Antifouling Manganese Oxide Nanoparticles: Synthesis

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Letter

Antifouling manganese oxide nanoparticles: Synthesis, characterization, and applications for enhanced MR imaging of tumors Peng Wang, Jia Yang, Benqing Zhou, Yong Hu, Lingxi Xing, Fanli Xu, Mingwu Shen, Guixiang Zhang, and Xiangyang Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13844 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 24, 2016

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ACS Applied Materials & Interfaces

Antifouling Manganese Oxide Nanoparticles: Synthesis, Characterization, and Applications for Enhanced MR Imaging of Tumors

Peng Wang,a, 1 Jia Yang,b, 1 Benqing Zhou,a Yong Hu,a Lingxi Xing,b Fanli Xu,a Mingwu Shen,a Guixiang Zhang,*b Xiangyang Shi*a

a

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of

Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China b

Department of Radiology, Department of Ultrasound, Shanghai General Hospital, School of

Medicine, Shanghai Jiaotong University, Shanghai 200080, People’s Republic of China

___________________________________________________________________________________________

* To whom correspondence should be addressed. E-mail: [email protected] (G. Zhang), [email protected] (X. Shi) 1

Authors contributed equally to this work.

1

ACS Paragon Plus Environment


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Abstract Antifouling manganese oxide nanoparticles were synthesized by solvothermal decomposition of tris-(2,4-pentanedionato) manganese(III) in the presence of trisodium citrate, followed by surface modification with polyethylene glycol and L-cysteine. The as-prepared nanoparticles have a uniform size distribution, good colloidal stability and good cytocompatibility. The modification of L-cysteine rendered the particles with much longer blood circulation time (half-decay time of 28.4 h) than those without L-cysteine modification (18.5 h), and decreased macrophage cellular uptake. Thanks to desirable antifouling property and relatively high r1 relaxivity (3.66 mM-1s-1), the L-cysteine-modified Mn3O4 NPs can be used for enhanced tumor magnetic resonance imaging applications.

Keywords: Antifouling; manganese oxide nanoparticles; L-cysteine; blood circulation time; tumor magnetic resonance imaging

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Molecular imaging technologies such as magnetic resonance (MR) imaging, computed tomography (CT), single-photon emission computed tomography (SPECT), positron emission tomography (PET), ultrasound imaging and optical imaging are becoming routine diagnosis techniques in hospitals.1-2 Among them, MR imaging is a powerful noninvasive medical diagnosis tool that can provides images with superb anatomical details based on the soft tissue contrast and the real-time monitoring style. In order to improve the sensitivity of MR imaging, contrast agents (CAs) are usually required. Paramagnetic and superparamagnetic nanomaterials have been shown to be used as promising CAs, and can work at low concentrations to generate fine images by conjugation with affinity molecules.3 Currently, the most commonly used MR CAs include paramagnetic Gd(III)- and Mn(II)-based chelates for positive T1-weighted MR imaging, and superparamagnetic iron oxide nanoparticles (NPs) for negative T2-weighted MR imaging.4-5 In general, the use of iron oxide NPs for clinical T2-weighted MR imaging applications has been hampered due to the intrinsic dark MR signals and magnetic susceptibility artifacts.6-7 Thus, T1-weighted MR CAs are desirable due to their predominant positive signal-enhancing ability to generate high spatial resolution. The clinically used MR CAs of chelator/Gd(III) complexes, such as Magnevist (Gd-DTPA) and Dotarem (Gd-DTOA) have shown to induce nephrogenic systemic fibrosis in patients with poor renal function.8 Manganese ions, like calcium ions, is a natural cellular constituent and a cofactor for enzymes and receptors, meaning that manganese-based agents have a low side effect.9 Hence, different Mn-based NPs such as MnO,4 Mn3O4,11 MnO2,10 or MnFe2O411 NPs have been investigated as CAs for favorable T1-weighted MR imaging, although in some cases, Mn3O4 NPs can also give T2-weighted contrast enhancement at higher concentrations.12 For example, polydopamine-coated Mn3O4 3



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NPs functionalized with polyethylene glycol (PEG) and folic acid (FA) can be loaded with doxorubicin for synergetic chemo-/photothermal therapy and MR imaging.13 SiO2-coated Mn3O4 NPs functionalized with rhodamine B isothiocyanate and FA can be used for targeted MR and fluorescence imaging of cancer cells.14 Hollow Mn3O4 NPs can be used for MR imaging of brain and kidneys as well as for intracellular drug delivery.15 In our previous work, Mn3O4 NPs with a uniform size distribution and desirable water-dispersibility were produced by a thermal decomposition method in the presence of polyethylenimine (PEI). The PEI-stabilized Mn3O4 NPs were conjugated with folic acid-linked polyethylene glycol (PEG) and used for targeted T1-weighted tumor MR imaging.16 In general, particles without desirable

antifouling

property

can

be

easily

uptaken

by

macrophages

in

the

reticuloendothelial systems (RES), thus having short blood circulation time and limited tumor accumulation, which is not desired for sensitive tumor imaging and diagnosis.17 NPs modified with antifouling property have been widely used in different biomedical applications, due largely to the fact that the antifouling property endows the NPs with prolonged blood circulation time and decreased uptake by macrophages. PEG as a common antifouling reagent has been extensively used in biomedical applications.18 Nevertheless, there is increasing evidence showing that PEGylated NPs can elicit antibody formation against PEG, which may limit their applications in vivo.19 Recently, literature reports show that zwitterionic molecules are a class of ideal antifouling material, and can be modified onto the surface of NPs to render the NPs with the desired antifouling property.20 The special structure of zwitterionic molecules consists of an equal molar ratio of positive and negative charges in close proximity, resulting in a thick hydrated shell onto the particle surface. Among various zwitterionic materials, cysteine (Cys) is of particular interest because of its 4


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ultra-low fouling property for resisting protein adsorption.21 For example, Cys has been successfully modified onto the surfaces of silica NPs,22 gold NPs,23 poly(2-oxazoline)s,24 polyethylene terephthalate films,25 and polyamide thin film,26 etc. to render the materials with desirable antifouling property. The prior work related to zwitterionic Cys surface modification and the use of Mn3O4 NPs for T1-weighted MR imaging application inspire us to develop antifouling Mn3O4 NPs for enhanced tumor MR imaging applications. Here, Mn3O4 NPs were prepared using a solvothermal method in the presence of sodium citrate (Cit). The Cit-stabilized Mn3O4 NPs were then modified with dual functional PEG with one end of maleimide group and the other end of amine group (Mal-PEG-NH2), followed by modification with Cys via thiol-maleimide coupling (Figure 1). The formed Mn3O4-Cit-PEG-Cys NPs were fully characterized in terms of their structure, composition, size, morphology, r1 relaxivity, and macrophage cellular uptake. The cytotoxicity of the NPs was evaluated by cell viability assay and cell morphology observation. Pharmacokinetic studies were carried out by inductively coupled plasma-optical emission spectroscopy (ICP-OES) to evaluate the half-decay time of the particles. Furthermore, the synthesized Mn3O4-Cit-PEG-Cys NPs were applied for MR imaging of cancer cells in vitro and a xenografted tumor model in vivo. To the best of our knowledge, this is the first example relating to the synthesis of Cys-functionalized Mn3O4 NPs with antifouling property for enhanced tumor MR imaging applications.

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Figure 1. Schematic illustration of the preparation of Mn3O4-Cit-PEG-Cys NPs for tumor MR imaging applications.

Similar to our previous work regarding the creation of PEI-coated Mn3O4 NPs,16 Mn(acac)3 salt was subjected to solvothermal decomposition in the presence of Na3Cit·2H2O to create Cit-coated Mn3O4 (Mn3O4-Cit) NPs. The formed Mn3O4-Cit NPs were subsequently functionalized Cys through a PEG spacer to afford Mn3O4 NPs with antifouling property (Figure 1). In our study, we used L-cysteine instead of D-cysteine to render the Mn3O4 NPs with antifouling property, since L-cysteine has been reported and used to endow the particles with antifouling property.26 XRD was first utilized to determine the crystalline phase of the as-prepared Mn3O4-Cit NPs (Figure S1, Supporting Information). All characteristic peaks in the pattern are in accordance with the standards of bulk Mn3O4 crystals (JCPDS card: 24-0734), and the lattice spacing at 2θ of 29.1, 32.6, 36.4, 44.8, 51.4, 58.5, and 60.3o corroborate the (112), (103), (211), (220), (105), (321), and (224) planes of the magnetic Mn3O4 crystals, in nice agreement with the literature.27 FTIR spectra were also used to characterize the as-prepared Mn3O4-Cit NPs before and after surface modification (Figure 2a). Clearly, the absorption peaks at 3437 cm-1, 862 cm-1 and 624 cm-1 correspond to O-H vibration in Na3Cit. The peaks 6


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at 2923 cm-1, 2853 cm-1 and 1433 cm-1 can be ascribed to C-H stretching vibration in Na3Cit. The peak at 1433 cm-1 correspond to -O-C=O stretching vibration in Na3Cit. The peak at 1075 cm-1 can be assigned to C-O vibration, and the peak at 1634 cm-1 is associated to the C=O vibration in Na3Cit. These characteristic peaks of Mn3O4-Cit NPs are all similar to those of the free Na3Cit in terms of peak positions, although the peaks related to Na3Cit for the Mn3O4-Cit NPs have different intensities and are much smoother than those of free Na3Cit. Furthermore, the characteristic peak of Mn-O vibration at 521 cm-1 can also be clearly observed. The FTIR data suggest the existence of Na3Cit onto the surface of Mn3O4 NPs. After modification of PEG, the Mn-O vibration peak shifted from 521 cm-1 to 469 cm-1 and the intensity of the peak at 1075 cm-1 corresponding to the PEG C-O vibration was obviously enhanced, proving that PEG was successfully modified onto the surface of Mn3O4 NPs. In addition, further conjugation of Cys resulted in a significant intensity increase of a peak at 1433 cm-1, which is associated to the carboxyl groups of Cys. Therefore, the FTIR data indicated that Cys was successfully modified onto the surface of Mn3O4 NPs. The amount of PEG and Cys conjugated onto the surface of Mn3O4 NPs was quantified by thermogravimetric analysis (TGA, Figure 2b). Obviously, the Mn3O4-Cit NPs show a weight loss of 40.91% at 750 oC, which is attributed to the coating of Na3Cit on the particle surface. Subsequent modification with the PEG and Cys afforded the Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs with weight losses of 45.94% and 47.12%, respectively at the same temperature. The grafting percentages of the PEG and Cys were then calculated to be 4.83% and 1.18%, respectively. The successful surface modification of the Mn3O4 NPs was then confirmed by zeta potential measurements (Table S1, Supporting Information). Clearly, the pristine Mn3O4-Cit 7



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NPs display a quite negative surface potential of -25.8 mV, which is due to the presence of Na3Cit. Subsequent modification of PEG and Cys leads to the increase of the surface potential of the NPs (-21.5 mV for the Mn3O4-Cit-PEG NPs, and -16.5 mV for the Mn3O4-Cit-PEG-Cys NPs). Furthermore, zeta potentials of both Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs were measured under different pH conditions (Figure S2, Supporting Information). It seems that with the increase of pH, both particles display decreased surface potentials, likely due to the deprotonation of the particle surface carboxyl groups. We also analyzed the hydrodynamic sizes of the Mn3O4 NPs prior to or after surface modification (Table S1 and Figure S3, Supporting Information). The hydrodynamic sizes of the Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs are 175.9 and 193.5 nm, respectively, which are much bigger than that of the pristine Mn3O4-Cit NPs. This should be attributed to the surface grafting of PEG or PEG/Cys. In addition, both Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs are quite colloidally stable in water, normal saline (NS), and cell culture medium (Figure S4a, Supporting Information) and no precipitation occurred after 30 days’ continuous monitoring. Furthermore, the hydrodynamic sizes of both Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs do not display appreciable changes after 30 days’ storage in water at 4 oC, suggesting their good stability (Figure S4b, Supporting Information). Transmission electron microscopy (TEM) was used to observe the morphology and size of the as-prepared Mn3O4-Cit-PEG-Cys NPs (Figures 2c and 2d). It can be seen that the particles with a mean core size of 2.7 ± 0.5 nm display a spherical or quasi-spherical shape with a fair narrow size distribution. The diffused TEM image of the Mn3O4-Cit-PEG-Cys NPs could be due to the thick polymer coating onto the particle surface. Owing to the diffused image, it is difficult to confirm exactly the specific shape of the Mn3O4 core 8


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particles. Our results suggests that the presence of Na3Cit effectively restricts the size growth of the Mn3O4 NPs, in agreement with the literature.16 Notably, the measured size by TEM is much smaller than that by DLS. This is because TEM is used to measure the size of the single Mn3O4 core particles, while DLS is used to measure the size of aggregated particles dispersed in aqueous solution. The aggregated particles likely consist of many single Mn3O4 NPs, in accordance with the literature data.28

Figure 2. (a) FTIR spectra of Na3Cit, Mn3O4-Cit, Mn3O4-Cit-PEG and Mn3O4-Cit NPs; (b) TGA curves of Mn3O4-Cit, Mn3O4-Cit-PEG, and Mn3O4-Cit-PEG-Cys NPs, respectively. (c) and (d) show that TEM images and size distribution histogram of the Mn3O4-Cit-PEG-Cys NPs, respectively. 9



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In order to investigate the feasibility to use the Mn3O4-Cit-PEG-Cys NPs for MR imaging in vivo, the T1-weighted MR phantom studies were performed. As shown in Figure 3a, the solutions of both Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs display increased MR contrast enhancement with the increase of Mn concentration. By linear fitting the relaxation rate (1/T1) versus Mn concentration, the r1 relaxivity of the Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs were calculated to be 3.47 and 3.66 mM-1s-1, respectively (Figure 3b), which are both comparable to that of DTPA(Gd) chelates and much higher than that reported in our previous work.16 The relatively high r1 relaxivity could be due to the small size and surface coating of the particles. Overall, the T1-weighted MR phantom studies show that the formed PEGylated Mn3O4 NPs with or without Cys could be used as potential T1-weighted MR CAs. We next tested the cytocompatibility of the Mn3O4-Cit-PEG-Cys NPs via CCK-8 cell viability assay of C6 glioma cells. As shown in Figure S5a (Supporting Information), both Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs are non-cytotoxic in the Mn concentration range of 0-400 µM. Even at the highest Mn concentration tested, the cell viability is still up to 76%. The cytocompatibility of the Mn3O4-Cit-PEG-Cys NPs was further confirmed by cell morphology observation (Figure S5b). In comparison with the NS control, C6 glioma cells treated with both Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs at the studied Mn concentrations (25-400 µM) do not show any significant morphological variations, further confirming that PEGylated Mn3O4 NPs with or without Cys have good cytocompatibility. With the known protein resistance ability of the zwitterionic Cys surface, it is expected that the Mn3O4-Cit-PEG-Cys NPs should have reduced macrophage cellular uptake. 10


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ICP-OES analysis of Mn concentration was conducted after the Raw 264.7 cells were incubated with the Mn3O4-Cit-PEG or Mn3O4-Cit-PEG-Cys NPs (Figure S5c, Supporting Information). Clearly, the Mn uptake by Raw 264.7 cells increases with the Mn concentration for both Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs regardless the negative surface potentials for both Mn3O4-Cit-PEG-Cys NPs (-16.5 mV) or Mn3O4-Cit-PEG NPs (-21.5 mV). The cellular uptake is likely through two distinct mechanisms: diffusion via cell walls and phagocytosis.28 However, at the same Mn concentrations ([Mn] = 15 µg/mL or above), the cellular uptake of the Mn3O4-Cit-PEG-Cys NPs is lower than that of the Mn3O4-Cit-PEG NPs. Our results suggest that the surface Cys modification renders the particles with good antifouling

property,

thereby

reducing

the

recognition

and

phagocytosis

of

the

Mn3O4-Cit-PEG-Cys NPs by macrophages. We then used the Mn3O4-Cit-PEG-Cys NPs for T1-weighted MR imaging of C6 glioma cells in vitro (Figures 3c and 3d). The MR signal intensity of the cells treated with either Mn3O4-Cit-PEG or Mn3O4-Cit-PEG-Cys NPs increases with the Mn concentration (Figure 3c), which can be further confirmed by quantification of T1 MR signal intensity (Figure 3d). Additionally, the MR signal intensity of the cells treated with the Mn3O4-Cit-PEG-Cys NPs is significantly higher than that treated with the Mn3O4-Cit-PEG NPs at the same Mn concentration (Figure 3d). This is likely due to the fact that a zwitterionic surface ligand has a greater hydrophilicity than PEG, thus having a greater tendency to reduce the spin-lattice relaxation of NPs (T1) and greater T1 contrast enhancement. We checked the cellular uptake of both NPs via ICP-OES analysis (Figure S6, Supporting Information). Clearly, similar to the macrophage cells, C6 glioma cells have much less uptake of the Mn3O4-Cit-PEG-Cys NPs than that of the Mn3O4-Cit-PEG NPs (p < 0.05). This is certainly contrary to the most 11



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common cases, where more cellular uptake of the CAs leads to a higher MR signal intensity of cells.29 The only explanation for now could be that the relaxation rate of the Mn3O4-Cit-PEG-Cys NPs is likely significantly different from that of the Mn3O4-Cit-PEG NPs after they are uptaken by cancer cells.

Figure 3. (a) T1-weighted MR images of the Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs at different Mn concentrations. (b) Linear fitting of 1/T1 of the corresponding particles as a function of Mn concentration. (c) and (d) shows the in vitro T1-weighted MR images and the quantitative MR signal intensity of C6 glioma cells treated with the Mn3O4-Cit-PEG or Mn3O4-Cit-PEG-Cys NPs for 6 h at the Mn concentration of 0.1, 0.2, 0.4, 0.8 and 1.6 mM, respectively (n = 3).

It is critical to investigate the pharmacokinetics of the Mn3O4-Cit-PEG-Cys NPs before MR imaging in vivo. We used ICP-OES to analyze the blood Mn concentration at different time points post intravenous injection of the Mn3O4-Cit-PEG or Mn3O4-Cit-PEG-Cys NPs to mice (Figure 4). The half-decay time (t1/2) of the Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys 12


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NPs were estimated to be 18.5 h and 28.4 h, respectively. Our results suggest that the Mn3O4-Cit-PEG-Cys NPs have sufficiently long circulation time in the blood, thanks to the Cys modification onto the particle surface. The longer t1/2 and less macrophage cellular uptake of the Mn3O4-Cit-PEG-Cys NPs than those of the Mn3O4-Cit-PEG NPs are greatly beneficial for the particles to be accumulated within tumors via the passive enhanced permeability and retention (EPR) effect.

20

Mn3O4-Cit-PEG Mn3O4-Cit-PEG-Cys

[Mn] (µg/g)

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15 10 5 0

0

10

20

30

40

50

60

70

Time (h) Figure 4. Blood circulation and pharmacokinetic data obtained for the Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs ([Mn] = 4 µg/µL, in 125 µL NS) in healthy mice (n = 3). Next, the potential to use the Mn3O4-Cit-PEG-Cys NPs for enhanced tumor MR imaging in vivo was tested (Figure 5). Clearly, the MR signal intensity reaches the highest at 90 min postinjection for tumors treated with either Mn3O4-Cit-PEG-Cys or Mn3O4-Cit-PEG NPs, and then levels off after 90 min (Figure 5a). In addition, the MR signal intensity of the C6 tumor treated with the Mn3O4-Cit-PEG-Cys NPs is higher than that treated with the Mn3O4-Cit-PEG NPs at the same time points. This can be further clarified by quantitative analysis of MR signal intensity (Figure 5b). The gradual decrease of the MR signal intensity after 90 min postinjection could be because the particles 13



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can be metabolized with time, resulting in gradually decreased tumor accumulation. The MR imaging results indicate that the Mn3O4-Cit-PEG-Cys NPs have more enhanced MR imaging contrast sensitivity than Mn3O4-Cit-PEG NPs without Cys modification, likely due to the extended blood circulation time and decreased macrophage cellular uptake. It is worth mentioning that the size of the tumors shown in Figure 5a may not be the same due to the fact that the images are not given in the same plane. In most of the cases, the mouse position could be slightly changed at different time points, hence it is very difficult to collect the MR images with the same tumor size at the same plane for the same mouse at different time points. In any case, the MR intensity data from the whole tumor at different slices were collected and used to create normalized statistical histograms of the signal intensity for all time points (Figure 5b), which is more reliable. In order to explore the in vivo metabolism of the Mn3O4-Cit-PEG-Cys NPs and Mn3O4-Cit-PEG NPs, we utilized ICP-OES to determine the distribution of the NPs within the tumor and organs including heart, liver, spleen, lung and kidney (Figure S7a&b, Supporting Information). At the earlier time points, both Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs were taken up predominantly by RES organs such as the liver, spleen and lung, while relatively small amount of Mn element was uptaken by other organs (heart, and kidney) and the tumor. The liver and spleen uptake of the Mn3O4-Cit-PEG-Cys NPs was lower than that of the Mn3O4-Cit-PEG NPs at the same time points. As time goes by, the Mn concentration in the tumor first increases, reaches the peak value at 1.5 h, and then decreases, which is consistent with the tumor MR signal intensity changes. Moreover, at the same time points the tumor uptake of the Mn3O4-Cit-PEG-Cys NPs is higher than that of the Mn3O4-Cit-PEG NPs (Figure S7c). These results suggest that the antifouling property of the Mn3O4-Cit-PEG NPs makes them have less RES uptake and more tumor accumulation than the NPs without Cys, thereby offering enhanced tumor MR imaging in vivo. Meanwhile, our data 14


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suggest that both Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs can be metabolized and gradually cleared out of body at 48 h postinjection. The long-term in vivo organ toxicity of the designed Mn3O4-Cit-PEG-Cys NPs was next evaluated histological examinations of the major organs of mice after intravenous injection (Figure S8, Supporting Information). The structure and morphology of all organs of the mice after intravenous injection of either Mn3O4-Cit-PEG-Cys or Mn3O4-Cit-PEG-Cys NPs are quite similar to those of the corresponding organs of the control mice. This suggests that the injection of both the Mn3O4-Cit-PEG-Cys and Mn3O4-Cit-PEG NPs does not lead to apparent tissue or cellular damages, and both NPs display desirable organ compatibility in vivo at a long time period postinjection (one month).

Figure 5. (a) In vivo T1-weighted MR images of the C6 glioma tumor before and after intravenous injection of the Mn3O4-Cit-PEG or Mn3O4-Cit-PEG-Cys NPs ([Mn] = 4 µg/µL, in 125 µL NS for each mouse, n = 1). The red circle points to the tumor site. (b) Quantitative MR signal intensity of the C6 glioma tumors before and after intravenous injection of the Mn3O4-Cit-PEG or 15



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Mn3O4-Cit-PEG-Cys NPs ([Mn] = 4 µg/µL, in 125 µL NS) (n=3, meaning that three different circular locations in the same mouse tumor were selected to capture the MR signal intensity, each circular position has a diameter of 4 mm).

To conclude, we have developed a facile approach to preparing Mn3O4-Cit-PEG-Cys NPs with good antifouling property for enhanced MR imaging of tumors. Cit-stabilized Mn3O4 NPs can be easily functionalized with Cys through a PEG spacer and the formed particles with a mean size of 2.7 nm show good water dispersibility, colloidal stability, and cytocompatibility in the given concentration range. With the excellent r1 relaxivity, decreased macrophage

cellular

uptake,

and

extended

blood

circulation

time,

the

formed

Mn3O4-Cit-PEG-Cys NPs can be used as an effective CA for enhanced T1-weighted MR imaging of cancer cells in vitro and the tumor model in vivo. Considering the possibility to functionalize the Mn3O4 NPs with other targeting ligands, the Mn3O4-Cit-PEG-Cys NPs may be developed as a promising antifouling nanoplatform for various biomedical applications.

Supporting Information Additional DLS, zeta potential, and XRD characterization data; stability assessment of Mn3O4-Cit-PEG and Mn3O4-Cit-PEG-Cys NPs; cytotoxicity assay and cell morphology observation of C6 cells; cellular uptake assays of C6 and Raw 264.7 cells; in vivo biodistribution; and histological examination data.

Acknowledgments This research is financially supported by the National Natural Science Foundation of China 16


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(21273032, 81501518, and 81371623), the Science and Technology Commission of Shanghai Municipality (15520711400 for M. Shen), the Sino-German Center for Research Promotion (GZ899), and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning.

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ToC Figure

21


Antifouling Manganese Oxide Nanoparticles: Synthesis

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