Funtionalization of magnetic nanoparticles for bio applications

Magnetite nanoparticles were modified to render them suitable for bio-applications, namely drug delivery and hyperthermia, using two different approaches.. These functionalized nanospher

BIO-APPLICATIONS

WUANG SHY CHYI

(B Eng (Hons), NUS)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR

ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE

2008

I would like to express my heartfelt gratitude to my supervisors, Professors K G Neoh, Daniel Pack, E.-T Kang and Deborah Leckband for their continued guidance, invaluable suggestions and profound discussion throughout this work Without their enthusiasm and help, this project would not be possible The knowledge gained under their supervision and the research experiences pave the way for my lifelong study

I would also like to thank the other members of my committee for their help and time,

as well as the research staff and laboratory officers, both in the National University of Singapore and the University of Illinois at Urbana-Champaign

Finally I thank my family, colleagues and numerous friends for their love, support and encouragement

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CHAPTER 5: ANTIBODY-ATTACHED MAGNETITE

NANOPARTICLES

5.1 Introduction 5.2 Methods and materials 5.3 Results and discussion 5.4 Chapter 5 Conclusion

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Magnetite nanoparticles were modified to render them suitable for bio-applications, namely drug delivery and hyperthermia, using two different approaches The first approach is to graft polymers onto the nanoparticles using surface-initiated atom transfer radical polymerization, followed by chemical linking of biomolecules onto the grafted polymers The monomers used include N-isopropylacrylamide, N-vinylformamide and methacrylic acid while the immobilized biomolecules include heparin, folic acid, doxorubicin and anti-HER2/neu antibodies It was found that the heparinized nanoparticles could reduce macrophage uptake, and at the same time inhibit plasma clotting The doxorubicin-bearing nanoparticles were able to release a greater amount of the drug under acidic conditions as opposed to physiological pH, and could potentially serve as drug depots Particles that were functionalized with anti-HER2/neu antibodies showed a preferential binding to cancer cells and may be useful for imaging purposes

The second approach was to encapsulate these magnetite nanoparticles into polypyrrole nanospheres via emulsion polymerization for potential use as hyperthermia causing agents The nanospheres were then functionalized with folic acid or herceptin to impart onto them cancer cell-targeting properties These functionalized nanospheres target cancer cells in vitro and possess good magnetization which is useful for magnetic fluid hyperthermia

Table 3.1 Dispersion of as-synthesized and functionalized magnetite in various

solvents at 25ºC

Table 3.2 Comparison of PRT obtained in the absence and presence of magnetic

nanoparticles

Table 6.1 Characteristics of the PPY nanospheres (Scale bar = 200nm)

Table 6.2 Properties of NS(PVA)

Table 6.3 Properties of NS(HA) and NS(HA)-HER2

Table 6.4 Amount of iron associated with SK-Br-3 and MDA-MB-231 cells after

incubation with NS(HA) and NS(HA)-HER2

Figure 3.1 Schematic representation of the process for preparing MNP-NP-He

Figure 3.2 XPS C 1s and S 2p core-level spectra of as-synthesized MNP (a, c),

magnetite-Cl (b, d) and N 1s core-level spectra of magnetite-Cl (e) and MNP-NP (f)

Figure 3.3 XPS C 1s and S 2p core-level spectra of MNP-NP (a, c) and

MNP-NP-He (b, d)

Figure 3.4 FTIR spectra of MNP (a), magnetite-Cl (b), NP(c) and

MNP-NP-He (d)

Figure 3.5 Room temperature magnetization curves of as-synthesized and

functionalized magnetite nanoparticles as a function of applied magnetic field: MNP (a), MNP-NP (b) and MNP-NP-He (c)

Figure 3.6 Optical microscopy images of macrophages cultured with control cells

(a, b), MNP (c, d), MNP-NP (e, f) and MNP-NP-He (g, h) after 2 and

24 h respectively Figure scale bar = 50μm, inset scale bar = 25μm Figure 3.7 Total uptake of as-synthesized and functionalized magnetite

nanoparticles by macrophages after 2, 8 and 24 h

Figure 3.8 Cytotoxicity of as-synthesized and functionalized magnetite

nanoparticles, as measured by the viability of macrophages grown in media containing 0.2 mg/ml of these nanoparticles relative to the non-toxic control T represents the results obtained with the toxic control Results are represented as mean ± standard deviation

Figure 4.1 Schematic for synthesis of doxorubicin-conjugated particles

Figure 4.2 XPS C 1s core-level spectra of MNP-P(MAA)-NHNH2 (a),

MNP-P(MAA)-NH-N=Dox (b) and doxorubicin hydrochloride (c)

Figure 4.3 Magnetization profiles of MNP-P(MAA)-NH-N=Dox in the solid state

(a), and as dispersed in 1% agarose (b)

Figure 4.4 In vitro doxorubicin release from MNP-P(MAA)-NH-N=Dox at 37°C

or 42°C in various pHs as indicated

Figure 4.5 In vitro doxorubicin release from MNP-P(MAA)-NH-N=Dox at 37°C

Arrow indicates point of pH change from 7.4 to 5.5 or 6.6

Figure 4.6 In vitro doxorubicin release from MNP-P(MAA)-NH-N=Dox Arrow

indicates point of temperature change from 37°C to 42°C and pH change from 7.4 to 5.5 or 6.6

MNP-P(MAA)-NH-N=Dox “*”, “#”, “**” and “##” denote statistical

differences (P < 0.05) between the similarly marked samples

Figure 4.8 Optical microscopy images of prussian blue staining of MDA-MB-231

cells cultured with MNP-P(MAA)-NH-N=Dox in pH 5.5 (a) and pH 7.4 (b) Scale bar = 25μm

Figure 5.1 Schematic for synthesis of MNP-NVAM-PEG-Ab

Figure 5.2 FTIR spectra of NVAM (a), NVAM-PEG (b) and

MNP-NVAM-PEG-Ab (c)

Figure 5.3 Optical microscopy images of SK-Br-3 cells after a 4 h incubation with

Ab (a) and co-treatment with

MNP-NVAM-PEG-Ab and free antibody (b) Scale bar = 25 µm

Figure 5.4 Prussian blue staining of the liver (a), bladder (b), heart (c), kidney (d),

spleen (e), lung (f), tumors (g-i) and the site of injection, tail (j) Scale bar = 50 µm

Figure 5.5 Prussian blue staining of the liver (a), bladder (b), heart (c), kidney (d),

spleen (e), lung (f), tumors (g-i) and the site of injection, tail (j) Scale bar = 50 µm

Figure 5.6 MMOCT spectra of negative control (a), and phantom with an

equivalent particle concentration of 23 µg Fe/ml (b) Scale bar = 250

μm

Figure 6.1 Schematic representation of the preparation route NS(PVA)-FA

Figure 6.2 (a) FTIR spectra of Fe3O4, PPY nanospheres and NS(PVA) (b) XRD

spectrum of NS(PVA)

Figure 6.3 Room temperature magnetization curves of NS(PVA) as a function of

applied magnetic field

Figure 6.4 FESEM and TEM images of NS(PVA) with (a, b) 0 %, (c, d) 23.5 %,

(e, f) 28.0 % and (g, h) 38.8 % Fe3O4 content respectively

Figure 6.5 XPS C 1s core-level and wide scan spectra of (a, b) NS(PVA) and (c, d)

NS(PVA)-FA

Figure 6.6 Viabilities of 3T3 fibroblasts incubated with medium containing 0.2

mg/ml of NS(PVA) with the indicated Fe3O4 content FA-(28.0%) denoted NS(PVA)-FA with 28.0% of Fe3O4 “*” denotes statistical

differences (P < 0.05) compared to the control experiment

statistical differences (P < 0.05) compared to the control experiment

Figure 6.8 Optical microscopy images of MCF-7 cells cultured with: (a) no

nanospheres (control) (b) NS(PVA) and (c) NS(PVA)-FA after 24 h Figure scale bar = 50μm

Figure 6.9 Schematic for preparation of NS(HA) and subsequent functionalization

with herceptin

Figure 6.10 Uptake of the nanospheres by HCC1954 cells: (a) Optical microscopy

images of cells after 24 h incubation with (i) NS(HA), and (ii) NS(HA)-HER1 Figure scale bar = 50 μm (b) Amount of iron in cells after 2 h and 24 h incubation with NS(HA) and NS(HA)-HER1 determined using ICP

Figure 6.11 Schematic representation of the preparation of NS(HA)-HER2

Figure 6.12 XPS C 1s core-level spectra of (a) NS(NH2), (b) NS(HA)-HER2 and (c)

herceptin

Figure 6.13 Amount of iron associated with SK-Br-3 cells after 2, 4 and 24 h

incubation with NS(HA), NS(HA)-HER2 and NS(HA)-HER2 with free herceptin Three sets of duplicates were done for each data point The

iron association of NS(HA)-HER2 is significantly higher (P < 0.01)

than those for NS(HA) and NS(HA)-HER2 with free herceptin at all time points

Figure 6.14 Transmission electron micrographs of cells cultured with (a)

NS(HA)-HER2 (b) NS(HA)-NS(HA)-HER2 with pre- and co-treatment of 200 µg/ml free herceptin, for 4h

Figure 6.15 Cytotoxicities of NS(HA)-HER2 and NS(HA) with various

concentrations of herceptin (HER), as measured by the viabilities of SK-Br-3 cells grown in media containing 0.2 mg/ml of these nanospheres relative to the non-toxic control Results are represented

as mean ± standard deviation “*” denotes statistical differences (P <

0.05) compared to the control experiment

Figure 6.16 Plot of viability of cells versus iron uptake by breast cancer cells

Tested cell lines include SK-Br-3 (■), MDA-MB-231 (♦) and MCF-7 (▲)

Figure 6.17 Magnetization curves of NS(HA)-HER2 in different environments (a)

NS(HA)-HER2 solid, (b) NS(HA)-HER2 dispersed in culture medium with 1% agarose and (c) endocytosed NS(HA)-HER2 dispersed in culture medium with 1% agarose

ATRP Atom-transfer radical polymerization

MNP-NVAM poly(N-vinylamine)-grafted MNP

MNP-NVAM-PEG-Ab Antibody-linked PEGylated MNP-NVAM

(3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide)

NS(HA)-HER1, NS(HA)-HER2 Herceptin-functionalized NS(HA)

NS(PVA) PVA-stabilized PPY-Fe3O4 nanospheres

NS(PVA)-FA Folic acid –functionalized NS(PVA)

scFv Single chain Fv

CHAPTER 1: INTRODUCTION

In recent years, magnetic nanoparticles have been proposed for use in a number of biomedical applications such as drug delivery, hyperthermia and chemotherapy and as radiotherapy enhancement agents because of their special physical properties [1-3] Magnetic nanoparticles have controllable sizes, smaller than cells and comparable to proteins and other biological entities, and hence they can be modified for interaction with these biological entities Through manipulation by an external magnetic field, magnetic nanoparticles are potentially very useful in the transport and immobilization

of magnetically tagged biological cargoes, and also in transferring energy from the exciting field The focus of this research project is to functionalize or modify magnetic nanoparticles to render them suitable for biomedical applications such as drug delivery, tumor-targeting and hyperthermia Two different approaches were used

Iron oxides, namely Fe3O4 and γ-Fe2O3, have high saturation magnetization values, and their potential uses in biomedical applications are widely investigated Magnetite (Fe3O4) nanoparticles offer distinct advantages in localized drug delivery They are superparamagnetic (i.e they retain no magnetic properties when the magnetic field is removed) and can be guided to the targeted area with external magnetic fields [4] They exhibit low toxicity [5-7] and can be made biocompatible Therefore, magnetite nanoparticles were chosen for use in this project

The first approach was to coat individual magnetite nanoparticles of about 6-8 nm with polymers to improve their biocompatibility, followed by chemical linking of biomolecules onto the grafted polymers In Chapter 3, the surface modification of

magnetite nanoparticles with heparin to increase their circulation time and for potential delivery of heparin locally to prevent the formation of blood clots was investigated Preliminary work has shown that the direct immobilization of heparin on the magnetite nanoparticles could not be easily achieved As such, a poly(N-isopropylacrylamide) (poly(NIPAAM)) layer was first formed on the surface of the magnetite nanoparticles via atom-transfer radical polymerization (ATRP) followed by the immobilization of heparin onto the poly(NIPAAM) shell The bioactivity of heparinized magnetite nanoparticles was assessed via their uptake by macrophages and plasma recalcification time Uptake by the mononuclear phagocytic system (MPS)

or reticuloendothelial system and complement activation leading to clot formation are two major challenges confronting the in vivo use of nanoparticles as drug delivery vehicles Heparin is hydrophilic and can inhibit coagulation by binding and thereby inhibiting thrombin Thus, the tested hypothesis was that heparin immobilized on the surface of nanoparticles would reduce or even eliminate the processes of macrophage uptake and clot formation

In Chapter 4, a cancer drug, doxorubicin, was functionalized onto poly(methacrylic acid)-grafted magnetite nanoparticles through the use of acid-sensitive hydrazone linkages With doxorubicin conjugated to the magnetic carriers, an external magnet can then direct the drug to the target site (usually cancerous tumors) With such a site-specific drug delivery system, the local concentration of the cytotoxic drug at the target site can be maintained at appropriate levels while reducing the overall dosage

or systemic concentration Moreover, the use of acid-sensitive linkages allows a greater amount of doxorubicin to be released at the acidic conditions of the tumor environment Next, using a similar approach, antibodies (namely anti-HER2/neu

antibodies) were attached onto poly(N-vinylamine)-grafted magnetite nanoparticles using a poly(ethylene glycol) bifunctional linker (Chapter 5) This imparts targeting capability onto the nanoparticles which can then be potentially used in the imaging of tumors

Another approach used in the modification of magnetite nanoparticles is to encapsulate these particles inside polypyrrole (PPY) nanospheres The PPY-Fe3O4nanospheres retained high levels of magnetization and electrical conductivity and hence are potentially useful for hyperthermia by deploying both the magnetic and conductive heating capacities To impart tumor-targeting capability onto these magnetic nanospheres, folic acid (a vitamin) and herceptin (a cancer antibody) were surface-immobilized onto the nanospheres As discussed in Chapter 6, these targeting moieties lead to an increased uptake of the nanospheres by cancer cells The encapsulation of magnetic nanoparticles inside electrically conducting PPY nanospheres imparts magnetic property onto the nanospheres In addition, the heating effect deployed in hyperthermia may be enhanced with conductive heating in addition

to magnetic heating When the magnetic nanospheres used in hyperthermia can specifically target cancer cells, the effectiveness of hyperthermia would be further enhanced

CHAPTER 2: LITERATURE REVIEW

2.1 MAGNETIC NANOPARTICLES

Magnetic nanoparticles are of great interest for researchers from a wide range of disciplines, including catalysis [8, 9], data storage [10], environmental remediation [11, 12] and more recently in biotechnology/biomedicine [13] Some of the more specific biomedical applications of magnetic nanoparticles include their use as magnetic contrast agents in magnetic resonance imaging (MRI), hyperthermia agents, where the magnetic particles are heated selectively by application of a high frequency oscillating magnetic field, and magnetic drug delivery

In most biomedical applications, magnetic nanoparticles perform best when the size

of the nanoparticles is around 10–20 nm [14] Each nanoparticle then becomes a single magnetic domain and shows superparamagnetic behavior when the temperature

is above the Curie temperature Superparamagnetism is a phenomenon by which magnetic materials may exhibit a behavior similar to paramagnetism even when at temperatures below the Curie or the Néel temperature The energy required to change the direction of the magnetic moment of a superparamagnetic particle is comparable

to the ambient thermal energy At this point, the rate at which the particles will randomly reverse direction becomes significant Such individual nanoparticles have a large constant magnetic moment and behave like a giant paramagnetic atom with a fast response to applied magnetic fields with negligible remanence (residual magnetism) and coercivity (the field required to bring the magnetization to zero) These features make superparamagnetic nanoparticles very attractive for a broad range of biomedical applications because the risk of forming agglomerates is

negligible at room temperature Superparamagnetic iron oxide (SPIO) nanoparticles are small synthetic γ-Fe2O3 or Fe3O4 particles with a core size of ~10 nm and an organic or inorganic coating Superparamagnetic magnetization is, compared to normal paramagnetic materials, much higher and can reach nearly the magnetization saturation of ferromagnetic iron oxide [15] This behavior allows the tracking of such particles in a magnetic field gradient without losing the advantage of a stable colloidal suspension

Nanotechnology has allowed for the production, characterization and functionalization of magnetic nanoparticles for specialized clinical applications Extensive research has been done on use of magnetic nanoparticles for bio-applications in recent years For instance, dendrimer modified magnetic nanoparticles have been synthesized to improve protein binding [16] Iron oxide nanoparticles coated with insulin have been prepared for exact drug delivery [17], while modified metal oxide-based nanoparticles were developed for conjugation with cell targeting agents [18] Others also demonstrated the use of magnetic nanoparticles in imaging [19] and gene delivery [20, 21]applications

The main advantages of magnetic nanoparticles in biomedicine are that they can be (i)

visualized by magnetic resonance imaging due to their ability to change the T 1 or T 2

relaxation times of the surrounding tissues; (ii) manipulated by means of a magnetic field (i.e in magnetic drug delivery); and (iii) heated in a magnetic field to trigger drug release or to produce hyperthermia/ablation of tissue

The common methods of synthesis of magnetic nanoparticles include co-precipitation, thermal decomposition, microemulsion and hydrothermal synthesis [14] Co-precipitation is a facile and reproducible way to synthesize iron oxides (γ-Fe2O3 or

Fe3O4) from aqueous Fe2+/Fe3+ salt solutions by the addition of a base under inert atmosphere at room temperature or at elevated temperature The size, shape, and composition of the magnetic nanoparticles depends greatly on the type of salts used, the Fe2+/Fe3+ ratio, as well as the reaction conditions (temperature, pH and ionic strength) It is, however, difficult to control the size and shape of the particles with the co-precipitation method

Monodisperse magnetic nanocrystals of smaller size can essentially be synthesized through the thermal decomposition of organometallic compounds in high-boiling organic solvents containing stabilizing surfactants Sun et al have demonstrated the synthesis of mondisperse MnFe2O4 and Fe3O4 nanoparticles via high-temperature organic phase decomposition [22, 23] whereas synthesis of CoFe2O4, MnO, CuO and other monodisperse nanocrystals of transition metal oxides was reported by Park and co-workers [24, 25] In principle, the ratios of the starting reagents including organometallic compounds, surfactant, and solvent are the decisive parameters for the control of the size and morphology of magnetic nanoparticles The reaction temperature, reaction time, as well as aging period may also be crucial for the precise control of size and morphology

Reverse micelles, which are water-in-oil droplets stabilized by a monolayer of surfactant, have been applied as nanoscale reactors for the synthesis of various nanoparticles [24, 26] In water-in-oil microemulsions, the aqueous phase is dispersed

as microdroplets surrounded by a monolayer of surfactant molecules in the continuous hydrocarbon phase The size of the reverse micelle is determined by the molar ratio of water to surfactant [27] On mixing two identical water-in-oil microemulsions containing the desired reactants, the microdroplets will continuously collide, coalesce, and break again, resulting in mixing of the reactants; finally a precipitate forms in the micelles By the addition of solvent, such as acetone or ethanol, to break the microemulsions, the precipitate can be extracted by filtering or centrifuging the mixture In this sense, a microemulsion can be used as a nanoreactor for the formation

of nanoparticles Using the microemulsion technique, metallic cobalt, cobalt/platinum alloys, and gold-coated cobalt/platinum nanoparticles have been synthesized in reverse micelles of cetyltrimethylammonium bromide, using 1-butanol as the cosurfactant and octane as the oil phase [28] MFe2O4 (M: Mn, Co, Ni, Cu, Zn, Mg, or

Cd, etc.) are among the most important magnetic materials and have been widely used for electronic applications

Under hydrothermal conditions a broad range of nanostructured materials can be formed Li et al reported a generalized hydrothermal method for synthesizing a variety of different nanocrystals by a liquid–solid–solution reaction [29] The system consists of metal linoleate (solid), an ethanol–linoleic acid liquid phase, and a water–ethanol solution at different reaction temperatures under hydrothermal conditions The strategy is based on a general phase transfer and separation mechanism occurring at the interfaces of the liquid, solid, and solution phases present during the synthesis

Fe3O4 and CoFe2O4 nanoparticles can be prepared in very uniform sizes of about 9 and 12 nm, respectively Hydrothermal synthesis is a relatively little explored method for the synthesis of magnetic nanoparticles, although it allows the synthesis of high-

quality nanoparticles To date, magnetic nanoparticles prepared from co-precipitation and thermal decomposition are the best studied, and they can be prepared on a large scale

2.2 MAGNETIC DRUG DELIVERY

Various organic materials (polymeric nanoparticless, liposomes, micelles) have been investigated as drug delivery nanovectors using passive targeting, active targeting with a recognition moiety or by a physical stimulus (e.g magnetism in magnetoliposomes) [30] However, these organic systems still present limited chemical and mechanical stability, swelling, susceptibility to microbiological attack, inadequate control over the drug release rate [31], and high cost Polymer nanoparticles also suffer from the problem of high polydispersity As-synthesized particles with a broad size distribution and irregular branching could lead to heterogeneous pharmacological properties Due to the disadvantages of organic nanoparticles for drug delivery, inorganic vectors are gaining much attention in research

In a general case of magnetic drug delivery, a drug or therapeutic radionuclide is bound to a magnetic compound, introduced in the body, and then concentrated in the target area by means of a magnetic field (using an internally implanted permanent magnet or an externally applied field) Depending on the application, the particles then release the drug or give rise to a local effect [30] Drug release can proceed by simple diffusion or take place through mechanisms requiring enzymatic activity or changes in physiological conditions such as pH, osmolality, or temperature [32]; drug

release can also be magnetically triggered from the drug-conjugated magnetic nanoparticles

In biomedicine, one major hurdle that underlies the use of nanoparticle therapy is the problem of getting the particles to a particular site in the body [1] A potential benefit

of using magnetic nanoparticles is the use of localized magnetic field gradients to attract the particles to a chosen site, to hold them there until the therapy is complete and then to remove them The particles may be injected intravenously, and blood circulation used to transport the particles to the region of interest for treatment Intravenous administration of drugs is the most versatile method to reach target organs and tissues since the blood circulation supplies all vital cells Alternatively, in many cases the particle suspension may be injected directly into the general area where treatment is desired Either of these routes has the requirement that the particles

do not aggregate and block their own spread

The size, charge, and surface chemistry of the magnetic particles are particularly important and strongly affect both the blood circulation time as well as bioavailability

of the particles within the body [33] In addition, magnetic properties and internalization of particles depend strongly on the size of the magnetic particles For example, following systemic administration, larger particles with diameters greater than 200 nm are usually sequestered by the spleen as a result of mechanical filtration and are eventually removed by phagocytic cells, resulting in decreased blood circulation times On the other hand, smaller particles with diameters of less than 10

nm are rapidly removed through extravasation and renal clearance Particles ranging

from circa 10 to 100 nm are optimal for in vivo injection and demonstrate the most

prolonged blood circulation times [34] The particles in this size range are small enough both to evade the MPS of the body as well as penetrate the very small capillaries within the body tissues and, therefore, may offer the most effective distribution in certain tissues [35]

The MPS is a cell family consisting of bone marrow progenitors, blood monocytes and tissue macrophages These macrophages are widely distributed and strategically placed in many body tissues to recognize and clear senescent cells, invading micro-organisms or particles [36] After particles are injected into the bloodstream they are rapidly coated by components of the circulation, such as plasma proteins This process

is known as opsonization, and is critical in dictating the fate of the injected particles [37] Normally opsonization renders the particles recognizable by the body’s MPS and results in their subsequent clearance by the macrophages As a result, the application of nanoparticles in vivo or ex vivo would require surface modification that ensures particles are non-toxic, biocompatible and stable to the MPS

As conventional colloidal drug delivery systems are rapidly removed from the blood stream after their intravenous administration, the fate of nanoparticles in the blood compartment have therefore been widely discussed, and the development of surface-modified nanoparticles appears crucial to increase circulation time [38] The first requirement for active targeting is minimizing or delaying the phagocytosis of the nanoparticles by the MPS Macrophage-evading particles increase the probability of attaining the desired target Modified nanoparticles should present surfaces which inhibit complement activation and opsonization by plasma proteins as these are the key factors involved in uptake of particles by the MPS Particles that have a largely

hydrophobic surface are efficiently coated with plasma components and thus rapidly removed from the circulation, whereas particles that are more hydrophilic can resist this coating process and are cleared more slowly [39] This has been used to an advantage when attempting to synthesize MPS-evading particles by sterically stabilizing the particles with a layer of hydrophilic polymer chains The most common coatings are derivatives of dextran, polyethylene glycol (PEG), polyethylene oxide (PEO), poloxamers and polyoxamines [40] The role of the dense brushes of polymers

is to inhibit opsonization, thereby permitting longer circulation times

Besides uptake by the MPS, there are other fundamental problems associated with the use of magnetic directed drug targeting Targeting to a specific cell type, for example, may be possible with directed coatings However, retaining the particles at the cell membrane for exact drug localization or magnetic cell separation and recovery [17] for any length of time is difficult as the cell often instigates receptor-mediated endocytosis In addition, the ability of magnetic particles to concentrate will depend

on both the blood flow rate and the intensity of the magnetic field The success therefore depends to a large extent on the construction of strong magnets able to produce high magnetic field gradients at the target sites It has been shown that most

of the available fields are only strong enough for the manipulation of particles against the diffusion and bloodstream velocities found in living systems over a distance of a few centimeters from the sharp end of a magnet pole [41] This means that it is difficult to build up and sustain field strength sufficient to counteract the linear blood flow rates in tissues so as to effectively retain the drug carrier at a specific location Improvements are needed to make magnetic directed drug targeting effective

2.3 CANCER TARGETING

Many bio-applications require magnetic particles to possess a cell targeting property especially in the case of cancer diagnosis and treatment Active targeting is based on the over or exclusive expression of particular epitopes or receptors in tumoral cells, and on sensitivity or response to stimuli such as temperature, pH, electric charge, light, sound or magnetism Active targeting may also be based on species which bind to over-expressed receptors These species include low molecular weight ligands (folic acid, thiamine, sugars), peptides (RGD, LHRD), proteins (transferrin, antibodies, lectins), polysaccharides (hyaluronic acid), polyunsaturated fatty acids, DNA, etc Efforts in conferring cell targeting property onto the magnetic nanoparticles include the modification of the particles with chemotherapeutic agents [32, 42], ligands [42, 43] and antibodies [44] These agents usually enter the target cells via endocytosis

Endocytosis is an important pathway by which drugs and chemotherapeutic agents enter cells It is also the most studied mechanism for cell targeting and uptake Cells will endocytose solutes from their extracellular environment through one of the following processes [45, 46]: (i) fluid-phase pinocytosis, where the solute to be endocytosed is present within the extracellular fluid bathing the cell surface and some

of the extracellular fluid is captured within the lumen of the budding endocytic vesicle; (ii) adsorptive endocytosis, where the solute that is to be endocytosed binds to the cell surface through non-specific mechanisms; or (iii) receptor-mediated endocytosis, where a solute will bind to its cognate cell membrane receptor to elicit either a constitutive or ligand-stimulated internalization

Human CD38 antigen, a 42–45 kDa type II transmembrane glycoprotein which is upregulated on cell surfaces in many lymphoid tumors, is a promising candidate in antibody therapy Orciani and co-workers have demonstrated that coupling liposome and an anti-CD38 antibody does not interfere with CD38 functionality [47] Their results showed a specific mechanism owing to CD38-mediated endocytosis of the immunoliposome

The human epidermal growth factor receptor (EGFR) is a group of transmembrane receptors consisting of four related members: HER1 (EGFR), HER2 (also known as

c-erbB-2 and neu), HER3, and HER4 EGFR and HER2 are two important receptors

frequently employed in the treatment of metastatic breast cancers The recombinant humanized monoclonal antibody trastuzumab (trade name: herceptin) was developed

as an immunotherapeutic agent against the HER2 extracellular domain It binds to the HER2 receptor and reduces tumor cell proliferation by interrupting the cellular pathway [48] When conjugated to poly(lactic acid) nanoparticles, an efficient internalization of the particles was observed in human ovarian carcinoma cells (SKOV-3) overexpressing HER2 [49] In a separate study by Germershaus et al., transfection experiments using human breast cancer cells (SK-Br-3) showed up to seven-fold higher luciferase expression with trastuzumab-conjugated complexes as compared to the complexes without trastuzumab at N/P=3.5 [50] Reporter gene expression was significantly inhibited by increasing trastuzumab concentrations This efficient inhibition with free trastuzumab verified the HER2-receptor dependency of the reporter gene expression

2.4 MAGNETIC FLUID HYPERTHERMIA

Magnetic fluid hyperthermia (MFH), which involves injecting biocompatible superparamagnetic nanoparticles (e.g iron oxides such as magnetite) into the target region and externally applying an alternating current (AC) magnetic field to selectively heat up the region, is gaining attention as a potential form of cancer treatment MFH is based on the transfer of power onto magnetic nanoparticles which

is determined by the type of particles, AC frequency and magnetic field strength Cancer cells generally perish at around 43 ºC due to insufficient oxygen supply via the blood vessels, whereas normal cells are more resistant to elevated temperatures In addition, tumors are more easily heated than the surrounding normal tissues, since the blood vessels and nervous systems are poorly developed in the tumor [51, 52] Therefore, hyperthermia is potentially very useful for the treatment of cancers with few side effects Magnetic fluids have been investigated as potential hyperthermia-causing agents due to their high specific power absorption rate (SAR) [53] Heat, by its very nature, can be applied locally with no systemic effects and reduced side effects, compared to traditional treatments (chemotherapy drugs have severe side effects on healthy organs, and radiotherapy adversely affects nearby tissues) [36] Today, hyperthermia remains a promising form of cancer therapy aside from the well-known methods of surgery, chemotherapy and radiotherapy Among the various types

of hyperthermia, including those utilizing radiofrequency, microwave and ultrasound, MFH is particularly attractive because it can offer localized heating with no systemic effect and reduced side effects as compared to traditional therapies Unlike the electric field used in radiofrequency hyperthermia, the AC magnetic field has negligible effects on healthy tissues The use of MFH can also avoid efficacy problems due to reflection and absorption phenomena, which can sometimes be observed with deep

regional hyperthermia using the microwave and ultrasound techniques [54, 55] With the use of magnetic nanoparticles, greater temperature homogeneity within the tumor can potentially be achieved and this is critical to the effectiveness of hyperthermia Ferrimagnetic microspheres have been prepared as promising thermoseeds for inducing hyperthermia in cancers [56] These microspheres are composed of small crystals of Fe3O4 and showed high heat generation at a magnetic field of 300 Oe and

100 kHz In its first clinical application, hyperthermia using magnetic nanoparticles was found to be feasible and well tolerated in previously irradiated and locally recurrent prostate carcinoma [54] Maximum intra-prostatic temperatures achieved in the study are in the thermoablative range Also, interstitial deposition of nanoparticles

in the pre-irradiated prostate is stable for several weeks, making sequential hyperthermia treatments possible without the need for repeated application of magnetic fluid into the prostate

2.5 MAGNETIC NANOPARTICLES IN IMAGING

Magnetic resonance imaging (MRI) is a powerful medical imaging technique used to visualize the structure and function of various tissues It can provide detailed anatomical images based on soft-tissue contrast and functional information in a non-invasive and real-time monitoring manner [57] MRI provides much greater contrast between the different soft tissues of the body than does computed tomography (CT), making it especially useful in neurological, musculoskeletal, cardiovascular, and oncological imaging Unlike CT it uses no ionizing radiation, but uses a powerful magnetic field to align the nuclear magnetization of hydrogen atoms in water in the body Radiofrequency (RF) fields are used to systematically alter the alignment of this magnetization, causing the hydrogen nuclei to produce a rotating magnetic field

detectable by the scanner This signal can be manipulated by additional magnetic fields to build up enough information to reconstruct an image of the body

Image contrast is therefore a result of the different signal intensity each tissue produces in response to the applied RF pulses This response is dependent on the proton density and magnetic relaxation times Since the proton density of soft tissues has a narrow range throughout most of the body, intrinsic contrast is primarily determined by the relaxation times which depend on the chemical and molecular

structure of the tissue [4] Magnetic relaxation is described by the time constants T 1 (longitudinal) and T 2 (transverse) that can be modified by the use of contrast agents, which generally include either paramagnetic complexes or magnetic nanoparticles Paramagnetic complexes, which are usually gadolinium or manganese chelates,

accelerate longitudinal (T 1) relaxation of water protons and exert bright contrast in regions where the complexes localize [58, 59] Magnetic nanoparticles as MRI contrast agents have gained much interest because they can create local field

inhomogeneity and hence shorten T 2

The popularity of SPIO particles can be attributed to several factors [60] Firstly, they provide the most change in signal (albeit hypointensity) per unit of metal, in particular

on T 2*-weighted images, and as they are composed of thousands of iron atoms they defeat the inherent low contrast agent sensitivity of MRI Secondly, they are composed of biodegradable iron, which is biocompatible and can thus be reused or recycled by cells using normal biochemical pathways for iron metabolism Their surface coating, usually dextran, allows straightforward chemical linkage of functional groups and ligands, and they can be easily detected by light and electron

microscopy Finally they can be magnetically manipulated and change their magnetic properties according to size, with the potential to reveal their structural conformation Among others, SPIO has been reported for use in the MRI of atherosclerotic plaques [61], tumors [62] and also in cardiovascular applications [63] as well as animal models [64]

However, the negative contrast effect and magnetic susceptibility artifacts of iron oxide nanoparticles are significant drawbacks of using SPIO in MRI The resulting dark signal can mislead the clinical diagnosis in weighted MRI because the signal is often confused with the signals from bleeding, calcification or metal deposits, and the susceptibility artifacts distort the image background [59, 60] Nonetheless, the recent development of molecular and cellular imaging to help visualize disease-specific biomarkers at the molecular and cellular levels has led to a great interest in SPIO

The availability of new imaging techniques appropriate to the microscale such as optical coherence tomography (OCT) can elucidate processes such as diffusion, which may be currently limiting our ability to target using magnetic nanoparticles Optical coherence tomography is a three-dimensional microstructural biomedical imaging modality using the coherence property of light to optically range light scattering structures [65] Various types of contrast agents have been developed for OCT, which may enhance its biomedical utility by providing molecular imaging [66, 67] The use

of plasmon-resonant nanoparticles in OCT has been of particular interest as they exhibit extremely high optical scattering or absorption cross-sections [68-70] Recently, a modulated OCT contrast method using plasmon-resonant nanoshells has been demonstrated using the resonant photothermal heating of nanoshells to modulate

the local refractive index [71] Modulated OCT contrast is important to successfully achieve background rejection in highly scattering tissues However, this method may

be limited by cumulative heating in the tissue, and has yet to be demonstrated in a biological sample Magnetic nanoparticles can be mechanically actuated with an externally applied magnetic field gradient, and novel magneto-optical particles have been designed for contrast in optical microscopy [72, 73] Magnetomotive OCT (MMOCT) is accomplished using an electromagnet which modulates a magnetic field within the tissue during OCT imaging [74] This provides a mechanical displacement

at the locations of the particles in the tissue, which is observed as a shift in the OCT interferogram

2.6 BIOCOMPATIBLE POLYMERS

Polymers that are commonly coated onto nanoparticles for use in biomedical applications include PEG, polyacrylamides and natural polysaccharides like dextran PEG is by far the most commonly used polymer for applications like drug delivery [75] to improve the saline compatibility of the nanoparticles and to increase the blood circulation times of the PEGylated particles or drugs Other polymers like polyvinylpyrrolidone, polylactic acid, polyglycolide and their copolymer poly(lactic-

co-glycolic acid) as well as polycaprolactone have also been used widely due to their

good biocompatibility Studies have also shown the potential of using natural polymers such as chitosan and collagen in gene delivery and tissue engineering applications [76]

Besides the commonly used polymers as mentioned above, a number of other

materials may find utility in biomedical application as well Poly(N-vinylformamide),

a water-soluble drag-reducing polymer [77] has potential industrial uses in water treatment, papermaking and as adhesives and coatings Its non-toxic hydrolysis products, such as polyvinylamine have potential for biomedical applications due to good water solubility and can offer convenience for chemical modifications [78] Similarly, poly(methacrylic acid) offers the benefits of being water soluble and bearing numerous carboxyl groups for functionalization of other molecules Due to its

pH responsiveness (pKa ~ 4.6), it has been investigated for use in hydrogels [79, 80] and drug delivery [81] Polypyrrole, a highly conducting polymer, has been studied for many industrial applications such as antistatics, electromagnetic shielding, actuators and polymer batteries PPY has also been widely studied for the immobilization of enzymes, antibodies and nucleic acids When combined with enzymes, PPY is used in biosensors to detect, for example, blood glucose [82] PPY is also suitable as a substrate for cell attachment and proliferation and possesses excellent biocompatibility in vivo [83] It was found to enhance neurite outgrowth [84], bind DNA [85] and allow controlled release of drugs [82]

CHAPTER 3: HEPARINIZED MAGNETITE NANOPARTICLES

3.1 INTRODUCTION

Superparamagnetic magnetite nanoparticles are of great interest due to their numerous existing and potential biomedical applications A crucial issue facing the use of magnetic nanoparticles for active targeting in vivo is the minimization or delaying of phagocytosis of these nanoparticles by the MPS The nanoparticles have to be modified to present surfaces which inhibit complement activation and opsonization by plasma proteins as these are the key factors involved in uptake of particles by the MPS Surface modification with heparin appears particularly appealing, as heparin binding was shown to inhibit human complement activation [86] The presence of heparin on polymeric nanoparticles has also been reported to increase their circulation time [87] Furthermore, heparin can be used for local applications to prevent blood clotting on stents and the vessel wall [88] Thrombosis plays an important role in the development of several complications of angioplasty, including closure and restenosis Studies have shown that human endothelial cells are stimulated and vascular smooth muscle cells are inhibited in their proliferation and migration by heparins [89] and local delivery of low molecular weight heparin promotes re-endothelialization and contributes to the inhibition of smooth muscle cell proliferation after balloon angioplasty [90]

Complement activation leading to uptake by the MPS, and clot formation are two major challenges confronting the in vivo use of nanoparticles as drug delivery vehicles The goal of this study was to test the hypothesis that heparin immobilized on the surface of magnetite (Fe3O4) nanoparticles (MNP) would reduce or even eliminate

these processes via their inhibitory effect on the complement system which reduces macrophage uptake, and at the same time prevents coagulation by binding and thereby inhibiting thrombin As direct immobilization of heparin on the magnetite nanoparticles could not be easily achieved, a poly(NIPAAM) layer was first formed

on the surface of the magnetite nanoparticles via ATRP followed by the immobilization of heparin onto the poly(NIPAAM) shell The poly(NIPAAM) layer also allows for the dispersion of the nanoparticles in formamide, the medium used for heparin immobilization Recent trends have shown that poly(NIPAAM) can be used for a number of potential biomedical applications such as for drug release and drug delivery [91-95]and as possible embolic agents [96] The bioactivity of heparinized magnetite nanoparticles in our study was assessed via their uptake by macrophages and plasma recalcification time (PRT) The results indicate that the above-mentioned surface modifications of the magnetite nanoparticles are effective in delaying phagocytosis and preventing blood clotting in vitro Such properties can be expected

to increase their potential for biomedical applications

3.2 METHODS AND MATERIALS

3.2.1 Materials

Benzyl ether (99%), 1,2-hexadecanediol (97%), oleic acid (90%), oleylamine (70%), N-isopropylacrylamide and copper (I) chloride were purchased from Aldrich Chemical Co Iron (III) acetylacetonate was from Strem Chemicals, Inc The ATRP initiator, 2-(4-chlorosulfonylphenyl) ethyltrichlorosilane (CTCS), was obtained from United Chemical Technologies, Inc in 50 wt% solution in dichloromethane Heparin sodium salt from porcine intestinal mucosa was from Sigma Chemical Co The other solvents and reagents were of analytical grade and used without further purification

Macrophage cells from mouse (RAW 264.7) were purchased from ATCC

RPMI-1640 media was from Sigma-Aldrich Co

3.2.2 Synthesis of heparinized nanoparticles

Figure 3.1 shows the schematic representation of the functionalization steps in the preparation of the heparinized magnetite nanoparticles (magnetite-poly(NIPAAM)-Heparin or MNP-NP-He) The as-synthesized MNP were first immobilized with the ATRP initiator, CTCS, and surface-functionalized with poly(NIPAAM) via a surface-initiated ATRP to give poly(NIPAAM)-grafted MNP (MNP-NP), followed by immobilization of heparin

H e rin

Hepar

n

CH2CH(CONHCH(CH3)2) Cl

Figure 3.1: Schematic representation of the process for preparing MNP-NP-He

3.2.2.1 Synthesis of magnetite nanoparticles

MNP were synthesized according to the method reported in the literature [23] Iron (III) acetylacetonate (2 mmol), 1,2-hexadecanediol (10 mmol), oleic acid (6 mmol), oleylamine (6 mmol) and benzyl ether (20 ml) were mixed and magnetically stirred under a flow of argon The mixture was heated to 200 ºC for 2 h and refluxed at 300

ºC for 1 h The black mixture was cooled to room temperature before ethanol was added for precipitation The precipitate was dissolved in hexane in the presence of oleic acid and oleylamine Centrifugation at 6000 rpm for 10 min was used to remove

any undispersed residue and the product was then precipitated with ethanol The average magnetite nanoparticle size was about 6-8 nm

3.2.2.2 Immobilization of initiator on magnetite and subsequent ATRP

The as-synthesized MNP were washed twice in ethanol, centrifuged and dried under reduced pressure CTCS was immobilized on the surface of the MNP following a self-assembled monolayer-deposition method reported in the literature [97] with some modifications MNP (100 mg) were mixed with 20 ml of dehydrated toluene to which 0.13 mmol of CTCS was added The reaction mixture was placed in an ultrasonic bath for 3 hours The obtained magnetite-Cl particles were washed with tetrahydrofuran (THF) and dried under reduced pressure In this method, the silane groups on CTCS condense with hydroxyl groups on the surface of MNP to form Si-O linkages

For the preparation of MNP-NP, the magnetite-Cl particles were added to 3 ml dimethyl sulphoxide (DMSO) and sonicated for 1 h NIPAAM (14.5 mmol) and 2-2’bipyridine (0.29 mmol) were then added The reaction mixture was stirred and purged with argon for 20 min CuCl (0.145 mmol) was then added to the mixture The reaction tube was sealed and kept at 40°C for 10 h with continuous stirring After the reaction, the particles were washed with DMSO and distilled water

3.2.2.3 Heparinization of MNP-NP nanoparticles

Heparin was immobilized onto MNP-NP using a previously reported protocol [98] In this protocol, the preserved active chlorine groups on the MNP-NP after ATRP of NIPAAM may serve to link up with the heparin molecules Heparin was dissolved in formamide to a concentration of 3 mg/ml, followed by the addition of the

nanoparticles The solution was kept at room temperature for 3 days under stirring The heparinised nanoparticles were collected by centrifugation and washed with distilled water and acetone to remove the solvent and excess heparin The washing was repeated until heparin was no longer present in the wash water, as confirmed with toluidine blue [99] The washed particles were then dried under reduced pressure

3.2.3 Characterization of functionalized MNP

X-ray photoelectron spectroscopy (XPS) analysis of the as-synthesized and functionalized MNP (MNP, MNP-NP, MNP-NP-He) was made on an AXIS HSi spectrometer (Kratos Analytical Ltd) using the monochromatized Al Kα X-ray source (1486.6 eV photons) at a constant dwell time of 100 ms and a pass energy of 40 eV The anode voltage was 15 kV and the anode current was 10 mA The pressure in the analysis chamber was maintained at 7x10-6 Pa or lower during measurement The core-level signals were obtained at a photoelectron take-off angle of 90° (with respect

to the sample surface) To compensate for surface charging effect, all core-level spectra were referenced to the C 1s hydrocarbon peak at 284.6 eV In spectral deconvolution, the linewidth (full width at half-maximum) of the Gaussian peaks was maintained constant for all components in a particular spectrum

Fourier transform infrared (FTIR) spectra of the nanoparticles, dispersed in KBr and pelletized, were obtained using a Bio-Rad FTIR Model FT135 spectrometer under ambient conditions The magnetic properties of the as-synthesized and functionalized MNP were recorded in a vibrating sample magnetometer (VSM, LakeShore 450-10) with a saturating field of 1T The magnetization values were normalized to the mass

of nanoparticles to yield the specific magnetization (emu/g particle)

3.2.4 Uptake of nanoparticles by macrophages

Mouse macrophage cells (RAW 264.7) were used in these experiments Cells were routinely cultured at 37°C in a humidified atmosphere with 5% CO2 (in air), in 25 cm2flasks containing 10 ml of RPMI-1640 media, supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and penicillin-streptomycin (100 U/ml) For subculture, the cells were washed once with phosphate-buffered saline (PBS) and incubated with trypsin-EDTA solution (0.25% trypsin, 1mM EDTA) for 10 min at 37°C to detach the cells, and complete media were then added in the flask at room temperature to inhibit the effect of trypsin The cells were collected by centrifugation and resuspended in the complete media for reseeding and growth in new culture flasks Cell viability was determined through staining with Trypan Blue and cells were counted using a hemocytometer Cell density was estimated using a 0.9 mm3 counting chamber

To study cellular uptake of nanoparticles via optical microscopy, the nanoparticles were added to the cell culture media at a particle concentration of 0.2 mg/ml The cells were cultured in 24-well plastic dishes with normal media for 20 h, after which the media was replaced with the nanoparticle-dispersed culture media After various periods of incubation at 37°C and 5% CO2, the cells were washed with PBS and viewed under the optical microscope

For quantification of the intracellular uptake of the nanoparticles, cells were grown in 24-well culture plates, with approximately 105 cells in 1 ml of media After incubation

at 37°C for 20 h, the media was replaced with that containing magnetic nanoparticles

at a concentration of 0.2 mg/ml The nanoparticles were sterilized with UV irradiation

for 30 min before use In control cultures, the cells were seeded at the same cell density and grown in 1 ml media without the nanoparticles The intracellular iron concentration was quantified using inductively coupled plasma spectroscopy (ICP) (Perkin Elmer Optima 3000 DV) after various periods of incubation (2, 8 or 24 h) Cells were washed thrice with PBS, detached, resuspended, counted, centrifuged down, and the cell pellet was dissolved in 37% HCl solution at 40°C for 30 min The samples were diluted to a final iron concentration of 1.0–4.0 µg/ml for ICP analysis Three replicates were measured and the results were averaged

3.2.5 Stability of immobilized heparin

To examine the stability of the immobilized heparin in the cell culture media

(RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 100 U/ml penicillin-streptomycin) at 37ºC, MNP-NP-He were dispersed in the culture media and the absorbance of the solution was measured, at a wavelength of 350 nm, at regular intervals Since MNP-NP-He disperse well in this solution, a decrease in absorbance would indicate the settling of the nanoparticles This could only happen if heparin is lost from MNP-NP-He, resulting in aggregation and sedimentation of the larger aggregates (similar to MNP-NP)

3.2.6 Cytotoxicity of nanoparticles

The cytotoxicity of the as-synthesized and functionalized MNP was evaluated by determining the viability of the macrophages after incubation with the media containing the nanoparticles Cell viability testing was carried out via the reduction of the MTT reagent (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma) The MTT assay was performed in a 96-well plate following the standard

procedure with minor modifications The nanoparticles were sterilized with UV irradiation for 30 min before use Control experiments were carried out using the complete growth culture media only (non-toxic control), and with 1% Triton X-100 (Sigma) (toxic control) Macrophages were seeded at a density of 104 cells/well for 20

h before the media was replaced with one containing the nanoparticles at 0.2 mg/ml The macrophages were incubated at 37°C and 5% CO2 for 2, 8 and 24 h The culture media from each well was then removed and 90 µl of media and 10 µl MTT solution (5 mg/ml in PBS) were then added to each well After 2 h of incubation at 37 °C and 5% CO2, the media were removed and the formazan crystals were solubilized with

100 µl dimethyl sulfoxide (DMSO) for 15 min The optical absorbance was then measured at 560 nm on a microplate reader (Tecan GENios) The results were expressed as percentages relative to the results obtained with the non-toxic control The differences in the results obtained from MNP, MNP-NP, MNP-NP-He and the

controls were analyzed statistically using the two sample t-test The differences observed between samples were considered significant for P < 0.05

3.2.7 Plasma Recalcification Time (PRT)

Fresh blood collected from a healthy rabbit was immediately mixed with 3.8 wt% sodium citrate solution at a dilution ratio of 9:1 It was then centrifuged at 3000 rpm, 8°C, for 20 min to obtain the platelet-poor plasma (PPP) 0.1 ml of the PPP was transferred to a clotting tube at 37ºC and 0.1 ml saline solution (0.9% NaCl) was added to the PPP 0.1 ml of 0.025 M CaCl2 solution was then added, and the stopwatch started The clotting tube was tilted at 15 sec intervals until a firm clot was formed For investigating the effect of the as-synthesized and functionalized MNP on the plasma clotting time, 0.1 ml of water containing 1 mg/ml nanoparticles was added

in place of the saline solution At least three experiments were carried out for each sample and the mean clotting time reported Different concentrations of MNP-NP-He

in water were also tested to determine the minimum concentration required to prevent clotting The differences between the PRT obtained with saline and as-synthesized

and functionalized MNP were analyzed statistically using the two-sample t-test The differences observed between the samples were considered significant at P < 0.05

3.3 RESULTS AND DISCUSSION

3.3.1 Characterization of functionalized MNP

The surface immobilization of the ATRP initiator, CTCS, onto the MNP was first ascertained using XPS The C 1s and S 2p core-level spectra of the as-synthesized and initiator-immobilized MNP (magnetite-Cl) are compared in Figure 3.2 (a to d) and the

N 1s core-level spectra of magnetite-Cl and MNP-NP are also shown in Figure 3.2 (e, f) The C 1s core-level spectrum of MNP (Figure 3.2a) consists of three peak components with binding energies at about 284.6, 285.8 and 288.6 eV, which can be fitted to C-C/C-H, C-C=O and O-C=O respectively[100] These species are due to the presence of oleic acid on the surface of MNP