用于肿瘤靶向性MRI对比剂双亲性超顺磁复合物的制备

doi:10.3969/j.issn.2095-4344.2014.30.011

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (30): 4823-4830.doi: 10.3969/j.issn.2095-4344.2014.30.011

• 药物控释材料 drug delivery materials • 上一篇下一篇

用于肿瘤靶向性MRI对比剂双亲性超顺磁复合物的制备

顾隽珩¹，张庆云²，张伟³，杨新林³

1天津市胸科医院影像科，天津市 300222
2武警后勤学院基础部化学教研室，天津市 300309
3南开大学化学学院高分子所教育部功能高分子材料重点实验室，天津市 300071

修回日期:2014-06-24 出版日期:2014-07-16 发布日期:2014-08-08
通讯作者: 杨新林，博士，副教授，南开大学化学学院高分子所教育部功能高分子材料重点实验室，天津市 300071
作者简介:顾隽珩，男，1965年生，浙江省绍兴市人，汉族，1989年天津医科大学毕业，副主任医师，主要从事影像学诊断和对比剂的应用研究。
基金资助:
国家自然科学基金(21374049)；武警后勤学院创新团队基金(WHTD201307-2)

Preparation of amphiphilic superparamagnetic composite particles with tumor targeted MRI contrast agent

Gu Jun-heng¹, Zhang Qing-yun², Zhang Wei³, Yang Xin-lin³

1 Department of Radiology, Tianjin Chest Hospital, Tianjin 300222, China
2 Department of Chemistry, Basic Section, Logistics University of Chinese People’s Armed Police Force, Tianjin 300309, China
3 Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, China

Revised:2014-06-24 Online:2014-07-16 Published:2014-08-08
Contact: Yang Xin-lin, M.D., Associate professor, Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, China
About author:Gun Jun-heng, Associate chief physician, Department of Radiology, Tianjin Chest Hospital, Tianjin 300222, China
Supported by:
the National Natural Science Foundation of China, No. 21374049; Innovation Team Fund of the Logistics University of Chinese People’s Armed Police Force, No. WHTD201307-2

摘要/Abstract

摘要：

背景：超顺磁四氧化三铁纳米粒子(Fe₃O₄ NPs)被广泛应用于MRI成像，为防止其聚集和实现高精度肿瘤诊断，制备高度稳定性、生物相容性和肿瘤靶向性的超顺磁MRI对比剂至关重要。
目的：合成具有基于叶酸受体靶向的肿瘤靶向性双亲性超顺磁复合粒子。
方法：首先通过化学共沉淀法制备出Fe₃O₄ NPs，再用N，N’-二环己基碳二亚胺作脱水剂，通过酯键将双亲性高分子Pluronic-F127(PF127)与叶酸(FA)分子连接，从而形成PF127-FA偶联物，最后用PF127-FA包裹Fe3O4纳米粒子，形成稳定的具有肿瘤靶向功能的双亲性超顺磁复合粒子。分别采用透射电镜、傅里叶红外光谱、紫外可见吸收光谱、热重分析、振动样品磁强计和T2加权成像对其进行表征，通过细胞毒性实验初步表征其细胞毒性。
结果与结论：通过酯化反应制备了Pluronic-F127与FA偶联物，再用其包裹Fe₃O₄纳米粒子，成功制备出具有良好水溶性和生物相容性的超顺磁性复合粒子。该PF127-FA-Fe₃O₄复合粒子透射电镜观察到该复合粒子大部分粒径小于200 nm，Fe₃O₄核心大小为10-20 nm，傅里叶红外光谱和紫外可见吸收光谱结果证明了叶酸被成功修饰到超顺磁性复合粒子表面。热重分析结果表明PF127-FA占PF127- FA-Fe₃O₄复合粒子总量的27.2 wt%。磁性检测结果表明该复合粒子饱和磁强度Ms为47.35 emu/g，核磁共振仪成像测得其弛豫率为0.025×10⁶ mol/s。细胞毒性实验表明显示了可以忽略的毒性。因此，实验成功制备了可用于肿瘤靶向性MRI对比剂的双亲性超顺磁复合物，实验所制备的PF127-FA-Fe₃O₄复合粒子有望用于肿瘤靶向性MRI对比剂。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

关键词: 生物材料, 纳米材料, 双亲性材料, 肿瘤靶向性, 超顺磁性, Fe3O4纳米粒子, MRI对比剂, Pluronic, 叶酸, 国家自然科学基金

Abstract:

BACKGROUND: Superparamagnetic iron oxide nanoparticles (Fe₃O₄ NPs) have been widely used in MRI. It is vital to prepare the superparamagnetic MRI contrast agent with high stability, biocompatibility and tumor targeting in order to prevent the aggregation of Fe3O4 NPs and realize the high-precision diagnose of tumor.
OBJECTIVE: To prepare the amphiphilic superparamagnetic composite particles with tumor targeting mediated by folate receptor.
METHODS: The stable amphiphilic superparamagnetic composite particles with tumor targeting function were prepared by coating the Fe₃O₄ NPs with a Pluronic F127-folic acid conjugate, which was synthesized via an esterification reaction between the carboxyl group of the tumor targeting molecule, folic acid and the hydroxyl group of an amphiphilic triblock copolymer, Pluronic F127. The resultant Pluronic F127-folic acid-Fe₃O₄ composite particles were characterized by transmission electron microscopy, Fourier transform infrared-spectra, UV-vis absorption spectra, thermal gravimetric analysis, vibrating sample magnetometer and T2-weighted imaging. WST assay was used to characterize their cytotoxicity preliminarily.
RESULTS AND CONCLUSION: The Pluronic F127-folic acid conjugates were prepared via esterification reaction. Then Fe₃O₄ NPs were wrapped with Pluronic F127-folic acid to result in the superparamagnetic composite particles with well dispersion and biocompatibility. The size of most superparamagnetic composite particles was less than 200 nm and the size of Fe3O4 core was 10-20 nm from the observation of transmission electron microscopy. The results from the Fourier transform infrared-spectra and UV-vis absorption spectroscop confirmed that folic acid molecules were modified on the surface of the superparamagnetic composite particles successfully. The mass ratio of Pluronic F127-folic acid conjugate was determined by thermal gravimetric analysis as 27.2 wt% in the resultant Pluronic F127-folic acid-Fe3O4 composite particles. The saturated magnetic intensity of the superparamagnetic composite particles was 47.35 emu/g by vibrating sample magnetometer and the relaxation rate was 0.025×106 mol/s from MRI. The WST assay showed the negligible cell cytotoxicity of Pluronic F127-folic acid-Fe₃O₄. The superparamagnetic composite particles have potential application as the MRI contrast agents with tumor targeting, and the Pluronic F127-folic acid-Fe₃O₄ composite particles is expected to be used as a MRI contrast agent for tumor targeting.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: tumor, molecular targeted therapy, metal nanoparticles, folic acid

中图分类号:

R318

顾隽珩，张庆云，张伟，杨新林. 用于肿瘤靶向性MRI对比剂双亲性超顺磁复合物的制备[J]. 中国组织工程研究, 2014, 18(30): 4823-4830.

Gu Jun-heng, Zhang Qing-yun, Zhang Wei, Yang Xin-lin. Preparation of amphiphilic superparamagnetic composite particles with tumor targeted MRI contrast agent[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(30): 4823-4830.

图/表（结果） 2

Preparation of PF127-FA-Fe₃O₄ composite particles and characterization of their structures

Since the magnetic nanoparticles without surface modification are prone to aggregate in the solvent due to their high specific surface area. Coating of magnetic nanoparticles with a polymer layer may prevent such aggregation, which also improves the chemical stability of the composite nanoparticles under rigid condition. In this work, magnetic Fe₃O₄ NPs were first prepared by the chemical co-precipitation of Fe³⁺/Fe²⁺ (2/1 in molar ratio) salts in an NH₄OH solution at 60 ℃ via a well-known sol-gel process. Then PF127 was conjugated with FA by ester bonding between the hydroxyl groups of PF127 and the carboxylic acid groups of FA to give PF127-FA. Finally, superparamagnetic Fe₃O₄ NPs were coated with PF127-FA spontaneously to endow a possible tumor targeting and prevent the aggregation, as shown in Figure 1.

The morphologies of superparamagnetic Fe₃O₄ NPs before and after coating with PF127-FA were characterized with transmission electron microscopy (Figure 2), which indicated that the size of Fe₃O₄ NPs was 10-20 nm (Figure 2A) and most of PF127-FA-Fe₃O₄ composite particles had the slightly aggregated size of below 200 nm (Figure 2B). Further, Fe₃O₄ NPs aggregation coated with a thin film of PF127-FA conjugates is clearly observed in Figure 2C with a higher magnification.

PF127-FA conjugate was synthesized via using the excessive molar ratios of FA to PF127 to ensure that at least one or two hydroxyl groups of PF127 were conjugated with the carboxylic acid groups of FA. The successful syntheses of PF127-FA conjugate and its coated superparamagnetic composite particles (PF127-FA-Fe₃O₄) were confirmed by Fourier transform infrared-spectra and Ultra-visible-near IR spectrophotometer in Figure 3.

Fourier transform infrared spectrum of the PF127-FA conjugate in Figure 3A indicated the presence of the characteristics peaks at 1 691, 1 648 and 1 605/cm corresponding to the stretching vibrations from FA component together with the peaks at 2 929, 2 866 and

1 105/cm assigning to the stretching vibrations of CH₂-bonds from PF127 chains. The Fourier transform infrared spectrum of PF127-FA-Fe₃O₄ composite particles in Figure 3A had a new peak at 577 /cm attributing to the stretching vibration of Fe-O bond in Fe₃O₄ core together with all the characteristic peaks from PF127-FA conjugate. The UV-vis spectrum of PF127-FA in Figure 3B had a peak at 280 nm attributing to the characteristic absorbance of FA component, which was also clearly observed in the UV-vis spectrum of PF127-FA-Fe₃O₄ composite particles. The mass ratio of PF127-FA conjugate was determined by thermal gravimetric analysis as 27.2 wt% in the resultant PF127-FA-Fe₃O₄ composite particles. All these results indicated that PF127-FA conjugate was successfully coated onto the Fe₃O₄ NPs.

The properties of PF127-FA-Fe3O4 composite particles were characterized

The presence of magnetite on PF127-FA-Fe₃O₄ was proven by a superconducting quantum interference device magnetometer. The magnetization curves of Fe₃O₄ and PF127-FA-Fe₃O₄ were measured at room temperature as shown in Figure 3A. The magnetic hysteresis loops were S-like curves, while the saturation magnetization of PF127-FA-Fe₃O₄ was 47.35 emu/g with near zero of magnetic remanence. This value was smaller than that of bulk Fe₃O₄ (53.9 emu/g), which was due to the coating of PF127-FA on Fe₃O₄. The results indicated that there was almost no remaining magnetization when the external magnetic field was removed, suggesting that PF127-FA-Fe₃O₄ composite particles exhibited a superparamagnetic behavior. These superparamagnetic PF127-FA-Fe₃O₄ composite particles could be dispersed stably as shown in the inserted photograph in Figure 4A.

To check the MRI efficiency of PF127-FA-Fe₃O₄ as a tumor targeting contrast agent, T2-weighted signal intensities were measured with a clinical 3.0 T magnetic resonance scanner using iron concentrations ranging from 0 to 0.14 mmol/L at room temperature. Relaxivity values are calculated through the curve fitting of 1/T2 relaxation time (/ms) versus the iron concentrations (mmol/L). The iron content of PF127-FA-Fe₃O₄was estimated as 32 wt% by atomic absorption spectrometry. The T2 relaxivity was increased dramatically with the increasing of the iron concentrations in PF127-FA-Fe₃O₄. The linear function equation of Y=0.124 667 ×10^-⁵ + 0.02494 X (R=0.9974, n=6) was obtained with iron concentration as x-axis and 1/T2 as y-axis. The transverse r2 relaxivities was obtained as 0.025×10⁶/mol/s from the slope of linear. Figure 4B showed the T2-weighted MRI images of PF127-FA-Fe₃O₄ with the various iron concentrations. PF127-FA-Fe₃O₄ had a significant change of light intensity with the changes of iron concentration. These superparamagnetic composite particles with FA conjugating may find a potential application for special detection of cancers.

The cytotoxicity of PF127-FA-Fe₃O₄ composite particles was valued by WST assay

As a MRI contrast agent, the potential toxicity PF127-FA-Fe₃O₄ composite particles should be concerned for its further application in biomedical fields. In order to examine the cytotoxicity of PF127-FA-Fe₃O₄ composite particles, Hela cells were incubated 24 hours with PF127-FA-Fe₃O₄ composite particles in the concentration

range from 0.1 to 200 mg/L. The result of WST-1 assay indicated that the PF127-FA-Fe₃O₄ composite particles showed no obvious toxicity at 200 mg/L to Hela cells for the cytotoxicity evaluation as shown in Figure 5. This confirmed their low levels of cytotoxicity.

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引言

Superparamagnetic Fe₃O₄ nanoparticles (Fe₃O₄ NPs) are emerging as a promising candidate for various biomedical applications, such as MRI^[1], targeted drug delivery^[2], hyperthermia treatment^[3], the labeling and sorting of cells^[4], and the separation of biochemical products^[5]. For these applications, it is important for the superparamagnetic nanoparticles to be biocompatible, water soluble, and physically and chemically stable in a physiological environment. In general, nanoparticles tend to flocculate due to van der Waals forces, but magnetic nanoparticles can in addition be magnetically attracted between each other and agglomerated. Therefore, it is necessary to modify the superparamagnetic nanoparticles to improve their biocompatibility, solubility and stability in physiological environments for various biomedical applications. Coating materials for biological applications include organic molecules (e.g. citrate)^[6], polymers (e.g. dextran^[7-8], poly(ethylene glycol)^[9], starch^[10], proteins

(human serum albumin^[11], protein corona^[12-13]) and inorganic molecules and shells (e.g. silica^[14], Au nanoparticles^[15-16]). The research interest of surface functionality of Fe₃O₄ NPs with polymeric shell owes to the flexibility in the control of the chemical structure, composition, and function of the polymers. Poly(ethylene glycol) is the most widely used synthetic biocompatible polymer for coating of magnetic nanoparticles to extend their circulation time, improve their hydrophilicity and biocompatibility and reduce the non-specific uptake by macrophage cells^{[9, 17]}. Moreover, magnetic nanoparticles covered with poly(ethylene glycol) are considered to be nonimmunogenic, nonantigenic, and protein resistant. Therefore, poly(ethylene glycol) has been one of the most popular polymers used for magnetic nanoparticles coating.

Magnetic nanoparticles composed of a magnetic core and a biocompatible polymeric shell can offer a potential method to get a targeted MRI reagent due to the EPR effect and the tumor targeting molecules conjugated with the polymeric shell. The methods of modification with a polymeric or silica shell over a magnetic core usually include in situ polymerization^[18-19], grafting of polymer^[20] or layer-by-layer self-assembly method^[21], etc. However, all these techniques not only involve the complicated chemical reactions but also decrease the magnetization of magnetic cores. Interestingly, it is a challenge to develop a simple method for getting a biocompatible polymeric shell via chemical modification of magnetic cores and keeping the magnetization of the resultant polymer/magnetite hybrid particles. Pluronic F127 (PF127), a block copolymer of poly(ethylene glycol) and poly(propylene glycol) with triblock structures of poly(ethylene glycol-b-propylene glycol-b-ethylene glycol), is highly possible for such a requirement for its amphiphilic block components. It has been widely used in pharmaceutics^[22], biomedical^[23-24] and personal care products due to its favorable toxicity profile^[25] and its action as ‘‘solubilizer’’. It can form ordered aggregates, most commonly as micelles in aqueous solutions, which show a good biocompatible character and prolong the circulation time in vivo due to its poly(ethylene glycol) shell. As a result, it behaves high biocompatiblity through effectively preventing aggregation, the adsorption of proteins and recognition by the reticulo-endothelial system in vivo. Further, it becomes possible and facile to coat the magnetic core nanoparticles with PF127 based on its unique self-assembly behaviors.

The development of tumor-targeted nanoparticles is essential for early detection of cancers. It is highly desirable that magnetic nanoparticles can provide sensitive and specific imaging information in cancer site. The magnetic nanoparticles conjugated with tumor-targeting ligands such as monoclonal antibodies^[26-27], peptides^[28-30], or small molecules, such as folic acid^[31-34], can be engineered to provide a MRI reagent for tumor selected imaging.

In order to prepare the superparamagnetic MRI contrast agent with high stability, biocompatibility and to realize the high-precision diagnose of tumors, a facile method was developed to synthesize a tumor targeting and stable magnetic nanoparticles in this work. PF127 is conjugated with folic acid (FA) at first since the folate receptors (FRs) is overexpressed in many malignant tissues and it can bind and internalize folic acid conjugates. Then the PF127-FA conjugate is used to coat superparamagnetic Fe₃O₄NPS via an efficient spontaneous interaction.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
全文链接：

材料方法

Design

The design of material and its structures and properties characterization.

Time and setting

The work was completed in the Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Nankai University, from February 2012 to December 2012.

Materials

Methods

Superparamagnetic Fe₃O₄ NPs were prepared by chemical co-precipitation method

Fe₃O₄ NPs were prepared by chemical co-precipitation of Fe³⁺ and Fe²⁺ under a basic condition. FeCl₃•6H₂O (270.0 mg, 1 mmol) and FeCl₂•4H₂O (99.5 mg, 0.5 mmol) were dissolved in 50 mL of deionized water with a mechanical stirring under argon for 30 minutes, then 2 mL (8 mol/L) of ammonium hydroxide was added. The reaction solution was then heated at 60 ℃ and stirred for 2 hours.

The resultant magnetite nanoparticles were washed with deionized water with the aid of a magnet. Deionized water was then added to disperse the resultant Fe₃O₄ NPs. The solid content of the magnetic fluid was adjusted to 4 mg/mL for further preparation.

The conjugates of PF127 and FA were prepared via an esterification reaction

88 mg (0.20 mmol) of FA was dissolved in 5 mL of dried dimethyl sulfoxide, then 45.3 mg (0.22 mmol) of 1,3-dicyclohexylcarbodiimide with catalytic amount of 4-dimethylaminopyridine was added. Finally 620 mg (0.05 mmol) of PF127 was added to the above solution. The reaction was allowed to proceed for 48 hours at room temperature. After reaction, the solution was condensed by a rotary evaporating. The condensed solution was precipitated in acetone and the precipitation was dissolved in water. The obtained solution was centrifuged at 5 000 r/min and the supernatant was placed into the dialysis chamber, which was dialyzed in PBS with pH 7.4 in order to remove the free FA since it can be dissolved under this condition. The yellow solution in dialysis chamber was lyophilization to afford PF127-FA conjugate.

Superparamagnetic composite particles PF127-FA-Fe₃O₄ with tumor targeting were prepared The above 5 mL of magnetic fluid was diluted into 195 mL of deionized water and stirred with a mechanical stirring under argon. Then PF127-FA (35 mg) dissolved in 5 mL of deionized water was dropped into the magnetic fluid and stirred for 6 hours continuously. Finally, the resulting product with 50% yield was washed with deionized water by magnetic separation and dried in a vacuum oven.

The structures and properties of PF127-FA-Fe₃O₄ composite particles were characterized

Transmission electron microscopy (FEI, TECNAI-20) was used to characterize the size and morphology of the samples. Fourier transform infrared spectra were obtained using a Bruker Tensor 27 spectrometer. UV spectra were determined on an Ultra-visible-near IR spectrophotometer (JASCO, V-570).

The composition of PF127-FA-Fe₃O₄ was determined with thermal gravimetric analysis using a Netzsch TG209 with a heating rate of 10 ℃/min from room temperature to 800 ℃ under N₂. The magnetization curves of Fe₃O₄ and PF127-FA-Fe₃O₄ were measured as a function of the applied magnetic field H with a 9 600 VSM (LDJ Co.) superconducting quantum interference device magnetometer. The hysteresis of the magnetization was obtained by changing H between +6 000 and -6 000 Oe at 300 K. The iron content of PF127-FA-Fe₃O₄ was determined with atomic absorption spectrophotometer analysis (HITACHI, 180-80). T2-weighted signal intensities were measured with a clinical 3.0 T magnetic resonance scanner (Siemens, TrioTim) using iron concentrations ranging from 0 to 0.168 mmol/L at room temperature. The T2-weighted images were acquired using a fast gradient echo pulse sequence (repetition time/echo time/flip angle: 1 000/41.4/5.5). Relaxivity values were calculated through the curve fitting of 1/T2 relaxation time (/ms) versus the iron concentrations (mmol/L).

The cytotoxicity of PF127-FA-Fe₃O₄ composite particles against Hela cells was determined by a standard WST-1 assay using the WST-1 cell proliferation and cytotoxicity assay kit. In brief, Hela cells were seeded in 96-well plates at a density of 5 000 cells/well and cultivated with Dulbecco’s modified Eagle’s medium plus 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin (200 μL) and incubated at 37 ℃ for 24 hours. The wells were then replaced in a culture medium (200 μL) containing PF127-FA-Fe₃O₄ composite particles with the concentration of 0.1, 1, 10, 50, 100, 200, 500 μg/mL. After the incubation for 48 hours, the medium in each well was washed with PBS for five times and replaced with fresh Dulbecco’s modified Eagle’s medium without fetal bovine serum (100 μL) and WST-1 solution (10 μL). The plate was incubated for a further 1 hour at 37 ℃, which allowed viable cells to reduce WST-1 into the orange formazan crystal.

The plate was read at 450 nm on a Bio-Rad microplate reader. Data are presented as mean±SD, n=3. The relative cellular viabilities were obtained according to the equation (1).

Relative cellular viability = 1-A/A₀ (1)

Where, A is the absorbance value of experimental groups under different concentration of PF127-FA-Fe₃O₄ composite particles, and A₀ is the absorbance value of control groups.

Main outcome measures

Relative cellular viability of Hela cells after treatment with PF127-FA-Fe₃O₄ composite particles with different concentrations.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

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讨论

Fe₃O₄ NPs can be prepared by chemical co-precipitation. The reaction conditions, including the ratio of Fe³⁺ and Fe²⁺, the concentration of reaction solution and the reaction temperature, will have effects on their size and morphology. Therefore, it should be optimized to obtain the Fe₃O₄ NPs with advisable size.

Superparamagnetic Fe₃O₄ NPs are unstable when they are placed in the process of in vitro due to van der Waals forces and magnetic dipole-dipole interaction between each others. When they enter in vivo, they will easily absorb numerous biomacromolecules such as protein. This results in their enlarged size and they will be phagocytized by the reticuloendothelial system which shortens their half-life.

To improve their stability in solutions and biocompatibility and target the tumor site actively, in this study, amphiphilic block polymer PF127 with high biocompatibility was conjugated with FA molecules which can target most of the tumor cells via an esterification reaction. The PF127-FA conjugates were coated on the surface of Fe₃O₄ NPs and the superparamagnetic PF127-FA-Fe₃O₄ composite particles with well dispersion in solution were obtained.

The surface morphology and size of the resultant products were characterized by transmission electron microscopy. The size of Fe₃O₄ NPs are about 10-20 nm and most of PF127-FA-Fe₃O₄ composite particles are below 200 nanometers from the result of transmission electron microscopy. The structure of PF127-FA-Fe₃O₄ composite particles was confirmed by Fourier transform infrared-spectra and UV-vis absorption spectroscopy. The mass ratio of PF127-FA conjugate in the resultant PF127-FA-Fe₃O₄ composite particles was further determined as 27.2 wt% by thermal gravimetric analysis.

An S-like magnetic hysteresis loop in the magnetization curve of the resultant PF127-FA-Fe₃O₄ composite particles suggested that they were superparamagnetics. Their saturation magnetization was 47.35 emu/g, which met the demand them as the MRI contrast agent. The result of their T2-weighted MRI indicated that the T2 relaxivity was increased dramatically with the increasing of the iron concentrations in PF127-FA-Fe₃O₄. The transverse r2 relaxivities was obtained as 0.025×10⁶/mol/s from the linear relation of the change of T2-weighted light intensity with the various iron concentrations.The result shows that these superparamagnetic composite particles with FA conjugating may be used in the special detection of cancer potentially. The cytotoxicity PF127-FA-Fe₃O₄ composite particles were further valued by WST-1 assay preliminarily. The result showed they had low cytotoxicity. This indicates that it is possible to use them as an MRI contrast reagent mediated by folate receptor.

Superparamagnetic Fe₃O₄ nanopartiles with tumor targeting function were prepared by coating the Fe₃O₄ NPs with a PF127-FA conjugate via an esterification reaction between the hydroxyl group of amphiphilic triblock copolymer PF127 copolymer and the carboxyl group of a tumor targeting FA molecule. The size of Fe₃O₄ NPs was 10-20 nm and most of PF127-FA-Fe₃O₄ composite particles had the size of below 200 nm. Fourier transform infrared-spectra and UV-vis spectra were used to confirm the formation of PF127-FA-Fe₃O₄.

The saturation magnetization of the tumor targeting superparamagnetic PF127-FA-Fe₃O₄ composite particles was 47.35 emu/g with near zero magnetic remanence. The T2 relaxivity value of PF127-FA-Fe₃O₄ was 0.025×10⁶/mol/s, which illustrated the potential application for a MRI contrast agent that specifically targets folate receptor overexpressing tumor cells.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
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用于肿瘤靶向性MRI对比剂双亲性超顺磁复合物的制备

Preparation of amphiphilic superparamagnetic composite particles with tumor targeted MRI contrast agent

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