from the nanoparticles and the residual drug in
the dialysis cells were cultured in 8-chamber polystyrene vessel tissue culture treated glasses
at the density of 5 × 10 5 cells/chamber at 37°C for 12 h in a 5 % CO2 atmosphere.
The myeloid HL60 leukemia cell line was line was purchased from DSMZ
(Braunschweig, Germany). The cells were cultured in RPMI 1640 medium supplemented
with 90 % GlutaMAX™ and 10% heat-inactivated FBS at 37°C in 95% humidified 5 % CO2
atmosphere. HL60 cells were seeded in 96-well plate at the density of 10 5 cell/well.
2.6.2 Cellular uptake
The uptake of the SPION-PEG-PCL-NPs by J774A cells was investigated. The nanoparticles
were labeled with fluorescein dye and QDs in order to visualize the nanoparticles by
fluorescence microscopy. The fluorescent nanoparticles were dispersed in medium and
incubated with the cells at concentrations of 1000, 100, 50, 25, 12.5, 6.25 and/or 3μg/ml.
The cell culture medium was aspirated from each chamber and substituted with the medium
alone (control). And then, chambers were incubated at 37°C for 2 h, 4 h, and 24 h in an
atmosphere containing 5 % CO2. The cell uptake experiment was terminated at each time
point by aspirating the test samples, removing the chamber and washing the cell mono layers
with ice-cold PBS three times. Each slide was fixed with methanol-acetone (1:1, v/v),
followed by examination the uptake of fluorescein-PEG-PCL-NPs by employing Eclipse i80
(Nikon, Tokyo, Japan) fluorescent microscope at a wavelength of 520 nm.
2.6.3 Cell viability (MTT) assay
The biocompatibility of the PEG-PCL-NPs was studied by evaluating the cytotoxic effect of
the nanoparticles on HL60 cells using MTT assay. PEG-PCL-NPs were incubated with HL60
17
cells at different concentrations of in 96-well plate with medium (RPMI 1640 with
GlutaMAX™ and 10% FBS) for 48 h at 37°C, 5 % CO2. After the incubation period, MTT
reagent was added to each well. The cells were further incubated for 4 h at 37°C. The
solubilizing agent was added to each well and crystals were solubilized by pipetting up and
down. The absorbance was measured at 570 nm and 690 nm as reference. PBS was used as
blank and cells in medium as control. The cells viability percentage was calculated as (cells
treated with the nanoparticles/ non treated control cells)*100%.
2.7 MRI and fluorescence imaging
2.7.1 In vitro magnetic resonance imaging
MRI phantoms of SPION-PEG-PCL-NPs and SPION-Mn:ZnS-PLGA-NPs were made with
concentrations of (0.1, 0.3, 0.5,1mM) and (0.05, 0.1, 0.2, 0.3, 0.4 , 0.5, 1mM), respectively.
The phantoms were prepared by mixing the nanoparticles suspension with agarose in water
(3 w/v %), heating and then drying in 50 ml falcon tubes at slow rate over night to become a
gel. The phantoms were placed in the extremity coil of a 3 T MR scanner (Siemens, Erlangen,
Germany) and a gradient echo T2* sequence was applied. The repetition time was 2000 ms
and six stepwise increasing echo times (TEs) of 2-17.2 ms for SPION-PEG-PCL-NPs and 12
TEs of 2–22.9 ms for SPION-Mn:ZnS-PLGA-NPs were used to obtain the T2*-weighted
images of the phantoms. Regions of interest (ROIs) were manually placed on the images.
The relaxation time, T2*, was then calculated as the slope of a semi-log plot of the signal
intensity in the ROIs versus the TEs. R2* was calculated as 1/ T2*. All R2* values for the
phantoms were subtracted by R2* value for the control sample (plain agarose gel). A
standard curve was plotted with R2* (s-1, y-axis) versus concentration (mM, x-axis).
2.7.2 In vivo fluorescence imaging/ computed tomography (CT)
Animal studies were approved by the Stockholm Southern Ethical Committee and
performed in accordance with Swedish Animal Welfare law. The distribution of fluorescence
was observed by an IVIS® Spectrum (PerkinElmer, Waltham, MA, USA). Quantum FX
(Perkin Elmer, Waltham, MA, USA) was also used to co-register functional optical signals
with anatomical μCT. Balb/C mice (22 ± 2 g) were purchased from Charles River (Charles
River Laboratories, Sulzfeld, Germany) and kept for one week in the animal facility to
acclimatize before the experiments. The animals had free access to food and water, ad
libitum, and were kept in a 12 h light/dark cycle under controlled humidity (55 % ± 5 %) and
temperature (21°C ± 2°C). Prior to all experiments, mice were fed for three weeks on a
18
synthetic diet free of unrefined chlorophyll-containing ingredients (alfalfa free, Research
diets, Inc., USA) to minimize the fluorescence noise signal from the gastrointestinal tract. A
suspension of VivoTag 680XL-labeled PEG-PCL-NPs (0.2 ml) was intravenously injected
into the lateral tail vein of the mice. The mice (n= 2 per time point) were anaesthetized using
2-3 % isoflurane (Baxter Medical AB, Kista, Sweden) during the whole imaging procedure.
The 2D/3D fluorescence imaging and μCT scans were performed at 1 h, 4 h, 24 h and 48 h
post injection. The Mouse Imaging Shuttle (MIS, 25 mm high, PerkinElmer) was used to
transfer the mice from the IVIS Spectrum to the Quantum FX-μCT while maintaining their
positions. Mice were firstly imaged by 2D epi-illumination fluorescent imaging in a ventral
position. Subsequently, the mice were imaged in the MIS using the 3D Fluorescent Imaging
Tomography (FLIT) with trans-illumination in a dorsal position. The 3D FLIT imaging
sequence was set up and images were acquired at excitation 675 nm and emission 720 nm.
The mouse in the MIS was then transferred to the Quantum FX-μCT and subjected to a fast,
low dose CT scan with a field of view (FOV) at 60 mm and 17 second scan-time. All images
were generated using the Living Image ® 4.3.1 software (PerkinElmer, Waltham, MA, USA).
2.7.3 Necropsy and histology
The mice were sacrificed at pre-determined time points immediately after the imaging
procedure, and histological analysis of lungs, liver, spleen and kidneys was performed using
phase contrast and fluorescence microscopy. To verify the observations from the in vivo live
imaging, the organs were removed from the mice, fixed in paraformaldehyde (4 %) for 24 h,
then transferred to ethanol (70 %), routinely processed and embedded in paraffin. Later,
tissue sections (4 μm) were mounted on super frost glass slides. Slides were routinely stained
with 4',6-diamidino-2-phenylindole (DAPI, 300 nM) to produce nuclear counter stain for
fluorescence microscopic evaluation.
19
3
Results and Discussion
3.1 Synthesis and characterization of PEG-PCL drug carrier
An interesting method to synthesize poly (ester–ether) di-block copolymers is the ring
opening polymerization of lactones using tin (II) 2-ethylhexanoate as a catalyst.147, 148 Ring
opening polymerization is utilized to produce polyesters with controlled molecular weight
under mild reaction conditions and no byproducts.
FTIR spectra of PEG-PCL copolymers are shown in Figure 3.1a. A strong sharp
absorption band appears at 1721 cm-1 is characteristic to the stretching vibration of ester
carbonyl group (C=O) of PCL. PEG-PCL copolymers exhibit characteristic peaks of both PEG
and PCL. The (C-H) stretching bands in PEG and PCL are vibrating at 2863 and 2942 cm ‫־‬¹,
respectively. PEG-PCL copolymers with different Mw have been prepared (paper II) and the
FTIR shows that the intensity of the band of the carbonyl group (C=O) has increased by
increasing the length of PCL block in the prepared copolymer (Figure 1A, paper II). The
intensity of aliphatic (C-H) stretching band of PCL in the copolymer has increased gradually
by increasing molar feed ratio of monomer/ initiator. The FTIR signals of prepared PEGPCL are similar to the results reported by Gyun Shin et.al149 which indicate that the
copolymerization of PCL and PEG is successfully formed. Figure 3.1b shows the ¹H-NMR
spectrum of PEG-PCL copolymer exhibits sharp singlet peak at chemical shift δ ~ 3.63 ppm
which is corresponding to the methylene protons of the PEG blocks unit in PEG-PCL
copolymer. It is clearly seen from the spectrum that there are two equally intense triplet
peaks at peak δ ~ 2.26 and 4.01 ppm, assigned to the methylene protons in PCL chain.
Additionally, the structure of PEG-PCL copolymer is confirmed by thermogravimetric
analysis as shown in Figure 3.1c. PEG-PCL was thermally decomposed at two weight loss
events. These two stages of copolymer weight loss are equivalent to the hydrophilic and
hydrophobic polymers chains in the PEG-PCL copolymer.
3.2 Inorganic imaging probes
3.2.1 Microscopic studies of SPION and QDs
The morphology and crystal structure of SPION and Mn:ZnS QDs were investigated by
transmission electron microscopy (TEM). Figure 3.2a shows the average diameter of SPION
20
Figure 3.1 FTIR spectra of PEG-PCL copolymers (a), 1H-NMR spectrum (b) and thermogravimetic
analysis of PEG-PCL copolymer (c).
Figure 3.2 Field emission transmission electron microscope (FE-TEM) images of hydrophobic SPION (a),
Mn: ZnS QDs (b) with inset of electron diffraction patterns, and high resolution image of a single SPION(c)
and Mn: ZnS QDs (d).
21
which is 10.7 nm (standards deviation σ≈ 8 %) while average diameter of Mn:ZnS QDs is
3.1 nm (σ ≈ 10 %) (Figure 3.2b). High resolution TEM images (Figure 3.2c, d) show the single
crystalline nature of SPION and Mn: ZnS QDs, respectively.
3.2.2 Magnetic and optical properties
The magnetic property of PION was measured on field-dependent magnetization and no
hysteresis was observed (Figure 3.3a) demonstrating the superparamagnetic property
desired for T2* -MRI application. The saturation magnetization of SPION 10.7 nm is 52 A m2
kg-1 . Figure 3.3b shows the UV-Visible absorbance and fluorescence spectra of Mn: ZnS QDs.
The characteristic peak of fluorescence emission at 594 nm is due to doping Mn2+ ions into
the ZnS lattice (3 wt % of Mn to Zn according to ICP analysis).
Figure 3.3 Field-dependent magnetization measurement of SPION at ambient temperature (a) and
UV-Vis absorbance and fluorescence intensity for Mn: ZnS QDs (b).
3.3 Multifunctional DDS
3.3.1 Microscopic studies and size control
3.3.1.1 SPION-Mn:ZnS-PLGA NPs
Two multifunctional DDS have been designed consisting of SPION-Mn:ZnS-PLGA and
SPION-PEG-PCL-NPs. The polymeric nanoparticles were prepared by O/W emulsion
solvent evaporation technique. The oil phase containing polymer (PLGA or PEG-PCL) and
inorganic gradients (SPION and QDs) were emulsified into aqueous phase containing poly
(vinyl alcohol) (PVA) as a stabilizing agent.
The morphology of polymeric nanoparticles and SPION-Mn:ZnS-PLGA-NPs was
investigated by TEM. The loading of these particles in PLGA vesicles can be clearly seen by
TEM. The images of unstained and stained SPION-Mn:ZnS-PLGA vesicles with
phosphotungstic acid are shown in Figure 3.4 a and b, respectively. The mean hydrodynamic
22
hydrodynamic diameter of vesicles in volume distribution is 93 nm (σ ≈ 20 %) as shown in
Figure 3.4 c.
Figure 3.4 Field emission transmission electron microscope (FE-TEM) of unstained (a), stained
PLGA-SPION-Mn:ZnS NPs (b) and dynamic light scattering (c).
3.3.1.2 SPION-PEG-PCL-NPs
Figure 3.5 shows the TEM micrograph of PEG-PCL-NPs and SPION-PEG-PCL-NPs
dispersed in deionized water, in which spheres are formed and particles aggregation is not
observed (Figure 3.5 a and b respectively). According to the TEM micrograph of PEG-PCLNPs (Figure 3.5 a), the average diameter of the particles is 212 nm (σ ~ 30 %). The loading
of SPION into PEG-PCL nanocarriers is clearly seen in Figure 3.5 b. The contrast agent,
SPION, was entrapped into the polymeric NPs during nanoparticles formation. The
hydrodynamic size of SPION-PEG-PCL is shown in Figure S3 paper III. It has a negative zeta
potential of approximately -2.8 mV at neutral pH. We also prepared small nanoparticles
from PEG-PCL copolymer using nanoprecipitation method (paper II). SEM images showed
uniform size distribution of the nanoparticles (Figure 7, paper II). Hydrodynamic diameter
of PEG-PCL-NPs prepared by nanoprecipitation method is lower than 100 nm. AlPc, a model
of photosensitizer drug for PDT, was encapsulated into these NPs. The loading of AlPc into
these particles led to an increase in the mean particle diameter (66.5- 127.4 nm) (Table 2,
paper II).
Figure 3.5 Field emission transmission electron microscope (FE-TEM) images of PEG-PCL
nanoparticles. Stained PEG-PCL nanoparticles (a), stained SPION-PEG-PCL nanoparticles (b).
23
3.3.2 Biocompatibility evaluation of polymeric NPs
The biocompatibility and cytotoxicity of the PEG-PCL-NPs was assessed on HL60 cell line.
The cell viability was determined by MTT assay after incubation with PEG-PCL-NPs for 48
h and the results are plotted in Figure 3.6. The viability of the cells was calculated as the
percentage of living cells treated with nanoparticles to that of the non-treated cells. PEGPCL-NPs exhibited 100 % cells viability and didn’t induce major cytotoxicity up to 25 µg/ml.
Cell viability of 80 % was observed at PEG-PCL-NPs concentrations of 50 and 100 µg/ml.
The synthesized PEG-PCL nanoparticles showed no cytotoxicity which implied that the
PEG-PCL-NPs is safe for biomedical applications at the experimental concentrations.
Figure 3.6 Cytotoxicity of PEG-PCL-NPs in HL60 cell line.
3.3.3 Cell uptake assessment of polymeric NPs
Cellular uptake and cellular distribution of PEG-PCL-NPs were investigated by fluorescence
microscopy using fluorescein-labeled PEG-PCL-NPs. The carboxy-fluorescein was
conjugated with the amino-terminated PEG-PCL copolymer via APTMS linker using the
carbodiimide chemistry. Fluorescein conjugation was confirmed by measuring the
fluorescence intensity of the PEG-PCL-NPs after removing excess dye by dialysis. The
maximum absorbance and fluorescence intensity of the PEG-PCL-NPs are observed at 460
nm and 518 nm, respectively (Figure 5, paper III). Internalization of PEG-PCL-NPs is
examined in murine macrophage cell line J774A as shown in Figure 3.7. Figure 3.7 a and b
show the superimposed optical and fluorescence imaging of non-treated J774A cells,
respectively. Internalization of fluorescein-PEG-PCL-NPs was observed at 4 h and 24 h coculture as shown in Figure 3.7 c and e, respectively. It is worth mentioning that nanoparticles
were seen in the cell cytosol. The fluorescence images of the uptake of fluorescein-PEG-PCLNPs by the J774A cells at 4 h (Figure 3.7 d) and at 24 h (Figure 3.7 f) show initial and
24
maximum fluorescence intensity of the cells treated with fluorescein-PEG-PCL-NPs
compared with non-treated cells. Incubation 24 h of SPION-Mn:ZnS-PLGA-NPs with J774A
cell line (Figure 5, paper I), showed strong fluorescence intensity in the cell plasma
indicating high uptake efficacy of the SPION-Mn:ZnS-PLGA-NPs.
Figure 3.7 Superimposed fluorescence and light microscopy images of J774A incubated with
fluorescein-labeled PEG-PCL-NPs. Non-treated control J774A cells, overlay images and
fluorescence images, respectively (a, b). Overlay images of J774A incubated with
nanoparticles for 4 h and 24 h, respectively (c, e). Fluorescence images of J774A incubated
with nanoparticles for 4 h and 24 h, respectively (d, f).
3.4 Drug release
Lipophilic drug model has been entrapped into nanocarrier system for specific cancer
treatment. In this study, busulphan as anticancer drug was successfully encapsulated in two
polymeric drug carriers PEG-PCL and PLGA with high entrapment efficiency 83 % and 89
% respectively. The drug was loaded in to the hydrophobic core of nanoparticles during the
nanoparticles formulation using O/W emulsion solvent evaporation technique. The drug
release behavior of busulphan was studied from SPION-PEG-PCL and SPION-Mn:ZnSPLGA-NPs by dialysis method.
In Figure 3.8 the percentage of drug released, represent on the vertical axis, was
calculated by dividing the amount of busulphan diffused into dialysis media by the total
25
amount of busulphan loaded into the nanoparticles. Figure 3.8a shows a sustained drug
release pattern from PEG-PCL-NPs during 10 h and around 98 % drug was released. In PEGPCL-NPs, we control the burst effect by increase the initial concentration of the monomer
(105 mM of ε-caprolactone) during the polymerization reaction to produce long hydrophobic
PCL chain. It resulted in reducing the initial penetration of water into the copolymer, and
hence we were able to depress burst release. Another drug model, photosensitizer, AlPc was
loaded into the PEG-PCL-NPs during the particles formation by nanoprecipitation method.
The release profile of AlPc from PEG-PCL-NPs showed a sustained release pattern up to 7
days (Figure 3, paper II).
Unlike other drug carrier systems, where the drug release shows a burst model at the
beginning of the release process, our results showed a relatively constant release rate from
PLGA particles (Figure 3.8 b) during the first 2 h, after which drug release continued at a
lower rate for 5–6 h to release a total of 70–80 % of the loaded drugs. The burst effect is
attributed to diffusion of the drug located at the interface between the nanoparticle core and
the surface in to the dissolution medium as well as the hydrophobicity of the drug carriers.
The degradation of the drug carrier in PBS was also tested. Figure 7, paper I, shows the
percentage of lactic acid degraded from PLGA nanoparticles at pH 7.4 and 37 °C for a period
of 5 weeks. At the second week, about 12 % lactic acid was degraded and released. The
degradation was found to follow a quasi-linear trend and around 32 % lactic acid was
degraded after 5 weeks.
Figure 3.8 Profile of busulphan release from SPION-PEG-PCL-NPs (a) and from SPION–
Mn:ZnS- PLGA-NPs (b) in PBS solution at pH 7.4.
26
3.5 MRI and fluorescence imaging
3.5.1 MRI phantoms of SPION-Mn:ZnS-PLGA and SPION-PEG-PCL-NPs
The T2*-weighted MRI was measured on two different phantoms containing SPIONMn:ZnS-PLGA-NPs and SPION-PEG-PCL-NPs with different iron concentrations different
echo time (TEs). For PLGA vesicles phantoms, with increasing concentrations of PLGA
vesicles, the signal intensity of MRI decreased owing to the increase in loaded SPION
(Figure 3.9 a). It is noted that the signal intensity decreases more rapidly with increasing TE
for phantoms containing higher concentrations of iron oxide. These characteristics allow the
application of SPION-Mn: ZnS-PLGA as a negative contrast agent for MRI. The transverse
relaxivity (r2*) is obtained as the slope of linear fitting for the relaxation rate at different iron
concentrations. The calculated relaxivity for SPION-Mn:ZnS-PLGA vesicles (Figure 3.9 b)
and SPION-PEG-PCL phantoms (Figure 4, paper III) are 523 mM-1 s-1 and 103.3 mM-1 s-1
respectively which is much more higher than SPION alone, demonstrating the high
efficiency of SPION-Mn:ZnS-PLGA and SPION-PEG-PCL-NPs for MR T2*- imaging and
utilized in further in vivo MRI studies.
Figure 3.9 T2* -weighted MR phantom images of SPION-Mn:ZnS-PLGA at different TEs
(TR = 1200 ms; TE = 2 ms, 3.9 ms, 5.8 ms)(a). Proton transverse relaxation rate R2* = 1/ T2* of
phantom samples versus iron concentration (b).
27
3.5.2 In vivo MRI
We further investigated the effects of SPION-Mn:ZnS-PLGA-NPs on in vivo MR imaging
using a rat model. Figure 3.10 shows a coronal view of liver slices of T2*-weighted images at
different time points after intravenous tail injection of PLGA vesicles. The liver appears
white on T2*-weighted images before injection of PLGA vesicles. A rapid signal decrease was
observed in the liver and spleen post-injection and the liver become darker after 7 min postinjection, demonstrating the fast accumulation of iron-containing PLGA vesicles in the liver.
The signal continues to decrease and the imaged liver becomes very dark, especially on the
images taken with longer TEs. Minimal signal intensity appears at 4 h post-injection and
remains low until 24 h post-injection. SPION-Mn:ZnS-PLGA-NPs show high contrast
efficiency, which provided higher relaxation rate by using a dose of iron 20 times lower than
the previously reported dextran-coated SPION.150 The rapid decrease of signal intensity or
faster relaxation in the liver compared to other organs is attributed to the SPION-Mn:ZnSPLGA-NPs being taken up by hepatic macrophages. Other organs had fewer macrophage
cells able to take up the nanoparticles, and the signal intensity therefore exhibited less or no
change.
t= 0 (pre)
7min
2h 40min
4h
24h
TE= 2 ms
TE= 3.9 ms
TE= 5.8 ms
Figure 3.10 T2*-weighted in vivo MR images of rat before and after intravenous injection of
SPION-Mn: ZnS-PLGA-NPs at a dose of 0.42 mg Fe/kg. Images were taken pre-injection and at
7 min, 2 h 40 min, 4 h, and 24 h post-injection with different TEs (2 ms, 3.9 ms, and 5.8 ms). The
regions of liver in the coronal planes are encircled by white dashed lines.
28
3.5.3 In vivo fluorescence imaging/ Computed Tomography (CT)
The PEG-PCL-NPs were further functionalized for in vivo fluorescence imaging using a near
infrared (NIR) fluorescence probe, VivoTag 680XL. The near infra-red (NIR) property of
VivoTag 680XL-PEG-PCL-NPs permits in vivo fluorescence imaging due to its high
penetration in tissue at activation wavelength (Ex/Em 675/720 nm) allowing organ
resolution. After IV administration of PEG-PCL-NPs, the biodistribution was followed up to
48 h. Figure 3.11 shows whole body 3D fluorescence imaging. Images were acquired at 1 h, 4
h, 24 h and 48 h post injection. During the first hour, we noticed that NPs distributed
primarily into the lungs, liver and spleen. Despite the fact that lungs are the first capillary
organ, higher distribution was observed in the spleen and liver 1h post injection due to the
sequestration effect by the mononuclear phagocyte system (MPS). At 4 h post injection, the
fluorescence intensity was reduced in the lungs, and increased in the spleen and liver. After
24 h, fluorescence intensity in the lungs was further reduced, and signals with equal strength
were observed in the liver and spleen. At 48 h, almost no fluorescence signal was observed
in the lungs and high signal accumulation in the spleen and liver.
3.5.4 Histological analysis
The histological examination of lungs, liver, spleen and kidneys removed from the sacrificed
mice was performed at 1 h, 4 h, 24 h and 48 h respectively. Microscopic analysis showed the
presence of PEG-PCL-NPs in the lungs, liver and spleen as shown in Figure 3.12.
Fluorescence microscopy showed tissue structure in addition to a red signal for VivoTag
680XL-labeled PEG-PCL-NPs and a blue signal from DAPI stained nuclei. At early
investigation time points (1 h and 4 h), NPs are clearly present in the lungs as shown in
Figure 3.12. Moreover, single NPs are found in septal capillaries and as small endocytosed
clusters in alveolar/interstitial macrophages. Notably, at 24 h post injection, relatively larger
aggregates of NPs appear in larger arterioles without any sign of emboli/fibrillary material
(eosinophilic intravascular occlusive material causing fibrin thrombo-emboli). At 48 h post
injection, very few NPs were observed in the lungs. There was no sign of increased cellularity
in the pulmonary septa and no macrophage accumulation in any of the mice, nor any sign of
inflammation. To a higher extent, we noticed NPs presence in the liver. Histological
investigation shows NPs association with sinusoidal Kupffer cells. There is no remarkable
change in the amount of NPs in liver over the time. The spleen seems to be another major
target organ, which can be seen in histological examinations as well as in the fluorescence
imaging. Nanoparticles were observed mainly in association with macrophages, most
29
commonly in the marginal zone. Slight signs of clearance of NPs in the spleen and liver were
observed at 48 h. Very few NPs were observed in the kidneys (Figure S5, paper III).
Figure 3.11 3D fluorescence imaging-CT imaging co-registration of Balb/c mice at 1 h, 4
h, 24 h and 48 h after IV administration of VivoTag XL680-PEG-PCL-NPs , dorsal side
and left side as shown. Heat map represents the area with fluorescence signal and color
represents the intensity.
30
Figure 3.12 Fluorescence microscopy overlaid phase contrast images of lung, liver and
spleen harvested at 1 h, 4 h, 24 h and 48 h. Fluorescence images of VivoTag 680XL
PEG-PCL-NPs are shown in red (CY5) while nuclei are stained with (DAPI) using 4',6diamidino-2-phenylindole. Scale bar = 100 μm.
31
4 Conclusions
The current thesis presents the essential ingredients to develop multifunctional drug
delivery
system
(DDS)
including
biocompatible
and
biodegradable
polymers,
chemotherapeutic agents, and high performance MRI T2 contrast agent and fluorescent
probes through different methodologies.
Monodisperse superparamagnetic iron oxide nanoparticles (SPION) were synthesized
via high temperature decomposition of iron-oleate precursor in an organic solvent. The
nanoparticles showed uniform size distribution and no hysteresis were observed which
demonstrated the desired superparamagnetism for T2 MRI application. Mn:ZnS
nanocrystals showed strong fluorescence emission peak at 594 nm of 3.1 nm QDs size.
SPION and Mn:ZnS were encapsulated into a matrix of biodegradable polymer (PLGA) via
O/W emulsion method. The MRI phantom of PLGA vesicles displays enhancement of the
T2* MRI contrast in vivo as it showed in liver of animal model. The drug delivery capability
of PLGA vesicles was achieved by loading busulphan as antineoplastic agent into the polymer
matrix. Sustained drug release rate was achieved for 5-6 h with low burst effect.
Biodegradable amphiphilic polymers were selected as mainstay to form the nanocarrier
system. Multifunctional drug carrier has been prepared by introducing SPION and
anticancer drug to the hydrophobic layer of PEG-PCL-NPs. The drug carrier showed slow
drug release rate during 10 h. SPION-PEG-PCL phantoms were successfully enhanced the
T2* MRI contrast. The cytotoxicity studies showed that PEG-PCL-NPs didn’t cause severe
damage in the treated cells providing the safe use of PEG-PCL-NPs for biomedical
application. The in vivo fluorescence imaging of PEG-PCL-NPs showed uptake and
distribution of NPs in various tissues mostly in liver and spleen as target tissues.
32
Future work
Extensive studies have been devoted to design ideal therapeutic device for clinical
applications. It should be in a form of minimum toxicity with low administration frequency.
A multi-in-single agent with simultaneous diagnostic and therapeutic functions is required
to achieve maximum therapeutic efficacy with low dosage and minimal side effects. This
agent should deliver the payloads to the pathological sites and release them in a controlled
manner. Further biological studies on busulphan loaded-PEG-PCL and PLGA nanocarriers
for in vivo and preclinical applications have to be assessed as follows
The fast drug diffusion from the polymeric nanocarriers can be overcome by covalently
linking of the drug molecules with the building units of polymer chains. Sustain drug release
from the polymeric nanoparticles with limited burst effect can be achieved by developing
nanocarrier system based on non-biodegradable polymers including polyvinyl alcohol
(PVA) and ethylene vinyl acetate (EVA) and/or mesoporous silica coated the drug carrier
which are utilized as a controlled elution membrane around the release area of the drug to
minimize the burst effect.
The therapeutic efficacy of busulphan loaded -PEG-PCL and PLGA NPs will be
evaluated in vivo using tumor models such myeloid leukemia, lymphatic leukemia, etc.
Moreover, the photodynamic activity of the developed AlPc loaded-PEG-PCL-NPs as a
nanophotosensitizer is essential to be investigated in vitro and in vivo by measuring the
generated singlet oxygen species after light irradiation of the nanophotosensitizer.
For early detection of diseases and targeted drug delivery, SPION-PEG-PCL-NPs and
SPION-PLGA-NPs can be functionalized with targeting moieties. It can be used to load
anticancer drug and reach to the site of action which recognized by the specific receptors on
the surface of cancer cells.
33
Acknowledgements
First of all, I would like to thank my main supervisor, Prof. Joydeep Dutta for his supervision,
great help and scientific discussion. My deepest gratitude to Prof. Mamoun Muhammed, for
giving me the precious opportunity to join the Division of Functional Materials and leading
me to the world of nanotechnology. His enlightening ideas and kindness are always strong
supports during my study.
I would like to thank my co-supervisor Prof. Moustapha Hassan from Karolinska
Institute for his great help from research to everyday life, especially for his kind help in
animal studies. I wish him a great success in the scientific career.
Thanks to my co-supervisor Dr. Fei Ye for his great patience, scientific discussion and
professional guidance on my first step of research. My appreciation is extended to Prof.
Muhammet Toprak and Dr. Abdusalam Uheida for their help and precious advices. Many
thanks to the members in the Division of Functional Materials, Adrine, Carmen, Venkatesh,
Mohsen, Noroozi, Nader, Yichen, Adem, Mohsin, Bejan, Natalia, Wei, Tanjim, Felipe, Aqib
and Lorenzo for their friendship and support. I appreciate Wubeshet for his great work of
maintaining and helping on use of electron microscopes.
I am grateful to my coworkers from the department of Laboratory Medicine, Karolinska
Institutet for the fruitful collaborations: Assoc. Prof. Manuchehr Abedi-Valugerdi, Ibrahim,
Maria, Mona, Fadwa, Shiva, Svetlana, Ying and Wenyi I wish all of them a splendid future
for their career.
I would also like to thank Prof. Sherif Kandil and Prof. Moataz Soliman from Alexandria
University in Egypt for their productive collaboration, support and encouragement.
Last but not least, I would like to thank my family for their endless support and
encouragement and the most important person, my beloved husband Raul Molines, for his
deep love and strong support during my research studies.
34
References
1. Muhammed, M., Engineering of Nanostructured Materials. In Nanostructures: Synthesis,
Functional Properties and Applications, Tsakalakos, T., Ovid’ko, I., Vasudevan, A., Eds.
Springer Netherlands: 2003, Vol. 128, pp 37-79.
2.
Gao, J., Xu, B., Nano Today 2009, 4 (1), 37-51.
3.
Arico, A. S., Bruce, P., Scrosati, B., Tarascon, J.-M., van Schalkwijk, W., Nat Mater
2005, 4 (5), 366-377.
4.
Pumera, M., Energy Environ. Sci. 2011, 4 (3), 668-674.
5.
Thatai, S., Khurana, P., Boken, J., Prasad, S., Kumar, D., Microchem. J. 2014, 116, 6276.
6.
Savage, N., Diallo, M., J. Nanopart. Res. 2005, 7 (4-5), 331-342.
7.
Silvestre, C., Duraccio, D., Cimmino, S., Prog. Polym. Sci. 2011, 36 (12), 1766-1782.
8.
Neethirajan, S., Jayas, D., Food and Bioprocess Technology 2011, 4
(1), 39-47.
9.
Barreto, J. A., O’Malley, W., Kubeil, M., Graham, B., Stephan, H., Spiccia, L., Adv.
Mater. 2011, 23 (12), 18-40.
10.
Marchesan, S., Prato, M., ACS Med. Chem. Lett. 2013, 4 (2), 147-149.
11.
Di Corato, R., Bigall, N. C., Ragusa, A., Dorfs, D., Genovese, A., Marotta, R., Manna,
L., Pellegrino, T., ACS Nano 2011, 5 (2), 1109-1121.
12.
Wang, C., Irudayaraj, J., Small 2010, 6 (2), 283-289.
13.
Yoon, T.-J., Kim, J. S., Kim, B. G., Yu, K. N., Cho, M.-H., Lee, J.-K., Angew.Chem. Int.
Ed. 2005, 44 (7), 1068-1071.
14.
Zhang, J., Sun, H., Ma, P. X., ACS Nano 2010, 4 (2), 1049-1059.
15.
Lee, J.-H., Lee, K., Moon, S. H., Lee, Y., Park, T. G., Cheon, J., Angew.Chem. 2009,
121 (23), 4238-4243.
16.
Yu, M. K., Jeong, Y. Y., Park, J., Park, S., Kim, J. W., Min, J. J., Kim, K., Jon, S.,
Angew. Chem. Int. Ed. 2008, 47 (29), 5362-5365.
17.
Santra, S., Kaittanis, C., Grimm, J., Perez, J. M., Small 2009, 5 (16), 862-1868.
18.
Bridot, J.-L., Dayde, D., Riviere, C., Mandon, C., Billotey, C., Lerondel, S., Sabattier,
R., Cartron, G., Le Pape, A., Blondiaux, G., Janier, M., Perriat, P., Roux, S., Tillement,
O., J. Mater. Chem. 2009, 19 (16), 2328-2335.
35
19.
Bardhan, R., Chen, W., Perez-Torres, C., Bartels, M., Huschka, R. M., Zhao, L. L.,
Morosan, E., Pautler, R. G., Joshi, A., Halas, N. J., Adv. Funct.Mater. 2009, 19 (24),
3901-3909.
20.
Liong, M., Lu, J., Kovochich, M., Xia, T., Ruehm, S. G., Nel, A. E.,Tamanoi, F., Zink,
J. I., ACS Nano 2008, 2 (5), 889-896.
21.
Kim, J., Kim, H. S., Lee, N., Kim, T., Kim, H., Yu, T., Song, I. C., Moon, W.K., Hyeon,
T., Angew. Chem. Int. Ed. 2008, 47 (44), 8438-8441.
22.
Kam, N. W. S., O'Connell, M., Wisdom, J. A., Dai, H., Proc. Natl. Acad.Sci. U. S. A.
2005, 102 (33), 11600-11605.
23.
Kim, J., Lee, J. E., Lee, S. H., Yu, J. H., Lee, J. H., Park, T. G., Hyeon, T.,Adv.
Mater. 2008, 20 (3), 478-483.
24.
Liu, H., Chen, D., Li, L., Liu, T., Tan, L., Wu, X., Tang, F., Angew. Chem.2011, 123
(4), 921-925.
25.
Kievit, F. M., Zhang, M., Acc. Chem. Res. 2011, 44 (10), 853-862.
26.
Allen, T. M., Cullis, P. R., Science 2004, 303 (5665), 1818-1822.
27.
Langer, R. S., Peppas, N. A., Biomaterials 1981, 2 (4), 201-214.
28.
Huang, X., Brazel, C. S., J. Control. Release 2001, 73 (2–3), 121-136.
29. Cho, K., Wang, X., Nie, S., Chen, Z., Shin, D. M., Clin. Cancer Res. 2008,14 (5), 13101316.
30.
LaVan, D. A., Lynn, D. M., Langer, R., Nat. Rev. Drug Discov. 2002, 1(1), 77-84.
31.
Torchilin, V., Liposomes in Drug Delivery. In Fundamentals and Applications of
Controlled Release Drug Delivery, Siepmann, J., Siegel, R. A., Rathbone, M. J., Eds.
Springer US: 2012, pp 289-328.
32.
Patel, M. P., Patel, R. R., Patel, J. K., J. Pharm. Pharm. Sci. 2010, 13 (4),536-57.
33.
Yuan, L., Tang, Q., Yang, D., Zhang, J. Z., Zhang, F., Hu, J., The Journal of Physical
Chemistry C 2011, 115 (20), 9926-9932.
34.
Basu, M., Lala, S, Nanoparticulates as Drug Carriers. Imperial College Press:
London, GBR, 2006.
35.
Jeon, S. I., Lee, J. H., Andrade, J. D., De Gennes, P. G., J. Colloid Interface Sci.
1991, 142 (1), 149-158.
36.
Woodle, M. C., Adv. Drug Del. Rev. 1995, 16 (2–3), 249-265.
37.
Brigger, I., Dubernet, C., Couvreur, P., Adv. Drug Del. Rev. 2002, 54(5), 631-651.
38.
Nair, L., Laurencin, C., Polymers as Biomaterials for Tissue Engineering and
Controlled Drug Delivery. In Tissue Engineering I, Lee, K., Kaplan, D., Eds. Springer
Berlin Heidelberg: 2006, Vol. 102, pp 47-90.
36
39.
Edlund, U., Albertsson, A. C., Degradable Polymer Microspheres for Controlled Drug
Delivery. In Degradable Aliphatic Polyesters, Springer Berlin Heidelberg: 2002,
Vol.157, pp 67-112.
40.
Nair, L. S., Laurencin, C. T., Prog. Polym. Sci. 2007, 32 (8–9), 762-798.
41.
Wei, X., Gong, C., Gou, M., Fu, S., Guo, Q., Shi, S., Luo, F., Guo, G., Qiu, L.,
Qian, Z., Int. J. Pharm. 2009, 381 (1), 1-18.
42.
Okada, M., Prog. Polym. Sci. 2002, 27 (1), 87-133.
43.
Sisson, A. L., Schroeter, M., Lendlein, A., Polyesters. In Handbook of Biodegradable
Polymers, Wiley-VCH Verlag GmbH & Co. KGaA: 2011, pp 1-21.
44.
Yang, W.-W., Pierstorff, E., Journal of Laboratory Automation 2012, 17(1), 50-58.
45.
Albertsson, A.-C., Varma, I., Aliphatic Polyesters: Synthesis, Properties and
Applications. In Degradable Aliphatic Polyesters, Springer Berlin Heidelberg: 2002,
Vol.157, pp 1-40.
46.
Otsuka, H., Nagasaki, Y., Kataoka, K., Adv. Drug Del. Rev. 2003, 55 (3), 403-419.
47.
Lasic, D. D., Trends Biotechnol. 1998, 16 (7), 307-321.
48.
Schmaljohann, D., Adv. Drug Del. Rev. 2006, 58 (15), 1655-1670.
49.
Gil, E. S., Hudson, S. M., Prog. Polym. Sci. 2004, 29 (12), 1173-1222.
50.
Qiu, Y., Park, K., Adv. Drug Del. Rev. 2012, 64, Supplement (0), 49-60.
51.
Gupta, P., Vermani, K., Garg, S., Drug Discov. Today 2002, 7 (10), 569-579.
52.
Kwon, G. S., Kataoka, K., Adv. Drug Del. Rev. 1995, 16 (2–3), 295-309.
53.
Haag, R., Angew. Chem. Int. Ed. 2004, 43 (3), 278-282.
54.
Kabanov, A. V., Batrakova, E. V., Alakhov, V. Y., J. Control. Release, 2002, 82 (2–3),
189-212.
55.
Gaucher, G., Dufresne, M.-H., Sant, V. P., Kang, N., Maysinger, D.,Leroux, J.-C., J.
Control. Release 2005, 109 (1–3), 169-188.
56.
Wakebayashi, D., Nishiyama, N., Yamasaki, Y., Itaka, K., Kanayama, N., Harada, A.,
Nagasaki, Y., Kataoka, K., J. Control. Release 2004, 95 (3), 653-664.
57.
Itaka, K., Yamauchi, K., Harada, A., Nakamura, K., Kawaguchi, H., Kataoka, K.,
Biomaterials 2003, 24 (24), 4495-4506.
58.
Nishiyama, N., Kato, Y., Sugiyama, Y., Kataoka, K., Pharm. Res. 2001,18 (7), 10351041.
59.
Adams, M. L., Lavasanifar, A., Kwon, G. S., J. Pharm. Sci. 2003, 92 (7),1343-1355.
60.
Rösler, A., Vandermeulen, G. W. M., Klok, H.-A., Adv. Drug Del. Rev.2012, 64,
Supplement (0), 270-279.
37
61.
Allen, C., Maysinger, D., Eisenberg, A., Colloids Surf. B. Biointerfaces 1999,16 (1–
4), 3-27.
62.
La, S. B., Okano, T., Kataoka, K., J. Pharm. Sci. 1996, 85 (1), 85-90.
63.
Jones, M.-C., Leroux, J.-C., Eur. J. Pharm. Biopharm. 1999, 48 (2), 101-111.
64.
Yokoyama, M., Kwon, G. S., Okano, T., Sakurai, Y., Seto, T., Kataoka, K., Bioconjug.
Chem. 1992, 3 (4), 295-301.
65.
Patra, S., Roy, E., Karfa, P., Kumar, S., Madhuri, R., Sharma, P. K., ACS Applied
Materials & Interfaces 2015, 7 (17), 9235-9246.
66.
Kohler, N., Sun, C., Fichtenholtz, A., Gunn, J., Fang, C., Zhang, M., Small 2006, 2
(6), 785-792.
67.
Jain, T. K., Richey, J., Strand, M., Leslie-Pelecky, D. L., Flask, C. A., Labhasetwar,
V., Biomaterials 2008, 29 (29), 4012-4021.
68.
Shah, B. P., Pasquale, N., De, G., Tan, T., Ma, J., Lee, K.-B., ACS Nano 2014, 8 (9),
9379-9387.
69.
Lee, H., Yu, M. K., Park, S., Moon, S., Min, J. J., Jeong, Y. Y., Kang, H.-W., Jon, S.,
J. Am. Chem. Soc. 2007, 129 (42), 12739-12745.
70.
Yu, M. K., Park, J., Jeong, Y. Y., Moon, W. K., Jon, S., Nanotechnology 2010, 21
(41), 415102.
71.
Tassa, C., Shaw, S. Y., Weissleder, R., Acc. Chem. Res. 2011, 44 (10), 842-852.
72.
Zhu, A., Yuan, L., Jin, W., Dai, S., Wang, Q., Xue, Z., Qin, A., Acta Biomater. 2009, 5
(5), 1489-1498.
73.
Berry, C. C., J. Phys. D: Appl. Phys. 2009, 42 (22), 224003.
74.
Zheng, T., Pang, J., Tan, G., He, J., McPherson, G. L., Lu, Y., John, V. T., Zhan, J.,
Langmuir 2007, 23 (9), 5143-5147.
75.
Yezhelyev, M. V., Qi, L., O’Regan, R. M., Nie, S., Gao, X., J. Am. Chem. Soc. 2008,
130 (28), 9006-9012.
76.
Qi, L., Gao, X., ACS Nano 2008, 2 (7), 1403-1410.
77.
Derfus, A. M., Chen, A. A., Min, D.-H., Ruoslahti, E., Bhatia, S. N., Bioconjug. Chem.
2007, 18 (5), 1391-1396.
78.
Zhu, S., Zhang, J., Tang, S., Qiao, C., Wang, L., Wang, H., Liu, X., Li, B., Li, Y., Yu,
W., Wang, X., Sun, H., Yang, B., Adv. Funct. Mater. 2012, 22 (22), 4732-4740.
79.
Biju, V., Mundayoor, S., Omkumar, R. V., Anas, A., Ishikawa, M., Biotechnol. Adv.
2010, 28 (2), 199-213.
80.
38
Zhou, J., Yang, Y., Zhang, C.-y., Chem. Rev. 2015, 115 (21), 11669-11717.
81.
Srivastava, B. B., Jana, S., Karan, N. S., Paria, S., Jana, N. R., Sarma, D. D.,
Pradhan, N., The Journal of Physical Chemistry Letters 2010, 1 (9), 1454-1458.
82.
Chin, P. T. K., Stouwdam, J. W., Janssen, R. A. J., Nano Lett. 2009, 9 (2), 745-750.
83.
Erwin, S. C., Zu, L., Haftel, M. I., Efros, A. L., Kennedy, T. A., Norris, D. J., Nature
2005, 436 (7047), 91-94.
84.
Norris, D. J., Yao, N., Charnock, F. T., Kennedy, T. A., Nano Lett. 2001, 1 (1), 3-7.
85.
Bhargava, R. N., Gallagher, D., Hong, X., Nurmikko, A., Phys. Rev. Lett. 1994, 72
(3), 416-419.
86.
Yamashita, F., Hashida, M., Adv. Drug Del. Rev. 2013, 65 (1), 139-147.
87.
Uhrich, K. E., Cannizzaro, S. M., Langer, R. S., Shakesheff, K. M., Chem. Rev. 1999,
99 (11), 3181-3198.
88.
Gombotz, W. R., Pettit, D. K., Bioconjug. Chem. 1995, 6 (4), 332-351.
89.
Fu, Y., Kao, W. J., Expert opinion on drug delivery 2010, 7 (4), 429-444.
90. Jo, Y. S., Kim, M.-C., Kim, D. K., Kim, C.-J., Jeong, Y.-K., Kim, K.-J., Muhammed, M.,
Nanotechnology 2004, 15 (9), 1186.
91. Freiberg, S., Zhu, X. X., Int. J. Pharm. 2004, 282 (1–2), 1-18.
92. Singh, R., Lillard Jr, J. W., Exp. Mol. Pathol. 2009, 86 (3), 215-223.
93. Koo, O. M., Rubinstein, I., Onyuksel, H., Nanomed. Nanotechnol. Biol. Med. 2005, 1
(3), 193-212.
94. Liang, H.-F., Chen, C.-T., Chen, S.-C., Kulkarni, A. R., Chiu, Y.-L., Chen, M.-C.,
Sung, H.-W., Biomaterials 2006, 27 (9), 2051-2059.
95. Sudimack, J., Lee, R. J., Adv. Drug Del. Rev. 2000, 41 (2), 147-162.
96. Wang, S., Low, P. S., J. Control. Release 1998, 53 (1–3), 39-48.
97. Leamon, C. P., Low, P. S., Drug Discov. Today 2001, 6 (1),44-51.
98. Park, E. K., Kim, S. Y., Lee, S. B., Lee, Y. M., J. Control. Release 2005, 109 (1–3),
158-168.
99. Kim, S. H., Jeong, J. H., Chun, K. W., Park, T. G., Langmuir 2005, 21 (19), 88528857.
100. Sonvico, F., Mornet, S., Vasseur, S., Dubernet, C., Jaillard, D., Degrouard, J.,
Hoebeke, J., Duguet, E., Colombo, P., Couvreur, P., Bioconjug. Chem. 2005, 16
(5),1181-1188.
101. Zhang, Y., Zhang, J., J. Colloid Interface Sci. 2005, 283 (2), 352-357.
102. Morosini, V., Bastogne, T., Frochot, C., Schneider, R., Francois, A., Guillemin, F.,
Barberi-Heyob, M., Photochemical & Photobiological Sciences 2011, 10 (5), 842-851.
103. Lu, J., Li, Z., Zink, J. I., Tamanoi, F., Nanomed. Nanotechnol. Biol. Med. 2012, 8 (2),
39
212-220.
104. Massoud, T. F., Gambhir, S. S., Genes Dev. 2003, 17 (5), 545-80.
105. Lee, N., Hyeon, T., Chem. Soc. Rev. 2012, 41 (7), 2575-2589.
106. Blanquer, S., Guillaume, O., Letouzey, V., Lemaire, L., Franconi, F., Paniagua, C.,
Coudane, J., Garric, X., Acta Biomater. 2012, 8 (3), 1339-1347.
107. Ling, Y., Wei, K., Luo, Y., Gao, X., Zhong, S., Biomaterials 2011, 32 (29), 7139-7150.
108. Lee, J. E., Lee, N., Kim, T., Kim, J., Hyeon, T., Acc. Chem. Res. 2011, 44 (10), 893902.
109. Yang, X., Grailer, J. J., Rowland, I. J., Javadi, A., Hurley, S. A., Steeber, D. A., Gong,
S., Biomaterials 2010, 31 (34), 9065-9073.
110. Purcell, E. M., Torrey, H. C., Pound, R. V., Phys. Rev. 1946, 69 (1-2), 37-38.
111. Bloch, F., Hansen, W. W., Packard, M., Phys. Rev. 1946, 69 (3-4), 127-127.
112. Fullerton, G. D., Magn. Reson. Imaging 1982, 1 (1), 39-53.
113. Schaeffter, T., Dahnke, H., Magnetic Resonance Imaging and Spectroscopy. In
Molecular Imaging I, Semmler, W., Schwaiger, M., Eds. Springer Berlin Heidelberg:
2008, Vol. 185/1, pp 75-90.
114. Lu, A.-H., Salabas, E. L., Schüth, F., Angew. Chem. Int. Ed. 2007, 46 (8), 1222-1244.
115. Krishnan, K. M., Pakhomov, A. B., Bao, Y., Blomqvist, P., Chun, Y., Gonzales, M.,
Griffin, K., Ji, X., Roberts, B. K., Journal of Materials Science 2006, 41 (3), 793-815.
116. Bean, C. P., Livingston, J. D., J. Appl. Phys. 1959, 30 (4), S120-S129.
117. Dave, S. R., Gao, X., Wiley Interdisciplinary Reviews: Nanomedicine and
Nanobiotechnology 2009, 1 (6), 583-609.
118. Crangle, J., Domain magnetism. In Solid · State Magnetism, Springer US: 1991, pp
155-181.
119. Koksharov, Y. A., Magnetism of Nanoparticles: Effects of Size, Shape, and
Interactions. In Magnetic Nanoparticles, Wiley-VCH Verlag GmbH & Co. KGaA: 2009,
pp 197-254.
120. Frangioni, J. V., Curr. Opin. Chem. Biol. 2003, 7 (5), 626-634.
121. Rao, J., Dragulescu-Andrasi, A., Yao, H., Curr. Opin. Biotechnol. 2007, 18 (1), 17-25.
122. Michalet, X., Pinaud, F. F., Bentolila, L. A., Tsay, J. M., Doose, S., Li, J. J.,
Sundaresan, G., Wu, A. M., Gambhir, S. S., Weiss, S., Science 2005, 307 (5709),
538-544.
123. Chatterjee, D. K., Rufaihah, A. J., Zhang, Y., Biomaterials 2008, 29 (7), 937-943.
124. Resch-Genger, U., Grabolle, M., Cavaliere-Jaricot, S., Nitschke, R., Nann, T., Nat
Meth 2008, 5 (9), 763-775.
40
125. Petryayeva, E., Algar, W. R., Medintz, I. L., Appl. Spectrosc. 2013, 67 (3), 215-252.
126. Rao, J. P., Geckeler, K. E., Prog. Polym. Sci. 2011, 36 (7), 887-913.
127. Hans, M. L., Lowman, A. M., Curr. Opin. Solid State Mater. Sci. 2002, 6 (4), 319-327.
128. Feng, S.-s., Huang, G., J. Control. Release 2001, 71 (1), 53-69.
129. Fessi, H., Puisieux, F., Devissaguet, J. P., Ammoury, N., Benita, S., Int. J. Pharm.
1989, 55 (1), R1-R4.
130. Pinto Reis, C., Neufeld, R. J., Ribeiro, A. J., Veiga, F., Nanomed. Nanotechnol. Biol.
Med. 2006, 2 (1), 8-21.
131. Bee, A., Massart, R., Neveu, S., J. Magn. Magn. Mater. 1995, 149 (1–2), 6-9.
132. Kandori, K., Kawashima, Y., Ishikawa, T., J. Colloid Interface Sci. 1992, 152 (1), 284288.
133. Wan, S., Huang, J., Yan, H., Liu, K., J. Mater. Chem. 2006, 16 (3), 298-303.
134. Kim, D. K., Mikhaylova, M., Zhang, Y., Muhammed, M., Chem. Mater. 2003, 15 (8),
1617-1627.
135. Jiang, W., Yang, H. C., Yang, S. Y., Horng, H. E., Hung, J. C., Chen, Y. C., Hong, C.Y., J. Magn. Magn. Mater. 2004, 283 (2–3), 210-214.
136. Park, J., An, K., Hwang, Y., Park, J.-G., Noh, H.-J., Kim, J.-Y., Park, J.-H., Hwang, N.M., Hyeon, T., Nat Mater 2004, 3 (12), 891-895.
137. Sun, S., Zeng, H., Robinson, D. B., Raoux, S., Rice, P. M., Wang, S. X., Li, G., J. Am.
Chem. Soc. 2004, 126 (1), 273-279.
138. Redl, F. X., Black, C. T., Papaefthymiou, G. C., Sandstrom, R. L., Yin, M., Zeng, H.,
Murray, C. B., O'Brien, S. P., J. Am. Chem. Soc. 2004, 126 (44), 14583-14599.
139. O'Brien, S., Brus, L., Murray, C. B., J. Am. Chem. Soc. 2001, 123 (48), 12085-12086.
140. Farrell, D., Majetich, S. A., Wilcoxon, J. P., The Journal of Physical Chemistry B
2003, 107 (40), 11022-11030.
141. Jana, N. R., Peng, X., J. Am. Chem. Soc. 2003, 125 (47), 14280-14281.
142. Samia, A. C. S., Hyzer, K., Schlueter, J. A., Qin, C.-J., Jiang, J. S., Bader, S. D., Lin,
X.-M., J. Am. Chem. Soc. 2005, 127 (12), 4126-4127.
143. Li, Y., Afzaal, M., O'Brien, P., J. Mater. Chem. 2006, 16 (22), 2175-2180.
144. Liu, C., Zou, B., Rondinone, A. J., Zhang, Z. J., The Journal of Physical Chemistry B
2000, 104 (6), 1141-1145.
145. Deng, H., Li, X., Peng, Q., Wang, X., Chen, J., Li, Y., Angew. Chem. Int. Ed. 2005, 44
(18), 2782-2785.
146. del Monte, F., Morales, M. P., Levy, D., Fernandez, A., Ocaña, M., Roig, A., Molins, E.,
O'Grady, K., Serna, C. J., Langmuir 1997, 13 (14), 3627-3634.
41
147. Tian, H., Tang, Z., Zhuang, X., Chen, X., Jing, X., Prog. Polym. Sci. 2012, 37 (2),
237-280.
148. Seyednejad, H., Ghassemi, A. H., van Nostrum, C. F., Vermonden, T., Hennink, W. E.,
J. Control. Release 2011, 152 (1), 168-176.
149. Gyun Shin, I. L., Yeon Kim, S., Moo Lee, Y., Soo Cho, C., Yong Kiel, S., J. Control.
Release 1998, 51 (1), 1-11.
150. Kumar, M., Yigit, M., Dai, G., Moore, A., Medarova, Z., Cancer Res. 2010, 70 (19),
7553-7561.
42