# Synthesis of Superparamagnetic Iron Oxide Nanoparticles Modified with MPEG-PEI via Photochemistry as New MRI Contrast Agent

Authors: Yancong Zhang; Lianying Zhang; Xinfeng Song; Xiangling Gu; Hanwen Sun; Chunhua Fu; Fanzong Meng
Journal: Journal of Nanomaterials
 (2015)

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## Abstract

Novel method for synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylenimine (PEI) and modified with poly(ethylene glycol) methyl ether (MPEG), MPEG-PEI-SPIONs, was developed. PEI-SPIONs were successfully prepared in aqueous system via photochemistry, and their surface was modified with poly(ethylene glycol) methyl ether (MPEG). The so-obtained MPEG-PEI-SPIONs had a uniform hydrodynamic particle size of 34 nm. The successful coating of MPEG-PEI on the SPIONs was ascertained from FT-IR analysis, and the PEI and MPEG fractions in MPEG-PEI-SPIONs were calculated to account for 31% and 12%, respectively. Magnetic measurement revealed that the saturated magnetization of MPEG-PEI-SPIONs reached 46 emu/g and the nanoparticles showed the characteristic of being superparamagnetic. The stability experiment revealed that the MPEG-PEI modification improved the nanoparticles stability greatly.T2 relaxation measurements showed that MPEG-PEI-SPIONs show similar R2 value to the PEI-SPIONs. The T2-weighted magnetic resonance imaging (MRI) of MPEG-PEI-SPIONs showed that the magnetic resonance signal was enhanced significantly with increasing nanoparticle concentration in water. These results indicated that the MPEG-PEI-SPIONs had great potential for application in MRI.


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## Body

## 1. Introduction

Magnetic resonance imaging (MRI) is a noninvasive technique routinely used in clinics for diagnostic imaging. It has become very important diagnostic modality in hospitals [1]. The quality of the MRI images depends on three NMR main parameters (the proton spin density, the nuclear spin-lattice relaxation timeT1, and the spin-spin relaxation timeT2), and the contrast agents (CAs), based on the different distribution of nuclear-spin density along the body, are able to improve the image contrast by increasing (locally) the nuclear relaxation rates [2].Superparamagnetic nanoparticles are represented asT2 (transversal relaxation time) contrast agents as opposed toT1 (longitudinal relaxation time) contrast agents such as paramagnetic gadolinium(III) chelates [3, 4]. The superparamagnetic iron oxide nanoparticles (SPIONs) are the most commonly used superparamagnetic contrast agents. During the preparation, storage, and application of MR contrast agent, the stability and biocompatibility of SPIONs are important [5]. However, due to the high ratio of surface to volume and magnetization, SPIONs are prone to aggregate in water or tissue fluid which limits the application [6]. To reduce aggregation and enhance the biocompatibility, the coating of polymer onto SPIONs surface is indispensable [7, 8]. When coated with polymer, SPIONs have a reduced aggregation, improved biocompatibility, and longer half-life in circulation and are successfully used for MRI contrast agent and targeted gene and drug delivery [9–11].Polyethylenimine (PEI) is a water soluble cationic polymer which contains amino and imino groups in each polymer chain. When the polymer PEI is dispersed in aqueous solution, each polymer chain is positively charged by amino and imino groups [12]. The dispersibility of the composite nanoparticles is expected to be enhanced by the electrostatic repulsive force and steric hindrance of the PEI polymers [13]. Corti et al. [14] synthesized polyethylenimine-coated superparamagnetic nanoparticles and demonstrated their ability to be used as MRI contrast agents. However, the toxicity of PEI has limited its use, particularly forin vivo application [15].Poly(ethylene glycol) (PEG) has been employed extensively in pharmaceutical and biomedical fields because of its outstanding physicochemical and biological properties including hydrophilic property, solubility, nontoxicity, ease of chemical modification, and absence of antigenicity and immunogenicity [16]. Therefore, PEG is widely used as a pharmacological polymer with high hydrophilicity, biocompatibility, and biodegradability. It is ideal for prevention of bacterial surface growth, decrease of plasma protein binding and erythrocyte aggregation, and prevention of recognition by the immune system.In recent years, PEG modified PEI has been synthesized for gene delivery [17, 18]. However, to the best of our knowledge, the PEG modified PEI coated superparamagnetic iron oxide nanoparticles have not been synthesized for MRI contrast agents. In this report, we describe the synthesis of PEI coated SPIONs via photochemistry and surface modified with poly(ethylene glycol) methyl ether (MPEG). The physical properties, stability, and MRI of the MPEG-PEI-SPIONs are investigated and the results showed that these novel nanoparticles may be a good candidate for bioapplications utilizing MRI.


## 2. Materials and Methods

### 2.1. Materials

Polyethylenimine (PEI, average Mn 400) and 1,1′-carbonyldiimidazole (CDI), analytical grade, were purchased from Tokyo Chemical Industry Co. Ltd., Japan. Poly(ethylene glycol) methyl ether (MPEG, average Mn 550) was purchased from Sigma-Aldrich, USA. Ferrous chloride tetrahydrate (FeCl2·4H2O), ferric chloride hexahydrate (FeCl3·6H2O), and NH3 aqueous solution (25 wt%), analytical grade, were purchased from Shanghai Chemical Reagents Company (China) and used without further treatment. Nitrogen (99.99%) was available from Dezhou LongLi Company, China. All the other chemicals were of analytical grade.Stirrer (IKA Company, Germany) was used to synthesize the Fe3O4 nanoparticles (SPIONs). A photochemical reaction device equipped with two 8 W low-pressure mercury lamps, which had been described in detail in our previous papers [19, 20], was used as the UV source to synthesize PEI-SPIONs.


### 2.2. Synthesis of Fe3O4 Nanoparticles: SPIONs

Superparamagnetic magnetic nanoparticles were prepared via improved chemical coprecipitation method [21, 22]. Briefly, 3.91 g FeCl3·6H2O and 1.43 g FeCl2·4H2O were dissolved in 150 mL deionized water in a 500 mL beaker under nitrogen gas and vigorously stirred at 85°C until dissolved, after which 7 mL of NH3 aqueous solution was injected into the solution rapidly. After 1 hour of stirring at 85°C, the SPIONs precipitated and were isolated from the solution by a permanent magnet (4,000 Gaus). The SPIONs were washed five times with deionized water and separated by magnetic decantation. Finally, the SPIONs were dispersed in 100 mL deionized water and stored at 4°C.


### 2.3. Preparation of PEI-SPIONs

The PEI coated SPIONs were synthesized in a 150 mL interlayer quartz flask equipped with a stirrer and a N2 inlet cooling with cycled water. 100 μL of PEI and 10 mg SPIONs were mechanically mixed in 60 mL water for 30 min, after which 1 mL of H2O2 was added, and then the mixture was irradiated by two 8 W lamps for 20 min and N2 was bubbled throughout the preparation process. The PEI-SPIONs were collected with a permanent magnet and washed with deionized water for several times and at last redispersed in deionized water.


### 2.4. Modification of PEI-SPIONs with MPEG

To a solution of 400μL MPEG in 15 mL dry acetonitrile under N2 was added 1.5 g of CDI, and the solution was stirred at 40°C for 4 hours. 30 mg of PEI-SPIONs (dissolved in 30 mL water) was added into the solution and the mixture was stirred for another 2 hours. At last, the MPEG-PEI-SPIONs were collected with a magnet and washed with deionized water for 3 times.


### 2.5. Characterization

FT-IR spectra of MPEG-PEI-SPIONs were analyzed with a Nicolet Avatar 370 Fourier transform infrared spectrometer (FT-IR) in a wave number range of 4000–500 cm−1. Structure of the magnetic nanoparticles was observed with transmission electron microscopy (TEM, Philips, CM120). The average size in aqueous system was detected by photon correlation spectroscopy (PCS, Zetasizer Nano ZS, Malvern Instruments Ltd.). Thermogravimetric analysis (TG) was done by a thermal analyzer (DTG-60, Shimadzu) by heating the sample from room temperature to 900°C under N2 atmosphere at a heating rate of 10°C min−1. Saturated magnetization of the nanoparticles was determined on vibrating sample magnetometer (VSM, Model 155, Princeton Applied Research) at room temperature. The samples for FT-IR, TEM, and TG were vacuum-dried at room temperature. The sample for PCS was diluted to 10−5 molL−1.MRI experiments were performed at 25°C in a clinical magnetic resonance (MR) scanner (NIUMAG, 0.5T, Shanghai, China). To demonstrate theT2 effects in an aqueous solution, MPEG-PEI-SPIONs were suspended in tubes of water (20 mL) with different SPION concentrations. The tubes were placed into the MR scanner and a number of MR sequences were run, spin-echo forR2 (32 echoes, TR: 3000 ms, TE: 0~4000 ms) determination.


## 2.1. Materials

Polyethylenimine (PEI, average Mn 400) and 1,1′-carbonyldiimidazole (CDI), analytical grade, were purchased from Tokyo Chemical Industry Co. Ltd., Japan. Poly(ethylene glycol) methyl ether (MPEG, average Mn 550) was purchased from Sigma-Aldrich, USA. Ferrous chloride tetrahydrate (FeCl2·4H2O), ferric chloride hexahydrate (FeCl3·6H2O), and NH3 aqueous solution (25 wt%), analytical grade, were purchased from Shanghai Chemical Reagents Company (China) and used without further treatment. Nitrogen (99.99%) was available from Dezhou LongLi Company, China. All the other chemicals were of analytical grade.Stirrer (IKA Company, Germany) was used to synthesize the Fe3O4 nanoparticles (SPIONs). A photochemical reaction device equipped with two 8 W low-pressure mercury lamps, which had been described in detail in our previous papers [19, 20], was used as the UV source to synthesize PEI-SPIONs.


## 2.2. Synthesis of Fe3O4 Nanoparticles: SPIONs

Superparamagnetic magnetic nanoparticles were prepared via improved chemical coprecipitation method [21, 22]. Briefly, 3.91 g FeCl3·6H2O and 1.43 g FeCl2·4H2O were dissolved in 150 mL deionized water in a 500 mL beaker under nitrogen gas and vigorously stirred at 85°C until dissolved, after which 7 mL of NH3 aqueous solution was injected into the solution rapidly. After 1 hour of stirring at 85°C, the SPIONs precipitated and were isolated from the solution by a permanent magnet (4,000 Gaus). The SPIONs were washed five times with deionized water and separated by magnetic decantation. Finally, the SPIONs were dispersed in 100 mL deionized water and stored at 4°C.


## 2.3. Preparation of PEI-SPIONs

The PEI coated SPIONs were synthesized in a 150 mL interlayer quartz flask equipped with a stirrer and a N2 inlet cooling with cycled water. 100 μL of PEI and 10 mg SPIONs were mechanically mixed in 60 mL water for 30 min, after which 1 mL of H2O2 was added, and then the mixture was irradiated by two 8 W lamps for 20 min and N2 was bubbled throughout the preparation process. The PEI-SPIONs were collected with a permanent magnet and washed with deionized water for several times and at last redispersed in deionized water.


## 2.4. Modification of PEI-SPIONs with MPEG

To a solution of 400μL MPEG in 15 mL dry acetonitrile under N2 was added 1.5 g of CDI, and the solution was stirred at 40°C for 4 hours. 30 mg of PEI-SPIONs (dissolved in 30 mL water) was added into the solution and the mixture was stirred for another 2 hours. At last, the MPEG-PEI-SPIONs were collected with a magnet and washed with deionized water for 3 times.


## 2.5. Characterization

FT-IR spectra of MPEG-PEI-SPIONs were analyzed with a Nicolet Avatar 370 Fourier transform infrared spectrometer (FT-IR) in a wave number range of 4000–500 cm−1. Structure of the magnetic nanoparticles was observed with transmission electron microscopy (TEM, Philips, CM120). The average size in aqueous system was detected by photon correlation spectroscopy (PCS, Zetasizer Nano ZS, Malvern Instruments Ltd.). Thermogravimetric analysis (TG) was done by a thermal analyzer (DTG-60, Shimadzu) by heating the sample from room temperature to 900°C under N2 atmosphere at a heating rate of 10°C min−1. Saturated magnetization of the nanoparticles was determined on vibrating sample magnetometer (VSM, Model 155, Princeton Applied Research) at room temperature. The samples for FT-IR, TEM, and TG were vacuum-dried at room temperature. The sample for PCS was diluted to 10−5 molL−1.MRI experiments were performed at 25°C in a clinical magnetic resonance (MR) scanner (NIUMAG, 0.5T, Shanghai, China). To demonstrate theT2 effects in an aqueous solution, MPEG-PEI-SPIONs were suspended in tubes of water (20 mL) with different SPION concentrations. The tubes were placed into the MR scanner and a number of MR sequences were run, spin-echo forR2 (32 echoes, TR: 3000 ms, TE: 0~4000 ms) determination.


## 3. Results and Discussions

### 3.1. Synthesis of MPEG-PEI-SPIONs

According to our previous studies, the SPIONs could adsorb PEI molecules on their surface due to the high ratio of surface to volume when SPIONs were dispersed in aqueous solution containing PEI [19, 20]. The H2O2 could be decomposed into •OH, which could initiate the PEI to generate free radicals when the solution was exposed to UV irradiation [23]. PEI chains were grafted from the SPIONs via recombination of the PEI free radicals, and then the surface of Fe3O4 nanoparticles was coated by a cross-linked PEI shell via further cross-linking. The PEI-SPIONs were surface-modified with MPEG by covalently binding CDI-activated MPEG. The preparation route of MPEG-PEI-SPIONs was illustrated in Figure 1.Figure 1
Schematic illustration of the preparation route of MPEI-PEI-SPIONs.Figure2 shows the FT-IR spectra of (a) MPEG-PEI-SPIONs and (b) naked SPIONs. The 1644 cm−1 bond was correlated to the scissoring vibration of N–H bond of –NH2 from PEI, and the 1550 cm−1 bond was correlated to the carbodiimide. The bands of 1360 cm−1 and 1102 cm−1 were assigned to the C–H bond of –CH3 and C–O–C bound of –(OCH2CH2)nCH3, respectively. Furthermore, the peak of 593 cm−1 in curves (a) and (b) was the characteristic absorption of Fe–O bound. All what is above confirmed that the MPEG-PEI-SPIONs were successfully prepared under UV irradiation.Figure 2
FT-IR spectra of the (a) MPEG-PEI-SPIONs and (b) naked SPIONs.Thermogravimetric analysis (TG) was carried out for the SPIONs, PEI-SPIONs, and MPEG-PEI-SPIONs. The SPIONs, PEI-SPIONs, and MPEG-PEI-SPIONs gave their distinctive TG curves, which provide indications of the amount of PEI and MPEG on the SPIONs. In Figure3(a), the weight loss from 120 to 700°C was about 5% for SPIONs. In Figure 3(b), the weight loss from 120 to 700°C was about 36%. Considering the weight loss of SPIONs, the weight of the PEI fraction in PEI-SPIONs was calculated to account for 31% of the total weight. Figure 3(c) shows the TG curve of MPEG-PEI-SPIONs. The weight loss of the MPEG-PEI-SPIONs from 120 to 700°C was about 48%. Considering the weight loss of SPIONs and PEI-SPIONs, the weight of the MPEG fraction in MPEG-PEI-SPIONs was calculated to account for 12% of the total weight.Figure 3
The TG curve of the (a) naked SPIONs, (b) PEI-SPIONs, and (c) MPEG-PEI-SPIONs.


### 3.2. Structure, Size, and Magnetic Properties of MPEG-PEI-SPIONs

Morphology and structure of the MPEG-PEI-SPIONs were studied by TEM. As shown in Figure4, the MPEG-PEI-SPIONs were of nearly spherical shape. The average size in aqueous system measured by PCS was 34 nm with a polydispersion index of 0.276 (Figure 5). As the diameter is smaller than 50 nm, the MPEG-PEI-SPIONs can avoid the phagocytosis of reticuloendothelial system (RES) and are designed to have a much longer blood half-life and could rapidly circulate almost the whole body. Thus, the MPEG-PEI-SPIONs can penetrate deep tissue spaces (such as the interior of atherosclerotic plaque and the myocardium), and the hydrophilic surface may prolong the circulation in blood [24, 25].Figure 4
The TEM image of MPEG-PEI-SPIONs.Figure 5
Particle size of the MPEG-PEI-SPIONs measured by PCS.The magnetic properties of the naked SPIONs, PEI-SPIONs, and MPEG-PEI-SPIONs were studied by vibration sample magnetometer (VSM). The saturation magnetization (Ms) value of naked SPIONs was 68 emu/g. After PEI coating, the Ms decreased to 55 emu/g due to the decrease in SPIONs content of the nanoparticles. After MPEG modification, the Ms decreased to 46 emu/g. This level of Ms is deemed sufficient for bioapplications where Ms of 7–22 emu/g is usually adopted [26, 27]. No hysteresis was observed in Figure 6, indicating the nanoparticles are superparamagnetic at room temperature. Thus, the MPEG-PEI-SPIONs possess superparamagnetism which are suitable for MR contrast enhancement by alterations of proton relaxation in the tissue microenvironment [28].Figure 6
Magnetization curve of the (a) naked SPIONs, (b) PEI-SPIONs, and (c) MPEG-PEI-SPIONs.


### 3.3. Stability

High stability is an important parameter, which determined whether MPEG-PEI-SPIONs can be applied in tissue imaging. The magnetic nanoparticles dissolved in PBS (pH 7.0) were stored at 4°C and the supernatant was sampled at different intervals to investigate the stability using ultraviolet-visible spectrophotometry (UV-1800, Shimadzu). The optical density (OD) was detected at 660 nm [29], at which the OD value was linearly related to the concentrations of magnetic nanoparticles. Stability was calculated according to
(1)Stability%=ODsODf×100%,
where ODs was OD value of nanoparticles stored for certain time and ODf was OD value of the freshly prepared sample. The result was shown in Figure 7. The PEI coating could improve the stability of the SPIONs in the observed 178 hours. After MPEG modification, the MPEG-PEI-SPIONs have a higher stability in PBS (pH 7.0), as they could retain 88.7% in aqueous suspension over a week. The high stability is due to the electrostatic repulsive force and steric hindrance of the PEI polymers against aggregation, and the high hydrophilicity of MPEG may also contribute to the stability.Figure 7
Stability of the (a) MPEI-PEI-SPIONs, (b) PEI-SPIONs, and (c) naked SPIONsin vitro (retention ratio versus stored time).


### 3.4. MR Imaging

To investigate theT2 relaxation properties of SPIONs, PEI-SPIONs, and MPEG-PEI-SPIONs,T2 relaxation measurements had been made using 0.5T MR imager. Figure 8 shows theT2 relaxation curves for the three samples. The naked SPIONs showed fasterT2 decay curve compared to PEI-SPIONs and MPEG-PEI-SPIONs. The MPEG-PEI-SPIONs had a similarT2 decay curve. Thus, the MPEG-PEI-SPIONs nanoparticles show similar R2(=1/T2) value with the PEI-SPIONs. This result indicates that the MPEG modification does not decrease theT2 relaxation properties compared with PEI-SPIONs.Figure 8
TheT2 relaxation curves for (a) MPEI-PEI-SPIONs, (b) PEI-SPIONs, and (c) naked SPIONs.Finally, we performed MRI experiments on a phantom composed of four sample tubes containing MPEG-PEI-SPIONs in water at different concentrations (spin-echo technique with TR = 3000 ms and TE = 100 ms). It can be seen from Figure9 that the MR signal intensity (related to theT2 relaxation time inT2-weighted image) for the samples of different concentrations is not identical. With increasing MPEG-PEI-SPIONs concentration in water, the MR signal was enhanced significantly (negative in brightness inT2-weighted image). This result indicates that the nanoparticles can generate high magnetic field gradients near the surface of the MPEG-PEI-SPIONs.Figure 9
T2-weighted MRI images of MPEG-PEI-SPIONs with different concentrations. (a) 0.063, (b) 0.125, (c) 0.250, and (d) 0.500 mg/mL.


## 3.1. Synthesis of MPEG-PEI-SPIONs

According to our previous studies, the SPIONs could adsorb PEI molecules on their surface due to the high ratio of surface to volume when SPIONs were dispersed in aqueous solution containing PEI [19, 20]. The H2O2 could be decomposed into •OH, which could initiate the PEI to generate free radicals when the solution was exposed to UV irradiation [23]. PEI chains were grafted from the SPIONs via recombination of the PEI free radicals, and then the surface of Fe3O4 nanoparticles was coated by a cross-linked PEI shell via further cross-linking. The PEI-SPIONs were surface-modified with MPEG by covalently binding CDI-activated MPEG. The preparation route of MPEG-PEI-SPIONs was illustrated in Figure 1.Figure 1
Schematic illustration of the preparation route of MPEI-PEI-SPIONs.Figure2 shows the FT-IR spectra of (a) MPEG-PEI-SPIONs and (b) naked SPIONs. The 1644 cm−1 bond was correlated to the scissoring vibration of N–H bond of –NH2 from PEI, and the 1550 cm−1 bond was correlated to the carbodiimide. The bands of 1360 cm−1 and 1102 cm−1 were assigned to the C–H bond of –CH3 and C–O–C bound of –(OCH2CH2)nCH3, respectively. Furthermore, the peak of 593 cm−1 in curves (a) and (b) was the characteristic absorption of Fe–O bound. All what is above confirmed that the MPEG-PEI-SPIONs were successfully prepared under UV irradiation.Figure 2
FT-IR spectra of the (a) MPEG-PEI-SPIONs and (b) naked SPIONs.Thermogravimetric analysis (TG) was carried out for the SPIONs, PEI-SPIONs, and MPEG-PEI-SPIONs. The SPIONs, PEI-SPIONs, and MPEG-PEI-SPIONs gave their distinctive TG curves, which provide indications of the amount of PEI and MPEG on the SPIONs. In Figure3(a), the weight loss from 120 to 700°C was about 5% for SPIONs. In Figure 3(b), the weight loss from 120 to 700°C was about 36%. Considering the weight loss of SPIONs, the weight of the PEI fraction in PEI-SPIONs was calculated to account for 31% of the total weight. Figure 3(c) shows the TG curve of MPEG-PEI-SPIONs. The weight loss of the MPEG-PEI-SPIONs from 120 to 700°C was about 48%. Considering the weight loss of SPIONs and PEI-SPIONs, the weight of the MPEG fraction in MPEG-PEI-SPIONs was calculated to account for 12% of the total weight.Figure 3
The TG curve of the (a) naked SPIONs, (b) PEI-SPIONs, and (c) MPEG-PEI-SPIONs.


## 3.2. Structure, Size, and Magnetic Properties of MPEG-PEI-SPIONs

Morphology and structure of the MPEG-PEI-SPIONs were studied by TEM. As shown in Figure4, the MPEG-PEI-SPIONs were of nearly spherical shape. The average size in aqueous system measured by PCS was 34 nm with a polydispersion index of 0.276 (Figure 5). As the diameter is smaller than 50 nm, the MPEG-PEI-SPIONs can avoid the phagocytosis of reticuloendothelial system (RES) and are designed to have a much longer blood half-life and could rapidly circulate almost the whole body. Thus, the MPEG-PEI-SPIONs can penetrate deep tissue spaces (such as the interior of atherosclerotic plaque and the myocardium), and the hydrophilic surface may prolong the circulation in blood [24, 25].Figure 4
The TEM image of MPEG-PEI-SPIONs.Figure 5
Particle size of the MPEG-PEI-SPIONs measured by PCS.The magnetic properties of the naked SPIONs, PEI-SPIONs, and MPEG-PEI-SPIONs were studied by vibration sample magnetometer (VSM). The saturation magnetization (Ms) value of naked SPIONs was 68 emu/g. After PEI coating, the Ms decreased to 55 emu/g due to the decrease in SPIONs content of the nanoparticles. After MPEG modification, the Ms decreased to 46 emu/g. This level of Ms is deemed sufficient for bioapplications where Ms of 7–22 emu/g is usually adopted [26, 27]. No hysteresis was observed in Figure 6, indicating the nanoparticles are superparamagnetic at room temperature. Thus, the MPEG-PEI-SPIONs possess superparamagnetism which are suitable for MR contrast enhancement by alterations of proton relaxation in the tissue microenvironment [28].Figure 6
Magnetization curve of the (a) naked SPIONs, (b) PEI-SPIONs, and (c) MPEG-PEI-SPIONs.


## 3.3. Stability

High stability is an important parameter, which determined whether MPEG-PEI-SPIONs can be applied in tissue imaging. The magnetic nanoparticles dissolved in PBS (pH 7.0) were stored at 4°C and the supernatant was sampled at different intervals to investigate the stability using ultraviolet-visible spectrophotometry (UV-1800, Shimadzu). The optical density (OD) was detected at 660 nm [29], at which the OD value was linearly related to the concentrations of magnetic nanoparticles. Stability was calculated according to
(1)Stability%=ODsODf×100%,
where ODs was OD value of nanoparticles stored for certain time and ODf was OD value of the freshly prepared sample. The result was shown in Figure 7. The PEI coating could improve the stability of the SPIONs in the observed 178 hours. After MPEG modification, the MPEG-PEI-SPIONs have a higher stability in PBS (pH 7.0), as they could retain 88.7% in aqueous suspension over a week. The high stability is due to the electrostatic repulsive force and steric hindrance of the PEI polymers against aggregation, and the high hydrophilicity of MPEG may also contribute to the stability.Figure 7
Stability of the (a) MPEI-PEI-SPIONs, (b) PEI-SPIONs, and (c) naked SPIONsin vitro (retention ratio versus stored time).


## 3.4. MR Imaging

To investigate theT2 relaxation properties of SPIONs, PEI-SPIONs, and MPEG-PEI-SPIONs,T2 relaxation measurements had been made using 0.5T MR imager. Figure 8 shows theT2 relaxation curves for the three samples. The naked SPIONs showed fasterT2 decay curve compared to PEI-SPIONs and MPEG-PEI-SPIONs. The MPEG-PEI-SPIONs had a similarT2 decay curve. Thus, the MPEG-PEI-SPIONs nanoparticles show similar R2(=1/T2) value with the PEI-SPIONs. This result indicates that the MPEG modification does not decrease theT2 relaxation properties compared with PEI-SPIONs.Figure 8
TheT2 relaxation curves for (a) MPEI-PEI-SPIONs, (b) PEI-SPIONs, and (c) naked SPIONs.Finally, we performed MRI experiments on a phantom composed of four sample tubes containing MPEG-PEI-SPIONs in water at different concentrations (spin-echo technique with TR = 3000 ms and TE = 100 ms). It can be seen from Figure9 that the MR signal intensity (related to theT2 relaxation time inT2-weighted image) for the samples of different concentrations is not identical. With increasing MPEG-PEI-SPIONs concentration in water, the MR signal was enhanced significantly (negative in brightness inT2-weighted image). This result indicates that the nanoparticles can generate high magnetic field gradients near the surface of the MPEG-PEI-SPIONs.Figure 9
T2-weighted MRI images of MPEG-PEI-SPIONs with different concentrations. (a) 0.063, (b) 0.125, (c) 0.250, and (d) 0.500 mg/mL.


## 4. Conclusions

In summary, PEI coated SPIONs were successfully synthesized via photochemistry method, and the MPEG-PEI-SPIONs were prepared with surface MPEG modification of PEI-SPIONs. The analyses of TEM and PCS indicated that the MPEG-PEI-SPIONs were nearly spherical in shape, and the mean diameter in aqueous solution was 34 nm. These novel nanoparticles are superparamagnetic with Ms of 46 emu/g and possess good stability in water. The surface modification by MPEG-PEI polymers could greatly enhance SPIONs’ stability. The MPEG-PEI-SPIONs showed similarR2 value with the PEI-SPIONs and could generate high magnetic field gradients near the surface of the MPEG-PEI-SPIONs. High stability, biocompatibility, and higherR2 value promised the MPEG-PEI-SPIONs would be an ideal candidate for potential MRI contrast agents. Further MRI tests on specific models of different pathologies are in progress.



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*Source: 417389-2015-02-15.xml*