Biomaterials 31 (2010) 13421348

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Size control of magnetic carbon nanoparticles for drug delivery

W.-K. Oh, H. Yoon, J. Jang*
School of Chemical and Biological Engineering, Seoul National University, 599 Gwanangro, Sillim-dong, Gwanak-gu, Seoul 151-742, Republic of Korea

a r t i c l e i n f o
Article history: Received 22 September 2009 Accepted 8 October 2009 Available online 29 October 2009 Keywords: Drug delivery Nanoparticle Magnetism Porosity Cell viablility

a b s t r a c t
Carbonized polypyrrole nanoparticles with controlled diameters were readily fabricated by the pyrolysis of polypyrrole nanoparticles. The carbonized polypyrrole nanoparticles showed narrow size distribution, large micropore volume, and high surface area. Magnetic phases were introduced into the carbon nanoparticles during the pyrolysis without sophisticated process, which resulted in useful magnetic properties for selective nanoparticle separation. Field emission scanning electron microscopy, Raman spectrometer, N2 adsorption/desorption, X-ray diffraction, and superconducting interference device were employed for characterizing the carbonized polypyrrole nanoparticles. Hydrophobic guest molecules were incorporated into the carbonized polypyrrole nanoparticles by surface adsorption, pore lling, and surface covalent coupling. The carbonized polypyrrole nanoparticles exhibited embedding capability using pyrene as a typical hydrophobic uorescent molecule. In addition, ibuprofen was incorporated into the carbon nanoparticles, and drug-loaded carbon nanoparticles sustained release property. In addition, the carbonized polypyrrole nanoparticles revealed low toxicity at concentrations below 100 mg mL1 via cell viability test and were uptaken inside the cells. These results suggest a new platform for the drug delivery using carbonized polypyrrole nanoparticles. 2009 Elsevier Ltd. All rights reserved.

1. Introduction Recent progress in synthesis of nanomaterials has made it possible to fabricate nanometer-sized materials with controlled structures and functionalities [13]. In particular, versatile porous materials with nanometer feature sizes have emerged as promising candidates for applications in the elds of catalysis [46], energy conversion and storage [7,8], separation [9], and biomedical science [10,11]. For example, as a typical microporous material, zeolite has been extensively utilized for molecular sieves, scaffolds, and templates for microporous replicas [12]. In addition, mesoporous carbons have been obtained using sacricial templates including colloidal silica nanoparticles and mesoporous silicas. Various precursors, such as sucrose, phenol resin, polypyrrole, polyacrylonitrile, and poly(furfuryl alcohol), were employed for obtaining porous carbon structures [13,14]. Carbon nanomaterials are of great interest in applications for biological elds [15,16]. Typically, carbon nanotubes (CNTs) have a feature of endohedral lling of 210 nm in diameter leading to encapsulation of small molecules. CNTs can be heterogeneously surface-functionalized and stained cytochemically with non-

* Corresponding author. Fax: 82 2 8881604. E-mail address: jsjang@plaza.snu.ac.kr (J. Jang). 0142-9612/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.10.018

quenching and non-photobleaching. Accordingly, CNTs may be suitable for bioapplications in biorecognition and drug delivery systems [1719]. However the biocompatibility of CNTs is still controversial and the tedious functionalization process of CNT surface remains major obstacles to practical applications [20,21]. The synthetic strategies towards carbon nanoparticles involve: i) pyrolysis of organic precursors under inert atmosphere [22] and ii) physical and chemical vapor deposition techniques [23]. While the pyrolysis approach is applicable to large-scale production, it offers very limited control on the carbon nanostructure. It has been well-known that carbon black is a mass-product manufactured by thermal decomposition or incomplete combustion of carbon hydrogen compounds [24]. However, carbon black consists of aggregates of spherical particles with the diameter of individual particles commonly ranging from 10 nm to 200 nm. Although the vapor deposition method allows precise control on the carbon nanostructure, it has also signicant drawbacks such as limited yield, high cost, and complex equipment. In general, carbon encapsulated magnetic nanoparticles were obtained by deposition of carbon onto Fe, Co, and Ni nanocatalysts using arc-discharge techniques [25]. However, it is known that the arc-discharge technique often gives an irreproducible and low yield due to the byproducts such as graphitic akes, carbon nanotubes, and carbides. Recently, we have explored the fabrication of several types of carbon nanoparticles from polymer nanoparticles as carbon

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precursors [26,27]. Direct carbonization of tailored polymer nanoparticles is favorable to the formation of carbon nanoparticles with the desired structure and composition. Furthermore, carbonization in mild temperature such as from 700 to 900 C remained their own biocompatibility of polymers, resulting in applying for biological elds. Notably, magnetic carbon nanospheres with controlled size and shape were fabricated via carbonization of iron-doped polymer nanomaterials [28,29]. Magnetic carbon nanostructures have aroused a great deal of interest due to their possible biological applications, including drug/gene delivery, thermal tumor therapy, invitro cell separation, and magnetic resonance imaging [30,31]. The geometry (size and shape) of the nanostructures is a key factor determining cellular uptake rate and mechanism. It has been found that an optimum geometry for endocytotic uptake is ca. 50 nm and spherical shape [32,33]. Here we report the fabrication of carbonized polypyrrole nanoparticles (CPyNs) with controlled diameters and their textural properties. Furthermore, we investigate the potential capability of CPyNs as imaging probes and drug carriers based on their porosity, magnetic property and biocompatibility. The guest moleculeloading of CPyNs was conducted with pyrene as a typical hydrophobic dye and the guest molecule-releasing test was performed with ibuprofen as a typical hydrophobic drug.
2. Materials and methods 2.1. Materials Dodecyltrimethylammonium bromide (DTAB, 99.0%), decyl alcohol (99.0%), pyrrole (98.0%), and ferric chloride (97.0%) were purchased from Aldrich. Ethanol (99.9%), tetrahydrofuran (THF, 99.9%), methanol (99.9%), and phosphate buffered solution (PBS: 0.1 M, pH 7.4) were also obtained from Aldrich. Pyrene (99%) and ibuprofen (98%) were purchased from Aldrich. A human broblast adherent cell line, which was derived from fetal lung tissue IMR90 (CCL-186), was purchased from American Type Culture Collection (ATCC). 2.2. Fabrication of CPyNs Polypyrrole (PPy) nanoparticles as the carbon precursor were prepared by micelle templating method and went through carbonization process in order to generate CPyNs. First, to prepare 60 nmdiameter PPy nanoparticles, DTAB (8 g) was dissolved in distilled water (160 mL) containing decyl alcohol (4.8 g) at 3 C. Pyrrole (1 g) was added dropwise into the DTAB/decyl alcohol solution and then ferric chloride (11.2 g) was added into the above solution. The chemical oxidation polymerization of pyrrole monomer was carried out for 1 h at 3 C. To prepare 80 nm/ 105 nmdiameter PPy nanoparticles, DTAB (8 g/8.4 g) was introduced into distilled water (160 mL) containing decyl alcohol (4.4 g/3.2 g). Pyrrole (1.6 g/2 g) was added dropwise into the surfactant solution, and ferric chloride (11.2 g) was added into the pyrrole/surfactant solution. The polymerization proceeded for 1 h at 3 C. The resulting products were washed with ethanol to remove the surfactant and other residual reagents and subsequently dried in a vacuum oven at room temperature. Carbonization of the PPy nanoparticles was conducted in a quartz tubular furnace under nitrogen atmosphere. The nanoparticles were heated to 800 C at a heating rate of 1 C min1, held for 3 h, and cooled to room temperature. CPyNs with controlled diameters could be obtained from the PPy precursors at approximately char yields of ca. 50%. Field emission scanning electron microscopy (FE-SEM) was performed with a JEOL 6330F at an acceleration voltage of 10 kV. The size distribution of CPyNs was calculated by software based on more than 100 particles in the images. Raman spectra were obtained in the range of 8001800 cm1 using a JobinYvon T64000 spectrometer. The BrunauerEmmettTeller (BET) nitrogen sorption experiments were conducted to calculate pore size distributions and cumulative pore volume with a Micromeritics ASAP 2020 at 77 K X-ray diffraction (XRD) measurement was performed using a M18XHF-SRA diffractometer (MAC Science Co.) equipped with a CuKa radiation source (l 1.5406 ) at 40 kV and 300 mA (12 kW). The magnetic property of CPyNs was measured using a superconducting interference device (SQUID) magnetometer (Quantum Design MPMS5). 2.3. Pyrene loading into CPyNs Pyrene was dissolved in THF solution at a concentration range of 5 102 to 25 mM and then mixed with an aqueous solution containing CPyNs for 12 h. The steady-state uorescence spectrum of each solution (lexc 334 nm) was obtained and were scanned in the range of 350450 nm using a Quanta Master Fluorescence Steady-State Spectrometer.

2.4. Surface modication with amino groups using plasma treatment The plasma reactor (Korea Vacuum Co.) is the parallelelectrode type with a 13.56 MHz radiofrequency generator. The diameter of the powered electrode on which the sample is placed is 35 cm and the distance between the two electrodes is 8 cm. The carbon nanoparticles were added into the plasma chamber, and the chamber was evacuated. When the chamber was evacuated below 103 Torr, the NH3 carrier gas was introduced into the chamber at rate of 30 cm3 min1 (operating pressure: 160 mTorr). The plasma output power was xed at 80 W and the plasma treatment was conducted in 2 min. This plasma treatment method and characterization were previously reported in the literature [34]. 2.5. Release prole of ibuprofen from CPyNs Ibuprofen (2 mg) was dissolved in methanol (2 mL) and then mixed into an aqueous solution (20 mL) containing CPyN-1 (10 mg). The CPyN-1 solution was stirred for 12 h, and subsequently it was separated by an external magnet and washed three times with water to remove residual drug. The drug-loaded CPyN-1 was dried in a vacuum oven at room temperature. To carry out time-dependent release tests, the following steps were employed: drug-loaded CPyN-1 was introduced into 0.1 M PBS (50 mL, pH) solution and incubated at 37 C. The samples (0.2 mL) were extracted at a constant time interval and dissolved in methanol (1.8 mL). The samples were quantitatively analyzed by HPLC (Waters). Separation of the analytes was achieved with an acetonitrile/water (1:1) containing 0.1% phosphate buffer, ow 1 mL min1, detection 228 nm and 270 nm, pressure 10,35,0001047000 psi, injected volume 50 mL, and C18 column. Linear regression coefcients from six-point linear calibration curves (concentration range 0.510 mg mL1) were between 0.9982 and 0.9999 for all compounds. 2.6. Cell viability assay IMR90 cells were cultured in Eagles Minimum Essential Medium (EMEM) containing sodium pyruvate (1 mM), Lglutamine (2 mM), 10% heatinactivated Fetal Bovine Serum (FBS), penicillin (10,000 IU mL1), and streptomycin (10 mg mL1). The cells were grown in a humidied incubator at 37 C (95% air, 5% CO2). Cell viability of the CPyN-treated cells was investigated using CellTiter glow luminescent cell viability assay (Promega, Madison, WI). For estimating viable cells, 10,000 cells per well were plated and treated with different concentrations of CPyNs (10, 25, 100, 250, and 500 mg mL1) for 24 h. 2.7. Transmission electron microscopy(TEM) of CPyNs-treated cells The ultrastructural alterations of IMR90 cell lines induced by the CPyNs were observed with TEM (JEM-2000EXII, JEOL). IMR 90 cells were incubated in Lab-Tek II chamber slides until 80% conuence. After a 24 h exposure of CPyNs, IMR90 was harvested by a cell scraper and prexed with 2% paraformaldehyde and 2% glutaraldehyde at 4 C for 4 h. After being washed with 0.05 M cacodylate buffer, IMR90 was postxed with 1% osmiumtetraoxide at 4 C for 2 h and washed with distilled water and then stained with 0.5% uranyl acetate at 4 C. The cells were dehydrated through a series of ethyl alcohol concentrations (30%, 50%, 70%, 80%, 90%, 100%, 100%, and dry alcohol). Then, the cells were treated with propylene oxide followed by 1:1 propylene oxide: spurrs resin for 2 h. The cells were inltrated in spurrs resin at 70  C for 24 h and ultramicrotome was performed with diamond knife, collected on carbon grids. Then, samples were observed with the TEM at 120 kV.

3. Results and discussion Polypyrrole (PPy) nanoparticles with controlled diameters were prepared by micelle templating in oil/water emulsions and used for carbon precursors in order to fabricate CPyNs. PPy consists of vemembered heterocyclic rings with cross-linking structures (a,a- and a,b-links) and includes iron cations as the dopant from the oxidizing agent (ferric chloride), which can act as the catalyst to facilitate the formation graphite structure [35]. During heat-treatment process under an inert atmosphere, the well-dened PPy nanoparticles can be readily converted into carbon nanoparticles and the doped iron cations are transformed into magnetic phases. As previously reported, spherical micelles were formed with DTAB in aqueous solution, and decyl alcohol as a cosurfactant was used to minimize the destabilization phenomenon such as Ostwald ripening effect to fabricate PPy nanoparticles with controlled diameters [2,25]. The size of micelles strongly depends on the concentrations of surfactant. In general, the size of nanoparticle decreases with increasing the surfactant concentration [35]. Under our experimental conditions, the diameter

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of PPy nanoparticle was effectively controlled with varying concentrations of DTAB and decyl alcohol. DTAB was employed to form micelles as a nanoreactor, and decyl alcohol was used as a cosurfactant. Decyl alcohol, a water-insoluble long chain alcohol, can retard or hinder the diffusion of pyrrole through the aqueous phase. Therefore, the size of the nanoparticles was controlled by the ratio between pyrrole and DTAB/decyl alcohol. When the monomer/surfactant ratio decreases, the diameter of PPy nanoparticles decreases. PPy nanoparticles were prepared with three different diameters (60, 80, and 105 nm) and were successfully transformed to CPyNs with uniform sizes by the carbonization procedure (char yield: ca. 50%). Fig. 1 represents FESEM images and size distribution histograms of CPyNs. The FESEM images exhibited spherical nanoparticles with reasonably uniform sizes and their diameters were 56 6 nm (CPyN1), 76 9 nm (CPyN2), and 99 8 nm (CPyN3), respectively.

The successful carbonization of CPyNs and the impregnation of iron oxides were conrmed by Raman spectroscopy. In Fig. 2a, the Raman spectrum of the CPyNs represented two distinct peaks at 1595 cm1 and 1350 cm1. The band at 1595 cm1 (G band) was assigned to the E2g vibration of graphitic carbon with sp2 conguration. On the other hand, the peak at 1350 cm1 (D band) was attributable to the A1g mode of an imperfection in the crystal structure of graphite (e.g., iron) with sp3 conguration. Although CPyNs were carbonized at mild temperature (800 C), the CPyNs represented the crystal structure and could be categorized as a glassy carbon structure. In addition, the XRD pattern of CPyNs conrmed the existence of graphite in the nanoparticles, originating the characteristic 002 and 100 Bragg reections of graphenes (Fig. 2b). Furthermore, the XRD pattern showed the presence of g-Fe2O3 and a-Fe (labeled F and A, respectively) as magnetic phases

Fig. 1. FE-SEM images and size distribution histograms of CPyNs with different diameters: CPyN-1(56 6 nm) (a), CPyN-2(76 9 nm) (b), and CPyN-3(99 8 nm) (c).

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Fig. 2. Raman spectrum of CPyN-1 measured between 800 and 1800 cm-1 a), powder XRD pattern of CPyN-1 (G: graphite, F: gFe2O3, A: aFe) b).

in the CPyNs. It is considered that iron-based complexes encapsulated in PPy nanoparticles formed iron oxides at 800 C. Nitrogen sorption experiments were performed to characterize the textural properties of CPyNs. The nitrogen adsorption desorption isotherms and the cumulative pore volume curves of CPyNs are displayed in Fig. 3. The isotherms represent an increase in the volume adsorbed up to the relative pressure in the range of 0.010.02, depending on the particular sample, and then leveled off. At low relative pressures, micropore lling mainly occurs due to the strong adsorbentadsorbate interactions and the amount adsorbed in the mesopores is negligible. Accordingly, this behavior is associated with adsorption in micropores and may also originate from adsorption in mesopores with sizes close to the micropore range. The CPyNs displayed a steep increase in the volume adsorbed at pressures close to the saturation vapor pressure. This phenomenon is due to the capillary condensation of nitrogen in interparticle pores with some contribution of multilayer adsorption on the surface of these pores [36]. BET surface areas were ca. 408 m2 g1, 235 m2 g1, and 164 m2 g1 for CPyN1, CPyN2, and CPyN3, respectively, which was in inverse proportion to the diameter of CPyNs. The pore size distributions of CPyNs derived from nitrogen adsorption isotherms was close to the micropore range of 0.02.5 nm in size. The total pore volumes of CPyNs were calculated to be ca. 0.62 cm3 g1, 0.39 cm3 g1, and 0.22 cm3 g1 for CPyN1, CPyN2, and CPyN3, respectively. The micropore volumes

Fig. 3. Nitrogen adsorption/desorption isotherms of CPyNs (insets: cumulative pore volumes of CPyNs at a pore diameter of less than 3 nm calculated by HK formalism): CPyN-1 (a), CPyN-2 (b), and CPyN-3 (c).

calculated from the HorvathKawazoe (HK) formalism were ca. 0.17 cm3 g1, 0.13 cm3 g1, and 0.10 cm3 g1, respectively (Fig. 3 insets). It is considered that the micropore volume increased in inverse proportion to the size of CPyNs because the heat conduction and mass transfer of CPyNs were varied with respect to the size of CPyNs, where the heat conduction proceeded from outside to inside of CPyNs and the mass transfer proceeded from inside to outside of CPyNs during carbonization process. Interestingly, the

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micropore volume of CPyNs was comparable to that of ZSM5 zeolite (micropore volume 0.14 cm3 g1, mesopore volume 0.02 cm3 g1), as a wellknown microporous material [37]. Magnetic properties of the CPyNs were investigated with an SQUID magnetometer. In Fig. 4, the hysteresis loop of the CPyNs at 300 K represented typical ferromagnetic behavior. Moreover, saturation of the magnetization from the hysteresis loop was found to be 12.5 emu g1. The coercivity (Hc) was observed at a eld of 25 Oe. Compared to the value of Hc for bulk iron (z1 Oe), a significant increase of coercivity was observed in the CPyNs. The magnetic property of CPyNs is useful for efcient targeting and separating of drug carriers. In general, carbon nanoparticles could load functional guest molecules by surface adsorption, pore lling, and surface covalent coupling [38]. As the diameter of a nanoparticle becomes smaller, it has more populations to contact with guest species due to the enhanced surface area. When the guest molecules reach to the nanoparticle surface, they can be incorporated into the nanoparticle by physical interactions such as van der Waals force and hydrophobic interaction. In order to gain a better insight into the adsorption behavior of guest molecules in CPyNs, pyrene was loaded into CPyNs by a phase separation method. Emission spectrum of pyrene shows characteristic the intensity ratio between the rst (369.5 nm) and the third (380 nm) monomeric peaks (Im1/Im3). The Im1/Im3 value is susceptible to the micro-environmental polarity of the solubilized pyrene molecules [39,40]. In general, the Im1/Im3 value increases as the solvent polarity increases. Accordingly, the location of pyrene in CPyNs could be traced on the molecular level by monitoring the Im1/Im3 value. Fig. 5a exhibits representative uorescence spectrum of pyrene/CPyN-1/water solution. The Im1/Im3 ratio was systematically investigated and normalized by dividing it by the initial value (the concentration of pyrene 5 102 mM). Fig. 5b displays changes in the normalized Im1/Im3 value for pyrene/CPyN/water systems as a function of pyrene concentration. The normalized Im1/Im3 value did not change up to a critical concentration (Ccrit), which was dependent on the size of CPyNs, and then gradually increased with further increasing pyrene concentration. At low concentrations, most pyrene molecules can exist in the hydrophobic pore or surface of CPyNs. The Ccrit increased in the order of CPyN-3 < CPyN-2 < CPyN-1. Because the pyrene amount in CPyN was determined by the pore volume and surface area of CPyNs, the Ccrit was strongly affected by the size of CPyNs. The increase in Im1/Im3 value at higher concentrations reects that pyrene locates a more hydrophilic microenvironment.

Fig. 5. Adsoption behavior of pyrene-loaded CPyN-1; Fluorescence spectrum of pyrene/CPyN-1/aqueous solution (a), and relative hydrophobic index of pyrene-loaded CPyN-1 as a function of pyrene concentration (b) (Im1/Im3 value: micro-environmental polarity of the solubilized pyrene molecules).

Fig. 4. Magnetic hysteresis loop of CPyN-1 measured at 300 K between 15 and 15 kOe (inset: magnied area between 50 and 50 Oe).

In other words, it is considered that an excess of pyrene molecules over the Ccrit exists in aqueous phase. These results conrm the fact that CPyNs have high afnity with hydrophobic molecules and can readily load a certain amount of guest molecules. Potential capability of CPyNs to load guest molecules provides the application to a drug carrier. Ibuprofen is a well known nonsteroidal antiinammatory drug and was investigated to releasing molecules of CPyNs in aqueous medium. Furthermore, a surface-modied CPyN-1 (termed CPyN-1-NH2) was prepared by further functionalization with ammonia plasma treatment for preparing the CPyN-1 with amino groups. Fig. 6 exhibits the in-vitro release proles of CPyNs for ibuprofen. CPyN-2 and CPyN-3 released over 70% of naproxen during 10 h, and more than 90% of naproxen was released within 50 h. In the case of CPyN-1, about 53% of ibuprofen released within 10 h, and CPyN-1 was sustained ca. 70% of ibuprofen during 50 h. CPyN-1 exhibits the relatively slow release because of higher surface area and pore volume. Notably, CPyN1-NH2 delayed the release of ibuprofen such as about 60% within 50 h, indicating that the ionic interaction between the amino group of the CPyN-1-NH2 and the carboxyl group of ibuprofen plays a crucial role of sustaining release property [41]. The biocompatibility of CPyNs is a key factor in their biological applications. The CPyNs were incubated with human lung broblast cell line (IMR90), and ATP assay was used for the study on the cell viability of CPyNs (Fig. 7a). The ATP assay is a homogeneous

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drastically beyond 100 mg mL1. The cell viability of carbon nanoparticles were concentrationdependent. Furthermore, cell viabilities decreased as the size of CPyNs decreased. As previously reported by Yen et al. for gold and silver nanoparticles, the smallest carbon nanoparticles exhibited most cytotoxic due to high surface area and large numbers [42]. The TEM images of IMR90 treated with CPyN-1 at 25 mg mL1 for 24 h are shown in Fig. 7V. CPyN-1 was taken by endocytosis inside the vesicles in the cytoplasm and morphology of cells remained normal. The CPyN-1 inside the vesicle formed a cluster. Magrez et al. reported carbon black nanoparticles showed hazardous effect compared to multiwalled carbon nanotubes and carbon nanobers [20]. In contrast, CPyNs represent relatively less cytotoxicity. It is thought that CPyNs are originated from non-cytotoxic precursor, polypyrrole nanoparticles. 4. Conclusion
Fig. 6. Cumulative ibuprofen release from ibuprofen loaded CPyN-1 in PBS solution (100 mM, pH 7.4, 37 C; n 3).

technique for determining the number of viable cells based on quantication of the ATP concentration. After 24 h treatments of CPyNs, the viability was declined to ca. 52%, 63%, and 72% for CPyN1, CPyN2, and CPyN3 at the highest concentration of CPyNs (500 mg mL1). Below 100 mg mL1, no signicant decrease was observed. However, ATP content of the cells decreased

PPy nanoparticles with controlled diameters were prepared by micelle templating in oil/water emulsions, and CPyNs with three different sizes (55, 76, and 99 nm) were successfully obtained by carbonization of the polymer precursors. CPyNs showed highly microporous compared to zeolite, resulting in loading guest molecules into CPyNs using phase separation. In addition, the magnetic property of CPyNs provided the selective separation and targeting. CPyNs sustained in-vitro drug release properties. Importantly, smaller size and amine surface modication of CPyNs provide an improved sustained property. Due to their superiorities such as microporous structure, monodispersity, magnetism, and biocompatibility, it is believed that the CPyNs open the way to use in elds such as biomaterials science, including bioimaging and magnetic induced drug carriers. Acknowledgements This work was supported by the Center for Advanced Materials Processing under the 21C Frontier Programs of the Ministry of Knowledge Economy, and WCU (World Class University) program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (400-20080230), Republic of Korea. Appendix Figures with essential color discrimination. Figs. 5 and 6 in this article are difcult to interpret in black and white. The full color images can be found in the on-line version, at doi:10.1016/j. biomaterials.2009.10.018. References
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Fig. 7. a) Cell viability of human broblast IMR90 cells incubated with CPyNs as a function of CPyN concentrations for 24 h (n 3). *Statistically signicant difference from control exposed to CPyNs (P < 0.05). b) TEM image of IMR90 cells incubated with CPyN-1 at 25 mg mL1 for 24 h. Circles exhibits the endocytotic CpyN-1 vesicle.

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