Folate–PEG–superparamagnetic iron oxide nanoparticles for lung cancer imaging

Acta Biomaterialia 8 (2012) 3005–3013

Contents lists available at SciVerse ScienceDirect

Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat

Folate–PEG–superparamagnetic iron oxide nanoparticles for lung cancer imaging Mi-Kyong Yoo a,b,1, In-Kyu Park c,1, Hwang-Tae Lim d, Sang-Joon Lee c, Hu-Lin Jiang d, You-Kyoung Kim a, Yun-Jaie Choi a, Myung-Haing Cho d,e,f,⇑, Chong-Su Cho a,⇑ a

Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea Department of Advanced Material Engineering for Information and Electronics, Kyung Hee University, Yongin 446-701, South Korea c Department of Biomedical Science, Chonnam National University Medical School and Center for Biomedical Human Resources (BK-21 program), Gwangju 501-746, South Korea d Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea e Department of Nano Science and Technology, Graduate School of Convergence Science and Technology, Advanced Institute of Convergence Technology, Seoul National University, Suwon 443-270, South Korea f Graduate Group of Tumor Biology, Seoul National University, Seoul 151-742, South Korea b

a r t i c l e

i n f o

Article history: Received 1 December 2011 Received in revised form 23 March 2012 Accepted 17 April 2012 Available online 24 April 2012 Keywords: Magnetic nanoparticles Folate Poly(ethylene glycol) Lung cancer Imaging

a b s t r a c t While superparamagnetic iron oxide nanoparticles (SPIONs) have been widely used in biomedical applications, rapid blood clearance, instability and active targeting of the SPIONs limit their availability for clinical trials. This work was aimed at developing stable and lung cancer targeted SPIONs. For this purpose firstly folic acid (FA)-conjugated poly(ethylene glycol) (FA–PEG) was synthesized, and FA– PEG–SPIONs were subsequently prepared by the reaction of FA–PEG with aminosilane-immobilized SPIONs. FA-PEG-SPIONs were labeled with Cy5.5 for optical imaging. The intracellular uptake of FA– PEG–SPIONs–Cy5.5 was evaluated in KB cells and lung cancer model mice to confirm active targeting. The sizes of the FA–PEG–SPIONs were little changed after up to 8 weeks at 4 °C, suggestive of very stable particle sizes. The results of fluorescent flow cytometry and confocal laser scanning microscopy suggest that the intracellular uptake of FA–PEG–SPIONs–Cy5.5 was greatly inhibited by pre-treatment with free folic acid, indicative of receptor-mediated endocytosis. Stronger optical imaging was observed in the lung cancer model mice for FA–PEG–SPIONs–Cy5.5 than PEG–SPIONs–Cy5.5 6 and 24 h post-injection through the tail vein, due to receptor-mediated endocytosis. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Nanoparticles have attracted much attention owing to their unusual electronic [1], optical [2], and magnetic [3] properties. Especially, nanoparticles have been used for various biomedical applications, such as diagnostics and therapeutics, due to their unique capabilities and their few side-effects. Among nanoparticles, superparamagnetic iron oxide nanoparticles (SPIONs) offer high potential for several biomedical applications, such as magnetic resonance imaging [4], tissue repair [5], hyperthermia treatment [6] and as drug delivery systems (DDSs) [7], owing to their biocompatibility, appropriate surface architecture, and easy conjugation with targeting ligands [8]. In particular, drug-loaded SPIONs can be directed to the desired target site by an external magnetic field while ⇑ Corresponding authors. Address: Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea. Tel.: +82 2 880 1276; fax: +82 2 875 8842 (M.-H. Cho), Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea. tel.: +82 2 880 4636; fax: +82 2 875 2494 (C.-S. Cho). E-mail addresses: [email protected] (M.-H. Cho), [email protected] (C.-S. Cho). 1 The first two authors contributed equally to this work.

simultaneously tracking the distribution of SPIONs in the body [9]. Application of SPIONs as DDSs requires that the SPIONs be stable under physiological conditions and that the circulation half-lives of the SPIONs in vivo should be long, as they are mainly captured by the reticuloendothelial system (RES) [10]. Many researchers have used poly(ethylene glycol) (PEG) to stabilize SPIONs, because a PEG shell on nanoparticles (NPs) facilitates their steric stabilization [11,12] and increases their circulation time in the bloodstream, because PEG minimizes protein adsorption and recognition by macrophages of the mononuclear phagocyte system [13,14]. The introduction of a targeting moiety onto SPIONs is aimed at increasing selective cellular binding and internalization through receptor-mediated endocytosis [15]. Folate receptors (FARs) on the cell membrane are a potential molecular target for tumor imaging because the FAR is highly expressed in a number of epithelial carcinomas [16] and the FAR provide highly selective sites that differentiate tumor cells from normal cells [17]. FARs are often present in large numbers on cancer cells with their limited expression on normal cells [18]. FARs are also over-expressed on activated and non-resting macrophages, although functional FARs are not expressed on resting macrophages or normal epithelial cells, which helps in targeting genetically altered cells [19].

1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.04.029

3006

M.-K. Yoo et al. / Acta Biomaterialia 8 (2012) 3005–3013

There are previously reported studies on conjugating folic acid (FA) directly to PEG and other carriers for imaging [20–23]. However, the use of PEG to stabilize FA-conjugated iron oxide nanoparticles for lung cancer targeting in vitro and in vivo has not been studied thus far. 2. Materials and methods

suka Electronics, Osaka, Japan) with scattering angles of 90° and 20° at 25 °C. The morphology of the SPIONs and FA–PEG–SPIONs was observed by transmission electron microscopy (TEM) using a JEOL model JEM 1010. The crystallinity of prepared SPIONs was confirmed by X-ray diffractometry (XRD) using a Bruker-AXS GmbH D8 Advance (Karlsruhe, Germany) equipped with a rotating anode, Sol-X energy dispersive detector, and CuK radiation source (k = 0.1542 nm).

2.1. Materials 2.7. Cell viability assay Ferric chloride hexahydrate (FeCl3
6H2O, >97% pure), ferrous chloride tetrahydrate (FeCl2
4H2O, >99% pure), 1-ethyl-3[3dimethylaminopropyl] carbodiimide hydrochloride (EDC), dimethyl sulfoxide (DMSO), N-hydroxysuccinimide (NHS), dicyclohexyl carbodiimide (DCC), pyridine and 3-aminopropyl triethoxysilane (APTES) were provided by Sigma-Aldrich Co. (St Luois, MO). Bifunctional PEG (molecular weight 5 kDa) was purchased from NOF Corp. (Tokyo, Japan). Cy5.5 NHS ester was purchased from GE Healthcare, formerly Amersham Biosciences (Amersham, UK). 2.2. Preparation of SPIONs The SPIONs were prepared by alkaline co-precipitation of FeCl3
6H2O (3.255 g, 12.04 mmol) and FeCl2
4H2O (1.197 g, 6.02 mmol) in 70 ml of deoxygenated water upon addition of NH4OH as previously reported [24]. Amino groups in the SPIONs were introduced by reaction of the SPIONs with APTES at room temperature for 24 h. 2.3. Synthesis of FA-conjugated PEG FA conjugation to PEG was carried out via the DCC/NHS chemistry. Briefly, folic acid (70.6 mg, 0.16 mmol) was activated in DMSO by DCC (65.92 mg, 0.32 mmol) and NHS (36.83 mg, 0.32 mmol) at room temperature for 12 h. The activated FA in DMSO was coupled with bifunctional PEG in basic pH using pyridine at room temperature for 4 h. After reaction the mixture was dialyzed using Spectra/PorÒ (molecular weight cut-off 1 kDa) followed by lyophilization. 2.4. Preparation of FA–PEG–SPIONs The FA–PEG (180 mg) was then coupled to amine-terminated SPIONs (36 mg) using EDC and NHS as activation agents at room temperature for 12 h. The resulting reaction mixture was then dialyzed against water at 4 °C for 48 h, lyophilized and characterized. 2.5. Labeling of FA–PEG–SPIONs with Cy5.5 The FA–PEG–SPIONs (50 mg) dispersed in 2.5 ml of DMSO were then labeled with Cy5.5 NHS ester (2.25 mg) dissolved in 500 ll of DMSO with 8 ll of triethyl amine in the dark for 12 h. After labeling the mixture was dialyzed using a 6.5 kDa molecular weight cut-off membrane, followed by lyophilization. PEG–SPIONs labeling with Cy5.5 was performed similarly. The content of Cy5.5 label in FA–PEG–SPIONs–Cy5.5 was estimated from the extinction coefficient (molar coefficient 2.5  105 M 1 cm 1) at 675 nm. 2.6. Characterization Fourier transform infrared (FT-IR) spectra were measured using a Nicolet Magna 550 Series II spectrometer (Midac, Atlanta, GA). The sizes of SPIONs and FA–PEG–SPIONs were measured using an electrophoretic light scattering spectrophotometer (ELS 8000, Ot-

Cell viability was assayed according to a previously reported method [16]. 2.8. Evaluation of intracellular uptake Intracellular uptake of FA–PEG–SPIONs–Cy5.5 in vitro was performed by competition assay using fluorescent flow cytometry similar to a method previously reported [24]. The intracellular uptake of FA–PEG–SPIONs–Cy5.5 was also checked by confocal laser scanning microscopy (Micro Systems LSM 410, Carl Zeiss, Germany) equipped with a 488 nm argon/krypton laser. 2.9. Tumor model 6-week-old male A/J mice were given a single intraperitoneal injection of urethane (1 mg g 1 body weight) dissolved in 0.9% saline as previously reported [25]. 6 weeks after the urethane injection the mice were divided into four groups (five mice per group). The animals were kept in the laboratory animal facility with the temperature and relative humidity maintained at 23 ± 2 °C and 50 ± 20%, respectively, under a 12 h light/dark cycle. The method used in this study was approved by the Animal Care and Use Committee of Seoul National University (SNU-081009-02). 2.10. In vivo optical imaging In vivo optical imaging was performed by intraveneous injection of FA–PEG–SPIONs–Cy5.5 (or PEG–SPIONs–Cy5.5) (5 mg kg 1) into lung cancer model mice through the tail vein using a whole body animal imaging system (IVIS-100 Spectrum, Caliper Life Sciences), equipped with 10 narrow band excitation filters (30 nm bandwidth) and 18 narrow band emission filters (20 nm bandwidth) that assist in reducing autofluorescence according to a method similar to one previously reported [26]. 2.11. Prussian blue staining Prussian blue staining of the SPIONs in the lungs of urethane-induced lung cancer model mice was performed as previously reported [24]. 3. Results and discussion 3.1. Synthesis of FA–PEG and FA–PEG–SPONs FA–PEG was synthesized by the reaction of activated folic acid using DCC/NHS with bifunctional PEG having amino and carboxylic acid groups, through amide linkage between the carboxylic acid groups of FA and the amine groups of bifunctional PEG, as shown in Fig. 1. The overnight activation of FA using excess DCC and NHS in DMSO yielded dicyclohexyl urea. The activated FA was then reacted with bifunctional PEG (molecular weight 5 kDa) in the presence of pyridine. The reaction mixture was dialyzed, initially using DMSO for 2 days and H2O for 3 days, and then lyophilized.

3007

M.-K. Yoo et al. / Acta Biomaterialia 8 (2012) 3005–3013 H N

H 2N

N

N

H N

N

H N

H2N

O

O

N

DCC/NHS

H N

COOH

N H N

N

DMSO

O

H N

O

COOH

O

O

O

N

COOH O

Folic acid

Activated folic acid O HO

n

O

NH2

Pyridine

NH2-PEG-COOH

H N

H 2N

O

N

N

H N

N

O

H N

O

O

COOH

O N H

O

O

n

OH

Folate-PEG Fig. 1. Proposed reaction scheme for the synthesis of FA–PEG. FA was chemically conjugated to PEG through an amide linkage between the amino group of bifunctional PEG and the carboxylic group in FA using NHS/EDC as the coupling agent.

H N

H2N N

N

a

H N

N O

b

c c

d

d

O

H N

O

e f COOH

g

N H

O

h

O

i

h

O

h OH

n

j

a c d

b

j i e

g f

Fig. 2. 1H NMR spectrum of folate–PEG in DMSO: d = 7.67, 6.68 (–C4H4–, p-phenyl in folate (c, d)), 3.63-3.33. (–CH2CH2O–, ethylene in PEG (h)). The substitution value of FA in PEG was determined to be 48.4 mol.% from the integral ratio between PEG protons (h) and the two protons (d) of the p-phenyl group in folate.

3008

M.-K. Yoo et al. / Acta Biomaterialia 8 (2012) 3005–3013

C2H5O

OH HO

Fe2O3

+

C2H5O

Si−CH2CH2CH2−NH2

C2H5O

OH

O

N2, 24 h

Fe2O3 O Si−(CH2)3−NH2

EtOH

O

3-Aminopropyltriethoxysilane (APTES)

SPIONs

APTES-SPIONs

PEG-FA

PEG-FA OH

OH

H2N

APTES

Fe2O3

FA-PEG-COOH

Fe2O3 H2N

OH

NH2

NHCO

Cy 5.5-NHS ester

Fe2O3

NHS/DCC

NH2

H2N

APTES-SPIONs

OH

SPIONs

H2N

H2N

NHCO Fe2O3

H2N

NH2

N Cy 5.5 H

FA-PEG-SPIONs-Cy5.5

FA-PEG-SPIONs

Fig. 3. Proposed reaction scheme for the fabrication of FA–PEG–SPIONs and labeling with Cy5.5. FA–PEG–SPIONs were prepared by the reaction of the carboxylic acid groups of FA–PEG and the amine groups of amine-terminated SPIONs through an amide linkage. Cy5.5 was anchored to the surface of SPIONs by reaction between the NHS groups in the monofunctional NHS ester of Cy5.5 and the amine groups of SPIONs.

100

% Transmittance

90

3425.6

1650.8

80

1466.8

636.5 582.4

2884.9

70

60

50 4000

3500

3000

2500

2000

1500

1000

500

Wavenumbers (cm-1) Fig. 4. FT-IR spectra of FA–PEG–SPIONs.

The conjugation of PEG with FA was confirmed by the appearance of characteristic peaks at 6.68 and 7.67 p.p.m. for four protons of the p-phenyl ring in FA (c, d), as shown in 1H NMR spectra (Fig. 2). The composition of FA in FA–PEG was also estimated by 1 H NMR measurement. The substitution value for FA in FA–PEG was 48.4 mol.% by calculating the integral ratio between the protons of PEG (h) and the two protons of the p-phenyl group in FA (d). FA–PEG–SPIONs were prepared by the reaction of carboxylic acid groups of FA–PEG and the amino groups of amine-terminated SPIONs through amide linkage, and were labeled with the nearinfrared (NIR) fluorescent dye Cy5.5 through reaction between the NHS group in the monofunctional NHS ester of Cy5.5 and the amino groups of SPIONs, as shown in Fig. 3. The preparation of FA–PEG–SPIONs was confirmed by FT-IR spectroscopy. As shown in Fig. 4, the peaks at 582.4 and 636.5, 1466.8, 1650 and 3425.6 cm 1 were assigned to the FeO of SPIONs, CH2 bending of PEG, C@O stretching of the amide bond and N–H stretching of the amide bond, respectively, suggestive of successful preparation of FA–PEG–SPIONs. TGA analysis of PEG–SPIONs with SPION:PEG

weight ratios of 1:2, 1:3, 1:5, and 1:7 were performed to determine the optimal conjugation ratio. The results showed that the PEG content in the PEG–SPIONs increased steadily up to a weight ratio of SPION to PEG of 1:5, whereas the amount of PEG did not increase much above a weight ratio of 1:7 (data not shown). When the PEG– SPIONs were synthesized at a weight ratio of 1:5 the major mass loss was approximately 36%, which occurred between 200 and 450 °C, and can be ascribed to the pyrolysis of PEG on the surface of the PEG–SPIONs. The same SPION:PEG ratio of 1:5 was also used for the preparation of FA–PEG–SPION in this study. 3.2. Characterization of SPIONs and FA–PEG–SPIONs The shape and size distributions of SPIONs and FA–PEG–SPIONs were checked by TEM and ELS, respectively. As shown in Fig. 5a and b, TEM images of SPIONs and FA–PEG–SPIONs indicated well-separated ellipsoidal iron oxide particles with average sizes of about 10 nm, suggesting that the shape and size of the FA– PEG–SPIONs were little changed compared with those of SPIONs.

3009

M.-K. Yoo et al. / Acta Biomaterialia 8 (2012) 3005–3013

Fig. 5. Morphological observations of (a) SPIONs and (b) FA–PEG–SPIONs by EF-TEM. Scale bar 50 nm.

Fig. 6. Particle size distribution of (a) SPIONs and (b) FA–PEG–SPIONs. The average particle sizes of SPIONs and FA–PEG–SPIONs were 8.8 ± 1.7 and 18.5 ± 3.4 nm, respectively.

100 PEG-SPIONs FA-PEG-SPIONs

90 80

Particle size (nm)

The size distributions of SPIONs and FA–PEG–SPIONs are shown in Fig. 6a and b, respectively. The size distributions of SPIONs and FA– PEG–SPIONs were unimodal. The average number particle sizes of SPIONs and FA–PEG–SPIONs were 8.8 ± 1.7 and 18.5 ± 3.4 nm, respectively, indicating that the particle sizes of SPIONs were increased by the introduction of FA–PEG into SPIONs. The average particle sizes obtained by ELS were larger than the sizes observed by TEM for corresponding samples due to the hydrodynamic diameter of the samples in aqueous solution. The size of SPIONs for biomedical applications should be small, uniform and stable. The change in particle size of PEG–SPIONs and FA–PEG–SPIONs in aqueous medium with time was investigated by ELS, as shown in Fig. 7. The results indicated that the sizes of PEG–SPIONs and FA–PEG–SPIONs were little increased up to 8 weeks at 4 °C, suggestive of very stable particle sizes due to the PEG brush effect, although the sizes of the FA–PEG–SPIONs increased more than those of the PEG–SPIONs due to the hydrophobic properties of FA. The stability of the PEGylated nanoparticles under physiological conditions was also measured for a period of 4 weeks (data not shown). The particles remained stable in physiological solutions and the average size of the PEG–SPIONs did not

70 60 50 40 30 20 10 0 0

1

2

3

4

5

6

7

8

Time (week) Fig. 7. Evaluation of the stability of FA–PEG–SPIONs in aqueous medium by ELS. The sizes of the PEG–SPIONs and FA–PEG–SPIONs were little increased up to 8 weeks at 4 °C.

3010

M.-K. Yoo et al. / Acta Biomaterialia 8 (2012) 3005–3013

(a)

120

PEG-SPIONs

130

FA-PEG-SPIONs

120

110

110

100

100

90

90

80

80

% of control

% of control

(b)

SPIONs

130

70 60 50

SPIONs PEG-SPIONs-Cy5.5 FA-PEG-SPIONs-Cy5.5

70 60 50

40

40

30

30

20

20

10

10 0

0 31.25

62.5

125

250

31.25

500

62.5

125

250

500

Concentration (µg/ml)

Concentration (µg/ml)

Fig. 8. Cytotoxicity of (a) FA–PEG–SPIONs and (b) FA–PEG–SPIONs–Cy5.5 to KB cells as determined by MTT assay. Percent viability of KB cells is expressed relative to control cells.

Fig. 9. FACS analysis of intracellular uptake of (a) PEG–SPIONs–Cy5.5 and (b) FA–PEG–SPIONs–Cy5.5 by KB cells in the absence and presence of folate in the medium.

Control

PEG-SPIONS (-) FA

(+) FA

FA-PEG-SPIONS (-) FA

(+) FA

Cy5.5

DAPI

Merge

Fig. 10. Confocal laser micrographs of KB cells incubated with PEG–SPIONs or FA–PEG–SPIONs for 1 h in the absence and presence of folate. Greater intracellular uptake of FA–PEG–SPIONs–Cy5.5 than PEG–SPIONs by KB cells was observed, and their intracellular uptake was greatly inhibited by pre-treatment with free folic acid. Scale bars 20 lm.

M.-K. Yoo et al. / Acta Biomaterialia 8 (2012) 3005–3013

3011

Fig. 11. Examination of the target specificity of FA–PEG–SPIONs–Cy5.5 using in vivo optical imaging in lung cancer model mice at 6 and 24 h after injection. NIR images of a mouse that received (a and c) PEG–SPIONs–Cy5.5 (5 mg kg 1) and (b and d) FA–PEG–SPIONs-Cy 5.5 (5 mg kg 1) via tail vein injection.

vary during that period, confirming stable dispersion and nonaggregation. 3.3. Cytotoxicity of FA–PEG–SPIONs and FA–PEG–SPIONs–Cy 5.5 Cell viability studies were performed by incubating SPIONs, PEG–SPIONs and FA–PEG–SPIONs with KB cells at different concentrations and assaying the cell metabolic activity after 24 h. The cell viability results for the particles are shown in Fig. 8. The PEG–SPIONs and FA–PEG–SPIONs showed low toxicity even at the highest concentration of 500 lg ml 1, although cell viability in the presence of the SPIONs was a little lower than that in the presence of the PEG–SPIONs and FA–PEG–SPIONs, whereas cell viability in the presence of the PEG–SPIONs–Cy5.5 and FA–PEG–SPIONs– Cy5.5 was decreased with increasing concentration, due to the cytotoxicity of Cy5.5 at high concentration. The excellent biocompatibility of the PEG–SPIONs and FA–PEG–SPIONs may be attributed to the introduction of biocompatible PEG into the SPIONs. 3.4. In vitro tumor targeting assay The ability of FAR-mediated endocytosis was evaluated by competition assay. The fluorescence intensity of PEG–SPIONs–Cy5.5 and FA–PEG–SPIONs–Cy5.5 in KB cells in the absence or presence of FA is compared in Fig. 9a and b. The fluorescence intensity of FA–PEG–SPIONs–Cy5.5 in KB cells was stronger than that of PEG– SPIONs–Cy5.5 and was drastically decreased in the presence of

FA, whereas the fluorescence intensity of PEG–SPIONs–Cy5.5 was unchanged in the presence of FA, indicative of FAR-mediated endocytosis of FA–PEG–SPIONs by the folate ligand. FAR-mediated endocytosis of FA-PEG–SPIONs–Cy5.5 compared with PEG–SPIONs–Cy5.5 was also investigated using confocal laser micrography (CLM). Fig. 10 shows the CLM of KB cells incubated with PEG–SPIONs–Cy 5.5 and FA–PEG–SPIONs–Cy 5.5 for 1 h in the absence or presence of FA. The results indicate that greater intracellular uptake of FA–PEG–SPIONs–Cy5.5 than PEG–SPIONs was observed in KB cells and intracellular uptake was highly inhibited by pre-treatment with free FA, resulting in much poorer FAR targeting, with a similar tendency as for flow cytometry, shown in Fig. 9. 3.5. In vivo tumor targeting assay The tumor uptake of PEG–SPIONs–Cy5.5 and FA–PEG–SPIONs– Cy5.5 was determined by near-IR optical imaging of lung cancer tumor model mice after injection of nanoparticles via the tail vein, because the Cy5.5 near-IR dye attached to PEG–SPIONs or FA–PEG– SPIONs allowed the generation of a near-IR fluorescence signal sufficient to detect the tumor-selective delivery of nanoparticles to the tumors. Fig. 11 shows in vivo near-IR fluorescence imaging of PEG–SPIONs–Cy5.5 (Fig. 11a and c) and FA–PEG–SPIONs–Cy5.5 (Fig. 11b and d) in mice 6 and 24 h post-injection through the tail vein. The radiance intensity obtained from the region of interest between FA–PEG–SPIONs–Cy5.5 and PEG–SPIONs–Cy5.5 was also compared 24 h after injection of the nanoparticles into the lung

3012

M.-K. Yoo et al. / Acta Biomaterialia 8 (2012) 3005–3013

(c) (a)

0x10

7

5x10

7

0x10

7

5x10

7

0x10

7

5x10

7

0x10

7

0x10

6

(b)

FA-PEG-SPIONs PEG-SPION

Liver

Spleen

Heart

Lung

Kidney

Intestine

Fig. 12. Ex vivo optical imaging of tumor and normal tissues confirmed the in vivo imaging results. Lung cancer model mice were injected with PEG–SPION–Cy5.5 (5 mg kg 1) or FA–PEG–SPION–Cy5.5 (5 mg kg 1) and then the heart, lung, intestine, liver, spleen and kidney were imaged using an IVIS-100 24 h after injection: (a) PEG–SPION–Cy5.5; (b) FA–PEG–SPION–Cy5.5. (c) Radiance was detected from the region of interest (ROI) using Living Imaging v. 2.5.

Fig. 13. Perl’s Prussian blue staining of lung cancer tissue after i.v. injection of (a) PEG–SPIONs and (b1, b2) FA–PEG–SPIONs. Magnification 40; scale bar 500 lm.

cancer model mice through the tail vein, as shown in Fig. 12. The results indicated that the radiance intensity due to FA–PEG–SPIONs–Cy5.5 was stronger than that due to PEG–SPIONs–Cy5.5 (as indicated by the scale bar) in all organs tested, although a stronger radiance intensity of the FA–PEG–SPIONs–Cy5.5 and PEG–SPIONs– Cy5.5 was obtained in the kidney compared with other organs. It was found that stronger fluorescence was observed in the lung for FA–PEG–SPIONs–Cy5.5 after 6 and 24 h than PEG–SPIONs– Cy5.5, suggestive of preferential accumulation of the nanoparticles in lung tumors, due to receptor-mediated endocytosis, although stronger fluorescence was observed in the liver and kidney for FA–PEG–SPIONs, due to opsonization of the nanoparticles and clearance by tissue macrophages of the RES [10,28,29]. FARs have also been found at significant levels in some normal epithelia involved in the retention and uptake of FA, primarily the choroid

plexus, placenta, lung, intestine, and kidney [27]. However, these FRs are mostly inaccessible to blood-borne folate conjugates, since they are localized at the apical surfaces of polarized epithelia. However, little difference in FA–PEG–SPIONs–Cy5.5 and PEG–SPIONs–Cy5.5 after Prussian blue staining was found, as shown in Fig. 13, although the reason is not at present clear.

4. Conclusion FA–PEG–SPIONs–Cy5.5 was prepared to develop a stable lung cancer imaging agent. The sizes of the FA–PEG–SPIONs were very stable up to 8 weeks. Intracellular uptake of FA–PEG–SPIONs– Cy5.5 occurred by a receptor-mediated mechanism in vitro. Stronger optical imaging was observed for FA–PEG–SPIONs–Cy5.5 than

M.-K. Yoo et al. / Acta Biomaterialia 8 (2012) 3005–3013

PEG–SPIONs–Cy5.5 in lung cancer model mice after injection via the tail vein. We believe that the characteristics of non-toxic and stable FA–PEG–SPIONs will be beneficial for the development of safer and more efficient lung cancer optical imaging agents. Acknowledgements This research was supported by a grant (08162KFDA553) from the Korea Food and Drug Administration in 2008. This work was also supported by the National Research Foundation (NRF-20110000380), Ministry of Education, Science and Technology, Korea. This study was also partially supported by the Research Institute for Veterinary Science of Seoul National University and the Bio Imaging Research Center at GIST. We also acknowledge the National Instrumentation Center for Environmental Management for permission to take NMR, FT-IR, EF-TEM and ELS measurements. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 2, 3 and 9–13, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2012.04.029. References [1] Poizot P, Lauelle S, Grugeon S, Dupont L, Tarascon JM. Nanosized transitionmetal oxides as negative-electrode materials for lithium-iron batteries. Nature 2000;407:496–9. [2] Mahmoudi M, Simchi A, Imani M, Stroeve P, Sohrabi A. Templated growth of superparamagnetic iron oxide nanoparticles by temperature programming in the presence of poly(vinyl alcohol). Thin Solid Films 2010;518:4281–9. [3] Tari A, Chantrell RW, Charles SW, Popplewell J. Magnetic properties and stability of a ferrofluid containing Fe3O4 particles. Physica B & C 1979;97: 57–64. [4] Cunningham CH, Arai T, Yang PC, McConnell MV, Pauly JM, Condly SM. Positive contrast magnetic resonance imaging of cells labeled with magnetic nanoparticles. Mag Reson Med 2005;53:999–1005. [5] Polyak B, Friedman G. Magnetic targeting for site-specific drug delivery: applications and clinical potential. Expert Opin Drug Deliv 2009;6:53–70. [6] Kawashita M, Kawamura K, Li Z. PMMA-based bone cements containing magnetic particles for the hyperthermia of cancer. Acta Biomater 2010;6: 3187–92. [7] Widder KJ, Senyei AE, Ranney DF. In vitro release of biologically active adriamycin by magnetically responsive albumin microspheres. Cancer Res 1980;40:3512–7. [8] Mahmoudi M, Sant S, Wang B, Laurent S, Sen T. Superparamagnetic iron oxide nanoparticles (SPIONs): development, surface modification and applications in chemotherapy. Adv Drug Deliv Rev 2011;63:24–46.

3013

[9] Shubayev VI, Pisanic TR, Jin S. Magnetic nanoparticles for theragnostics. Adv Drug Deliv Rev 2009;61:467–77. [10] Gupta AK, Gupta M. Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomateials 2005;26:3995–4021. [11] Yang X et al. Multifunctional SPIO/DOX-loaded wormlike polymer vesicles for cancer therapy and MR imaging. Biomaterials 2010;31:9065–73. [12] Geng Y, Discher DE. Hydrolytic degradation of poly(ethylene oxide)-blockpolycaprolactone worm micelles. J Am Chem Soc 2005;127:12780–1. [13] Cole AJ, David AE, Wang J, Galban CJ, Hill HL, Yang VC. Polyethylene glycol modified, cross-linked starch-coated iron oxide nanoparticles for enhanced magnetic tumor targeting. Biomaterials 2011;32:2183–93. [14] Sun S, Zeng H. Size-controlled synthesis of magnetic nanoparticles. J Am Chem Soc 2002;124:8204–5. [15] Wang M, Thanou M. Targeting nanoparticles to cancer. Pharmacol Res 2010;62:90–9. [16] Arote RB et al. The therapeutic efficiency of FP-PEA/TAM 67 gene complexes via folate receptor-mediated endocytosis in a xenograft mice model. Biomaterials 2010;31:2435–45. [17] Chem TJ, Cheng TW, Hung YC, Lin KT, Liu GC, Wang YM. Targeted folic acid– PEG nanoparticles for noninvasive imaging of folate receptor by MRI. J Biomed Mater Res 2008;87A:165–75. [18] Ross JF, Chaudhuri PK, Ratnam M. Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines. Physiologic and clinical implications. Cancer 1994;73(9):2432–43. [19] Turk MJ, Breur GJ, Widmer WR, Paulos CM, Xu LC, Grote LA, et al. Folatetargeted imaging of activated macrophages in rats with adjuvant-induced arthritis. Arthritis Rheum 2002;46(7):1947–55. [20] Meier R et al. Breast cancers: MR imaging of folate-receptor expression with the folate-specific nanoparticle P1133. Radiology 2010;255(2):527–35. [21] Lin JJ, Chen JS, Huang SJ, Ko JH, Wang YM, Chen TL, et al. Folic acid–Pluronic F127 magnetic nanoparticle clusters for combined targeting, diagnosis, and therapy applications. Biomaterials 2009;30(28):5114–24. [22] Rastogi R, Gulati N, Kotnala RK, Sharma U, Jayasundar R, Koul V. Evaluation of folate conjugated PEGylated thermosensitive magnetic nanocomposites for tumor imaging and therapy. Colloids Surf B Biointerfaces 2011;82(1): 160–7. [23] Chen TJ, Cheng TH, Hung YC, Lin KT, Liu GC, Wang YM. Targeted folic acid–PEG nanoparticles for noninvasive imaging of folate receptor by MRI. J Biomed Mater Res A 2008;87(1):165–75. [24] Yoo MK et al. Superparamagnetic iron oxide nanoparticles coated with mannan for macrophage targeting. J Nanosci Nanotech 2008;8:5196–202. [25] Xu CX et al. Poly(ester amine)-mediated, aerosol-delivered Akt1 small interfering RNA suppresses lung tumorigenesis. Am J Respir Crit Care Med 2008;178:60–73. [26] Kumar M, Yigit M, Dai G, Moore A, Medarova Z. Image-guided breast tumor therapy using a small interfering RNA nanodrug. Cancer Res 2010;70: 7553–61. [27] Weitman SD, Lark RH, Coney LR, Fort DW, Frasca V, Zurawski Jr VR, et al. Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues. Cancer Res 1992;52(12):3396–401. [28] Cyrille B, Michael RW, Volga B, Jingquan L, Thomas PD. The design and utility of polymer-stabilized iron-oxide nanoparticles for nanomedicine applications. NPG Asia Mater 2010;2(1):23–30. [29] Cyrille B, Priyanto P, Thomas PD, Dakrong P, Volga B, Maria K, et al. Anti-fouling magnetic nanoparticles for siRNA delivery. J Mater Chem 2010;20:255–65.