Surface-Modified Superparamagnetic Nanoparticles for Drug Delivery

66

IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 3, NO. 1, MARCH 2004

Surface-Modified Superparamagnetic Nanoparticles for Drug Delivery: Preparation, Characterization, and Cytotoxicity Studies Ajay Kumar Gupta* and Stephen Wells

Abstract—Superparamagnetic iron oxide nanoparticles have been used for many years as magnetic resonance imaging (MRI) contrast agents or in drug delivery applications. In this study, a novel approach to prepare magnetic polymeric nanoparticles with magnetic core and polymeric shell using inverse microemulsion polymerization process is reported. Poly(ethyleneglycol) (PEG)-modified superparamagnetic iron oxide nanoparticles with specific shape and size have been prepared inside the aqueous cores of AOT/n-Hexane reverse micelles and characterized by various physicochemical means such as transmission electron microscopy (TEM), infrared spectroscopy, atomic force microscopy (AFM), vibrating sample magnetometry (VSM), and ultraviolet/visible spectroscopy. The inverse microemulsion polymerization of a polymerizable derivative of PEG and a cross-linking agent resulted in a stable hydrophilic polymeric shell of the nanoparticles. The results taken together from TEM and AFM studies showed that the particles are spherical in shape with core–shell structure. The average size of the PEG-modified nanoparticles was found to be around 40–50 nm with narrow size distribution. The magnetic measurement studies revealed the superparamagnetic behavior of the nanoparticles with saturation magnetization values between 45–50 electromagnetic units per gram. The cytotoxicity profile of the nanoparticles on human dermal fibroblasts as measured by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that the particles are nontoxic and may be useful for various in vivo and in vitro biomedical applications. Index Terms—Biological cells, drug delivery systems, magnetic materials, nanotechnology.

I. INTRODUCTION

S

UPERPARAMAGNETIC iron oxide nanoparticles with tailored surface chemistry have been widely used experimentally for numerous in vivo applications such as magnetic resonance imaging (MRI) contrast enhancement, tissue repair, immunoassay, detoxification of biological fluids, hyperthermia, drug delivery, cell separation, etc. [1]–[4]. All these biomedical and bioengineering applications require that these nanoparticles have high magnetization values and size smaller than 100 nm with overall narrow particle size distribution, so that the particles have uniform physical and chemical properties. In addition, these applications need special surface coating of

Manuscript received July 3, 2003; revised August 19, 2003. This work was supported by EC Contract GRD5-CT2000-00375, project acronym MAGNANOMED. Asterisk indicates corresponding author. *A. K. Gupta is with the Centre for Cell Engineering, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, U.K. (e-mail: [email protected]). S. Wells is with Liquid Research Limited, Gwynedd LL5 7UP, U.K. Digital Object Identifier 10.1109/TNB.2003.820277

the magnetic particles, which has to be not only nontoxic and biocompatible but also allow a targetable delivery with particle localization in a specific area [5]. Because of large surface area to volume ratio, the magnetic nanoparticles tend to agglomerate and adsorb plasma proteins. The body’s reticuloendothelial system (RES), mainly the kupffer cells in the liver, usually take up these nanoparticles due to the hydrophobic surface. Surface coverage by amphiphilic polymeric surfactants such as poloxamers, poloxamines and poly(ethylene glycol) (PEG) derivatives over the nanoparticles significantly increases the blood circulation time by minimizing or eliminating the protein adsorption to the nanoparticles. PEG is widely used as a coating material for nanoparticles due to the following properties: 1) easy excretion through the kidney; 2) low interfacial free energy water; 3) excluded volume effect; 4) nonimmunogenic properties; and 5) nonantigenic properties. In addition, it has been demonstrated that PEG-modified nanoparticles can interact with cell membranes resulting in enhanced cellular response [6]. For biomedical applications, the size, charge, and surface chemistry of the magnetic particles is particularly important and strongly affects both the blood circulation time as well as bioavailability of the particles within the body [7]. In addition, magnetic properties and internalization of particles depend strongly on the size of the magnetic particles [8]. 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 the cells of the phagocyte system, 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 subcutaneous injection and demonstrate the most prolonged blood circulation times. The particles in this size range are small enough both to evade RES 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 [9]. An advance in the use of magnetic particles for biomedical applications depends on the new synthetic methods with better control of the size distribution and of particle surface characteristics. The most common method for synthesis of magnetite particles is by coprecipitation from a solution of Fe(II) and Fe(III) salts in an appropriate ratio using alkali metal hydroxides. Smaller and more uniform particles can be synthesized by using the microemulsion approach with a good

1536-1241/04$20.00 © 2004 IEEE

GUPTA AND WELLS: SURFACE-MODIFIED SUPERPARAMAGNETIC NANOPARTICLES FOR DRUG DELIVERY

control over iron oxide amount and magnetic properties [10]. Synthesis of hydrophilic magnetic polymeric nanoparticles with magnetite core and polymeric shell is possible using an inverse microemulsion polymerization process [11]. The strategy of utilizing inverse microemulsion approach to modulating the surface of magnetic nanoparticles with PEG is based on the following prior observations: 1) Preparation of hydrophilic nanoparticles is possible in the aqueous cores of reverse micellar droplets. 2) The size of the particles can be modulated down to 10-nm diameter by regulating the size of the aqueous core of reverse micelles. 3) Since the cross-linking and polymerization reactions take place in the aqueous core of reverse micelles, it is possible to coat the magnetic particles inside these nanoreactors [12]. Superparamagnetic iron oxide nanoparticles of narrow size range can be easily produced and coated with various polymers, providing convenient, readily targetable MRI agents. The object of this study is to present a novel approach to prepare more uniform magnetic polymeric nanoparticles with magnetite core and polymeric shell inside the aqueous cores of reverse micelles. The magnetic polymeric particles prepared have been characterized by various physicochemical methods and the cytotoxicity of the surface modified nanoparticles on human dermal fibroblasts in vitro has been assessed, as compared to those underivatized particles using standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Magnetic polymeric nanoparticles prepared using this method were found to be nontoxic and, thus, may serve as an improved way of drug delivery and targeting.

II. MATERIALS AND METHODS A. Materials All the chemicals were of reagent grade and were used without further purification. Ferric chloride hexahydrate FeCl .6H O 99 , ferrous chloride tetrahydrate FeCl .4H O , methoxy-PEG Mw 5000 D , maleic anhydride (MA), N,N’-methylene bis acrylamide (MBA), ammonium persulphate (APS), sodium bis(2-ethylhexyl sulphosuccinate) (AOT), n-hexane, MTT, and sodium oleate C H NaO 99 were obtained from Sigma (Dorset, 99 and hyU.K.) while sodium hydroxide NaOH 37 v/v were obtained from Fluka, drochloric acid HCl Dorset, U.K. Double-distilled water was used for all the experiments. B. Preparation of Polymerizable Derivative of PEG (i.e., MA Ester of PEG) The polyester was prepared by polycondensation reaction under N environment. For preparation of ester of PEG of maleic acid, 1 : 1 mole of MA and methoxy PEG (mPEG, MW 5000) was dissolved in chloroform with hydroquinone (to inhibit the free radical polymerization of MA). N gas was

67

passed and reaction was carried out at 37 C for 18–24 h. An CH-CO- O-CH CH -OCH , is ester bond, -HOOC-HC formed due to the chemical bonding between anhydride group of MA and hydroxyl group of mPEG. Greenish-yellow crystals were obtained. To remove the hydroquinone from the product, the MA ester of PEG (MA-PEG) crystals were recrystallized using ethanol. C. Synthesis of PEG-Coated Magnetic Nanoparticles 1) Synthesis of Magnetic Nanoparticles in Reverse Micelles: In order to achieve a narrow particle size distribution, magnetite nanoparticles were prepared by using an inverse microemulsion approach. Highly monodispersed iron oxide nanoparticles were synthesized by using the aqueous core of aerosol-OT (AOT)/n-Hexane reverse micelles (without microemulsions) in N atmosphere. The reverse micelles have an aqueous inner core, which can dissolve hydrophilic compounds, salts, etc. A deoxygenated aqueous solution of the ferric and ferrous salts (molar ratio 2:1, 1 M) was dissolved in the aqueous core of the reverse micelles formed by 0.05 M AOT in n-hexane. Chemical precipitation was achieved by using a 1 M deoxygenated solution of sodium hydroxide. The reaction was carried out in nitrogen atmosphere at low temperature (4 C–6 C) with vigorous stirring. The hexane was evaporated and the particles were recovered by precipitation in an excess of an acetone-methanol mixture (9 : 1 ratio), followed by dialysis using 12-kD cutoff dialysis membrane against double-distilled water to remove unreacted iron salts. The surface of the particles was neutralized with 0.01 M HCl, and the particles were dried in a vacuum oven at 70 C–80 C. Particles were then coated with PEG to form the stable dispersion of the magnetite nanoparticles. 2) Surface Modification of Magnetic Nanoparticles With PEG: PEG is hydrophilic and is widely used in biological research, as it protects surfaces from interacting with cells or proteins. Thus, coated particles may result in increased blood circulation time. Ten-milligram magnetite particles were dispersed in 1.0 ml of deoxygenated water by sonication for 30 min. The aqueous dispersion of magnetic nanoparticles was dissolved in the aqueous cores of reverse micelles together with polymerizable derivative of PEG as a monomer (i.e., MA-PEG) MBA as cross-linking agent under nitrogen gas. Additional amounts of water may be added in reverse micellar solution in order to get the host micellar droplet of desired size. In a typical experimental protocol, to a 50-ml 0.05-M AOT solution in hexane, 500 l of magnetite solution (10 mg/ml), 100 l of MA-PEG (5 mg/ml), and 10 l of MBA (0.5 mg/ml) were dissolved. The solution was stable and brownish transparent at this stage. The nitrogen gas was bubbled through this solution to remove the dissolved oxygen. After 30 min, 20 l of 2% ammonium persulphate as an initiator was added. The polymerization of monomers was carried out by free radical polymerization mechanism at 37 C for 8 h. After polymerization, the particles were purified from unreacted monomers and other toxic reactants by dialysis against distilled water and dried in an oven before characterization.

68


D. Characterization of Magnetic Nanoparticles

G. In Vitro Cell Viability/Cytotoxicity Studies

1) Fourier Transformed Infrared (FTIR) Spectral Studies: The FTIR spectrum was recorded in the transmission mode on a Nicolet Impact 410 spectrometer. The dried samples of magnetite or PEG-modified magnetic particles were grounded with KBr and the mixture was compressed into a pellet. The spectrum was taken from 4000 to 400 cm . 2) Transmission Electron Microscopy (TEM) Studies: The average particle size, size distribution, and morphology were examined using a Zeiss 902 TEM at a voltage of 80 kV. The aqueous dispersion of the particles was drop-cast onto a carboncoated copper grid, and the grid was air dried at room temperature before loading into the microscope. 3) Atomic Force Microscopy (AFM) Studies: The aqueous dispersion of the nanoparticles was put on a glass coverslip and the coverslip was air dried at room temperature. Once dry, the samples were analyzed using the Nanoscope III scanning probe microscope (Digital Instruments, Santa Barbara, CA). 4) Vibrating Sample Magnetometer (VSM) Analysis: Magnetic properties and magnetic particle size measurements have been done using a VSM (Liquid Research Ltd., Gwynedd, U.K.) on liquid samples. 5) Total Iron Determination: In order to determine the total concentration of iron present in the nanoparticles, the nanoparticles were completely dissolved in 30% v/v HCl for 2 h at elevated temperatures (50 C–60 C). The iron concentration was determined by spectrophotometric measurements at 340 nm using a Shimadzu UV-160 A UV-visible recording spectrophotometer.

The MTT assay is a simple nonradioactive colorimetric assay to measure cell cytotoxicity, proliferation, or viability. MTT is a yellow, water-soluble tetrazolium salt. Metabolically active cells are able to convert this dye into a water-insoluble dark blue formazan by reductive cleavage of the tetrazolium ring [13]. Formazan crystals, then, can be dissolved in an organic solvent such as dimethylsulphoxide (DMSO) and quantified by measuring the absorbance of the solution at 550 nm, and the resultant value is related to the number of living cells. To determine cell cytotoxicity/viability, the cells were plated at a density of 1 10 cells/well in a 96-well plate at 37 C in 5% CO atmosphere. After 24 h of culture, the medium in the wells was replaced with the fresh medium containing nanoparticles in varying concentrations. After 24 h, 20 l of MTT dye solution (5 mg/ml in phosphate buffer pH 7.4, MTT Sigma, Dorset, U.K.) was added to each well. After 4 h of incubation at 37 C and 5% for exponentially growing cells and 15 min for steady-state confluent cells, the medium was removed and formazan crystals were solubilized with 200 l of DMSO and the solution was vigorously mixed to dissolve the reacted dye. The absorbance of each well was read on a microplate reader (Dynatech MR7000 instruments) at 550 nm. The spectrophotometer was calibrated to zero absorbance, using culture medium without cells. The relative cell viability (%) related to control wells containing cell culture medium without nanoparticles was calculated by . test control

E. Cell Culture

The statistical analysis of experimental data utilized the student’s t-test and the results were presented as mean standard deviations. Statistical significance was accepted at a level of 0.05.

Infinity telomerase-immortalized primary human fibroblasts (hTERT-BJ1, Clontech Laboratories, Inc., Hampshire, U.K.) were seeded onto 13-mm glass coverslips in a 24-well plate at a density of 1 10 cells per well in 1 ml of complete medium for 24 h, after which the growth medium was removed and replaced with the medium containing nanoparticles. For control experiments, medium with no particles was used. The medium used was 71% Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, Dorset, U.K.), 17.5% Medium 199 (Sigma, U.K.), 9% fetal calf serum (FCS) (Life Technologies, Paisley, U.K.), 1.6% 200 mM L-glutamine (Life Technologies, Paisley, U.K.), and 0.9% 100 mM sodium pyruvate (Life Technologies, Paisley, U.K.). The cells were incubated at 37 C in a 5% CO atmosphere. F. Live–Dead Assay for Cell Viability The fibroblast cells were seeded onto 13-mm glass coverslips at 10 000 cells/ml in a 24-well tissue culture plate. After the cells were attached to the coverslips, cell medium was exchanged with the fresh medium containing nanoparticles, and cells were cultured at 37 C. After 24 h, medium was removed and the cells were washed with phosphate-buffered saline (PBS) followed by viability staining using calcein AM (2 M, Molecular Probes, Leiden, The Netherlands) and ethidium homodimer (4 M, Molecular Probes, Leiden, The Netherlands) for 1 h at room temperature. All samples were viewed on a fluorescence microscope.

H. Statistical Analysis

III. RESULTS AND DISCUSSION A. Synthesis of PEG-Coated Magnetic Nanoparticles The surfactant (for example, AOT) when dissolved in nonpolar solvents like hexane forms reverse micelles where hydrophobic tails of surfactants are assembled toward the bulk nonpolar solvent and the hydrophilic head is directed inside enclosing a aqueous core [14]. The magnetic nanoparticles were synthesized by coprecipitation of ferrous and ferric salts solution by concentrated sodium hydroxide solution inside the aqueous cores of reverse micellar droplets. Smaller and more uniform particles were prepared by precipitation of magnetite at low temperature in the presence of nitrogen gas. The synthesis of magnetic nanoparticles in oxygen-free environment not only protects the oxidation of iron oxide particles but also reduces the size of the particles as compared with methods without removing oxygen [15]. The size of the inner aqueous core of reverse micelles is in nanometer range, so the magnetic nanoparticles prepared inside these nanoreactors were found to be very small in size (less than 15 nm) with narrow size distribution. The advantage of utilizing this type of microemulsion system for nanoparticle formation is that the size of nanoparticles can be controlled by modulating the size of aqueous micellar core [12].


69

Fig. 2. FTIR spectra. (a) Magnetic nanoparticles. (b) PEG-coated magnetic nanoparticles.

Fig. 1.

FTIR spectra. (a) MA. (b) PEG. (c) MA-PEG.

Magnetic nanoparticles have a large ratio of surface area to volume and, therefore, tend to agglomerate in order to reduce their surface energy by strong magnetic dipole–dipole attractions between particles. The colloidal suspension of magnetite particles, however, can be stabilized by coating the particle surfaces with high molecular weight polymers such as PEG, polyvinylalcohol (PVA), dextran, etc. Such coatings have been postulated as necessary for effective stabilization for ferrofluids [16]. The aqueous dispersion of magnetic nanoparticles along with the aqueous solutions of MA-PEG and cross-linking agent were dissolved in the aqueous core of the reverse micelles, and since these cores are hydrophilic, the cross linking of MA-PEG with MBA and their subsequent absorption on the nanoparticles’ surface takes place inside these droplets, giving a uniform and highly stable polymeric coating. The magnetic polymeric particles synthesized in reverse micelles under controlled conditions of temperature and oxygen have shown fairly narrow size distribution. The colloidal solution of magnetic particles coated with PEG showed very high stability at neutral pH and no sedimentation was observed even after two months of storage at room temperature, whereas uncoated magnetic particles did not form a stable colloidal suspension and sedimented within a week. B. FTIR Spectral Studies FTIR spectra of MA-PEG along with those of MA and PEG are shown in Fig. 1. The figure shows the strong peaks in the range of 800–1000 cm corresponding to the stretching mode of -CH CH- of MA skeleton whereas the strong peak at 1781 cm has been attributed to the anhydride group of MA. The -C-O-C- ether stretch band at 1101.6 cm and the vibration band around 1342–1353 cm (antisymmetric stretch) appear

in the FTIR spectra of both PEG and MA-PEG. Similarly, the bands around 2890 cm and 945–962 cm correspond to -cm - stretching vibrations and -CH- out-of-plane bending vibrations, respectively. Being ethylene glycol derivative, the hydroxyl groups of the polymer gives rise to a broad and intense peak in the range 3400–3550 cm . The -C-O-C-, -CH -, -CH-, and hydroxyl group peaks strongly indicate the presence of PEG in the spectra. The infrared (IR) absorption band around 1467 cm may be due to the bending vibration of -CH - group, and the bending vibrations of -CH - groups can be attributed to the peaks in the slightly higher regions. In the spectra of MA-PEG, the new peak at 1735 cm is due to the presence of ester bond, -CH CH-CO-O CH CH formation in the structure, suggesting a chemical bonding between the anhydride group of MA and the hydroxyl group of PEG, while another peak around 1640 cm is due to the carbonyl stretching bonds. FTIR spectra of unmodified and PEG modified magnetite nanoparticles are shown in Fig. 2. The IR spectra of iron oxide exhibit strong bands in the low frequency region (1000–400 cm ) due to the iron oxide skeleton. In other regions, the spectra of iron oxide have weak bands. The spectrum is highly consistent with magnetite Fe O spectrum (bands at 408.9, 571.5, and 584.5 cm ). As shown in the figure, strong peaks in the range of 800–1000 cm corresponding to the stretching mode of vinyl double bonds disappeared in the spectrum of PEG-coated particles indicating that polymerization has taken place. The C-O–C ether stretch band at 1106 cm and the vibration band at 1344 cm (antisymmetric stretch) appear in the FTIR spectrum of the nanoparticles after PEG modification. Similarly, the bands around 2912 and 955 cm correspond to -CH stretching vibrations and -CH out-of-plane bending vibrations, respectively. The C-O-C, -CH , and -CH peaks are strong evidence that PEG was covered at the nanoparticle surface. The spectra of PEG-coated nanoparticles shows the small shift in the positions of the main peaks seen in the uncoated nanoparticles. This is due to the change in environment of the particle after polymer coating.

70


(a) Fig. 3.

(b)

TEM pictures of (a) magnetic nanoparticles and (b) PEG-coated magnetic nanoparticles.

Fig. 4. AFM picture showing core–shell structure of nanoparticles.

C. Determination of Average Size and Size Distribution TEM and AFM studies: TEM picture of the magnetic particles was taken to determine the shape, size, and uniformity of the particles. TEM picture of the magnetic nanoparticles synthesized in inner aqueous core of the water-in-oil microemulsions shows that these particles have a very small size of around 10- to 15-nm diameter with narrow size distribution [Fig. 3(a)]. The size of the particles after coating was about 40- to 50-nm diameter, as was determined by TEM studies [Fig. 3(b)]. AFM was performed to study the shape, size, and surface appearance of the nanoparticles. A drop of diluted aqueous solution was

placed on a glass coverslip and dried out at room temperature for 24 h. Fig. 4 represents the AFM image of PEG-modified nanoparticles after water evaporation showing the core shell structure and size homogeneity of the nanoparticles. The average particle diameter of the particles was found about 50 nm, which was in close agreement with the size obtained by TEM studies. D. Chemical Analysis Determination of total iron content in magnetic nanoparticles requires total dissolution of the oxides and is achieved by


71

Fig. 6. Cytotoxicity profiles of magnetic nanoparticles when incubated with human fibroblasts as determined by MTT assay. Percentage of viability of fibroblasts was expressed relative to control cells (n = 6). Results are represented as mean standard deviations.

6

shows no diamagnetic contribution and are small enough to exhibit superparamagnetic behavior; thus, they are of particular interest for drug targeting systems, as they do not retain any magnetism after removal of a magnetic field. F. Cytotoxicity Studies

Fig. 5. Relative magnetization M/Ms and magnetization versus applied magnetic field for uncoated magnetic nanoparticles.

treating the particles in the presence of concentrated HCl at elevated temperature for 2 h. Iron content in the nanoparticles was 340 nm and determined spectrophotometrically at was found to be more than 90% of the original iron salts. It was calculated that a total of 1.71 10 particles are present in one gram of magnetic nanoparticles, and each iron oxide nanoparticle contained 62 896 iron atoms. E. Measurement of Magnetic Properties Fig. 5 shows the relative magnetization curve as a function of magnetic field for the uncoated particles. From the figure, no hysteresis curve was observed, which indicates the characteristic superparamagnetic behavior of the particles. The saturation magnetization value of the magnetite nanoparticles was found between 45 and 50 electromagnetic units per gram (emu/g). In addition, the magnetization decreases from the plateau value and reaches zero when the magnetic field is removed. The behavior shows that the iron oxide particles correspond to the single-crystal domain exhibiting only one orientation of the magnetic moment and are magnetite in structure. The size distribution was calculated using the equation based on a log-normal function [17]. The average particle size 0.48 nm, which is in close was found to be around 12.92 agreement with the size obtained from TEM measurements. It was found from VSM studies that the magnetic particles

The cell viability staining using calcein AM/ethidium homodimer showed that the cells exposed to PEG-coated nanoparticles were more than 99% viable. Live–dead cell viability assay is a two-color fluorescence assay that is based on the simultaneous determination of the numbers of live and dead cells. Live cells have intracellular esterases that converts nonfluorescent, cell-permeable calcein acetoxymethyl (calcein AM) to the intensely fluorescent green calcein which is retained within the cells. On the other hand, ethidium homodimer enters the damaged membranes of dead cells and is fluorescent red when bound to nucleic acids. The MTT assay for cell viability evaluation has been described as a suitable method for detection of biomaterial toxicity [13]. The MTT assay relies on the mitochondrial activity of fibroblasts and represents a parameter for their metabolic activity. The proliferation/viability of fibroblasts was measured by MTT assay after culturing for 24 h and showed that cell proliferation was more favorable in case of PEG-coated particles than with uncoated ones. All nanoparticles affected the metabolic activity in concentration dependent manner when they were added in the concentration range of 0–1000 g/ml to the cells. Cytotoxicity of the nanoparticles increased in relation to increasing concentration, as shown in Fig. 6. PEG-coated nanoparticles revealed no cytotoxic effects to cells and they remained more than 100% viable relative to control at concentration as high as 1 mg/ml. These samples increased the viability of the cells to about 10%–40% depending on the nanoparticle concentration in the medium. The increased cell viability can be explained by nutrient effect [18]. Incubation with uncoated plain magnetic particles, however, the fibroblasts showed significant loss in viability of about 25%–50% observed at concentrations of 250 g/ml. Below this concentration, cellular metabolic activity did not change much in comparison with control cells.

72


Nanoparticle–cell interaction depends on the surface aspects of materials, which may be described according to their chemistry, hydrophilic/hydrophobic characteristics, or surface energy. These surface characteristics determine how the nanoparticles will adsorb to the cell surface and more particularly determine the cell behavior on contact. It was observed from cell viability studies that the plain uncoated nanoparticles reduced cell viability significantly as compared to the cells that were not exposed to the nanoparticles. One possible explanation for this large decrease in cell viability may be that these nanoparticles are taken up by the cells as a result of endocytosis or are promoting apoptosis (programmed cell death) due to weak cell adhesive interactions with the nanoparticles. The low toxicity of nanoparticles coated with PEG may be attributed to the fact that PEG is hydrophilic, and it protects surfaces from interacting with cells or proteins. It has been demonstrated that PEG-modified nanoparticles can interact with cell membranes, resulting in enhanced cellular response, as these coatings on the nanoparticles are biocompatible, nonimmunogenic, and nonantigenic [19]. PEGs at present are the most important material for tissue engineering and other biomedical applications including RES avoidance and blood residence prolongation of nanoparticles. PEG consists of hydrophobic parts and hydrophilic tails. The strong anchoring of the PEG molecules on the surface of a particulate carrier results in the steric stabilization of the particles. The steric stabilization improves the particle stability in the biological milieu against interactions with macromolecules (e.g., opsonins) and cells, thus imparting prolonged circulation in blood and reduced RES uptake to particulate carrier system [20]. Biodistribution studies performed in mice have shown that the blood circulation time increases as the molecular weight of the PEG increases (from 1900 up to 20 000 Da) [21]. A wide variety of superparamagnetic iron oxide nanoparticles have been prepared to date and tested for many preclinical applications and in clinical trials [22]–[25]. Most iron oxides have a relatively short blood half-life and their primary application is for imaging of the liver, spleen, and gastrointestinal tract. Surface-modified iron oxide nanoparticles having long blood circulation times, however, may prove very useful for imaging of the vascular compartment (magnetic resonance angiography), imaging of lymph nodes, perfusion imaging, receptor imaging, and target-specific imaging.

IV. CONCLUSION In this paper, PEG-modified superparamagnetic iron oxide nanoparticles about 50 nm in size with a narrow size distribution have been prepared using an inverse microemulsion polymerization method and characterized in vitro by various physicochemical means. The magnetic polymeric nanoparticles have a core–shell structure with magnetic core and polymeric shell. The colloidal solution of nanoparticles shows high stability. These nanoparticles exhibit superparamagentic behavior and are nontoxic and, hence, may be useful for relevant drug delivery and other biomedical applications.

ACKNOWLEDGMENT The authors would like to thank Prof. A. S. G. Curtis, University of Glasgow, Glasgow, U.K., for encouraging them to work in his laboratory.

REFERENCES [1] R. Weissleder, A. Bogdanov, E. A. Neuwelt, and M. Papisov, “Longcirculating iron oxides for MR imaging,” Adv. Drug Delivery Rev., vol. 16, pp. 321–334, 1995. [2] P. Reimer and R. Weissleder, “Development and experimental application of receptor-specific MR contrast media,” Radiology, vol. 36, pp. 153–163, 1996. [3] C. Chouly, D. Pouliquen, L. Lucet, J. J. Jeune, and P. Jallet, “Development of superparamagnetic nanoparticles for MRI: Effect of particle size, charge and surface nature on biodistribution,” J. Microencapsulation, vol. 13, pp. 245–255, 1996. [4] P. K. Gupta and C. T. Hung, “Magnetically controlled targeted microcarrier systems,” Life Sci., vol. 44, pp. 175–186, 1989. [5] Y. Zhang, N. Kohler, and M. Zhang, “Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake,” Biomaterials, vol. 23, no. 7, pp. 1553–1561, 2002. [6] C.-G. Gölander, J. N. Herron, K. Lim, P. Claesson, P. Stenius, and J. D. Andrade, “Properties of immobilized PEG films and the interaction with proteins,” in Poly(Ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications, J. M. Harris, Ed. New York: Plenum, 1992, pp. 221–45. [7] C. Chouly, D. Pouliquen, I. Lucet, J. J. Jeune, and P. Pellet, “Development of superparamagnetic nanoparticles for MRI: Effect of particle size, charge and surface nature on biodistribution,” J. Microencapsulation, vol. 13, pp. 245–255, 1996. [8] C. Chouly, D. Pouliquen, I. Lucet, J. J. Jeune, and P. Jallet, “Development of superparamagentic nanoparticles for MRI: Effect of particle size, charge and surface nature on biodistribution,” J. Microencapsulation, vol. 3, pp. 245–255, 1996. [9] S. Stolnik, L. Illum, and S. S. Davis, “Long circulating microparticulate drug carriers,” Adv. Drug Delivery Rev., vol. 16, pp. 195–214, 1995. [10] G. Mobe, K. Kon-No, K. Kyori, and A. Kithara, J. Colloid Interface Sci., vol. 93, pp. 293–293, 1983. [11] P. A. Dresco, V. S. Zaitsev, R. J. Gambino, and B. Chu, “Preparation and properties of magnetite and polymer magnetite nanoparticles,” Langmuir, vol. 15, pp. 1945–1951, 1999. [12] N. Munshi, T. K. De, and A. N. Maitra, “Size modulation of polymeric nanoparticles under controlled dynamics of microemulsion droplets,” J. Colloid Interface Sci., vol. 190, pp. 387–391, 1997. [13] T. Mosmann, “Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxic assay,” J. Immunol. Methods, vol. 95, pp. 55–63, 1993. [14] M. J. Hou, M. Kim, and D. O. Shah, “A light scattering study on the droplet size and interdroplet interaction in microemulsion of AOT-oil water systems,” J. Colloid Interface Sci., vol. 123, pp. 398–412, 1988. [15] D. K. Kim, Y. Zhang, W. Voit, K. V. Rao, and M. Muhammed, “Synthesis and characterization of surfactant-coated superparamagnetic monodispersed iron oxide nanoparticles,” J Magn. Magn. Mater., vol. 225, no. 1–2, pp. 30–36, 2001. [16] V. S. Zaitsev, D. S. Filimonov, I. A. Presnyakov, R. J. Gambino, and B. Chu, “Physical and chemical properties of magnetite and magnetitepolymer nanoparticles and their colloidal dispersions,” J. Colloid Interface Sci., vol. 212, pp. 49–57, 1999. [17] C. G. Granqvist and R. H. Buhrman, “Ultrafine metal particles,” J. Appl. Phys., vol. 47, pp. 2200–2219, 1976. [18] D. Fischer, Y. X. Li, B. Ahlemeyer, J. Krieglstein, and T. Kissel, “In vitro cytotoxicity testing of polycations: Influence of polymer structure on cell viability and hemolysis,” Biomaterials, vol. 24, no. 7, pp. 1121–1131, 2003. [19] M. Amimji and K. Park, “Prevention of protein absorption and platelet adhesion on surfaces by PEO/PPO/PEO triblock copolymers,” Biomaterials, vol. 13, pp. 682–692, 1992. [20] G. Storm, S. O. Belliot, T. Daemen, and D. D. Lasic, “Surface modification of nanoparticles to oppose uptake by mononuclear phagocyte system,” Adv. Drug Delivery Rev., vol. 17, pp. 31–48, 1995.


[21] S. E. Dunn, A. Brindley, S. S. Davis, M. C. Davies, and L. Illum, “Polystyrene-poly(ethyleneglycol) (PS-PEG-2000) particles as model system for site specific drug delivery. 2. The effect of PEG surface density on the in vitro cell interaction and in vivo biodistribution,” Pharmaceut. Res., vol. 11, no. 7, pp. 1016–1022, 1994. [22] T. A. Kent, M. J. Quast, B. J. Kaplan, R. S. Lifsey, and H. M. Eisenberg, “Assesment of a superparamagnetic iron oxide (AMI-25) as a brain contrast agent,” Mag. Reson. Med., vol. 13, pp. 434–443, 1990. [23] R. Weissleder, J. F. Heautot, B. K. Schaffer, N. Nossiff, M. I. Papisov, A. A. Bogdanov, and T. J. Brady, “MR lymphography: Study of a high efficiency lymphotropic agent,” Radiology, vol. 191, pp. 225–230, 1994. [24] R. Weissleder, “Editorial: Liver MR imaging with iron oxides: Toward consensus and clinical practice,” Radiology, vol. 193, pp. 593–595, 1994. [25] R. Weissleder, A. Lee, B. Khaw, T. Shen, and T. Brady, “Antimyosin labeled monocrystalline iron oxide allows detection of myocardial infarct: MR antibody imaging,” Radiology, vol. 182, pp. 381–385, 1994.

73

Ajay Kumar Gupta received the M.Sc. degree in chemistry and the Ph.D. degree from the University of Delhi, Delhi, India, in 1997 and 2001, respectively. He is currently with the Centre for Cell Engineering, University of Glasgow, U.K., working on the synthesis of superparamagnetic nanoparticles of a specific shape and narrow size distribution with tailored surface chemistry, which may be useful for various biomedical applications. He has worked on drug delivery systems using nanotechnology, and successfully devised a nanoparticulate carrier system using polymeric micelles for ophthalmic delivery of hydrophobic drugs. He has U.S. and European patents and has filed Indian patents for these formulations. The technical know-how has been transferred to a pharmaceutical company, Panacea Biotech, New Delhi, India, for further commercialization of the formulation.

Stephen Wells, photograph and biography not available at the time of publication.