Multifunctional Theranostic Nanoparticles Based on Exceedingly

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Multifunctional Theranostic Nanoparticles Based on Exceedingly Small Magnetic Iron Oxide Nanoparticles for T1Weighted Magnetic Resonance Imaging and Chemotherapy Zheyu Shen, Tianxiang Chen, Xuehua Ma, Wenzhi Ren, Zijian Zhou, Guizhi Zhu, Ariel Zhang, Yijing Liu, jibin song, Zihou Li, Huimin Ruan, Wenpei Fan, Lisen Lin, Jeeva Munasinghe, Xiaoyuan Chen, and Aiguo Wu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b04924 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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Multifunctional Theranostic Nanoparticles Based on Exceedingly Small Magnetic Iron Oxide Nanoparticles for T1-Weighted Magnetic Resonance Imaging and Chemotherapy

Zheyu Shen,†,‡,# Tianxiang Chen,†,# Xuehua Ma,† Wenzhi Ren,† Zijian Zhou,‡ Guizhi Zhu,‡ Ariel Zhang,‡ Yijing Liu,‡ Jibin Song,*,‡ Zihou Li,† Huimin Ruan,† Wenpei Fan,‡ Lisen Lin,‡ Jeeva Munasinghe,§ Xiaoyuan Chen,*,‡ Aiguo Wu*,†

†

CAS Key Laboratory of Magnetic Materials and Devices, & Key Laboratory of Additive

Manufacturing Materials of Zhejiang Province, & Division of Functional Materials and Nanodevices, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, 1219 Zhongguan West Road, Ningbo, Zhejiang 315201, China. ‡

Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging

and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, United States. §

Mouse Imaging Facility, National Institute of Neurological Disorder and Stroke, National

Institutes of Health, Bethesda, Maryland 20892, United States.

Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] Author Contributions #

Z.S. and T.C. contributed equally to this work.

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ABSTRACT The recently emerged exceedingly small magnetic iron oxide nanoparticles (ES-MIONs) (< 5 nm) are promising T1-weighted contrast agents for magnetic resonance imaging (MRI) due to their good biocompatibility compared with Gd-chelates. However, the best particle size of ES-MIONs for T1 imaging is still unknown because the synthesis of ES-MIONs with precise size control to clarify the relationship between the r1 (or r2/r1) and the particle size remains a challenge. In this study, we synthesized ES-MIONs with 7 different sizes below 5 nm and found that 3.6 nm is the best particle size for ES-MIONs to be utilized as T1-weighted MR contrast agent. To enhance tumor targetability of theranostic nanoparticles and reduce the non-specific uptake of nanoparticles by normal healthy cells, we constructed a drug delivery system based on the 3.6 nm ES-MIONs for T1-weighted tumor imaging and chemotherapy. The laser scanning confocal microscopy (LSCM) and flow cytometry analysis results demonstrate that our strategy of precise targeting via exposure or hiding of the targeting ligand RGD2 on demand is feasible. The MR imaging and chemotherapy results on the cancer cells and tumor-bearing mice reinforce that our DOX@ES-MION3@RGD2@mPEG3 nanoparticles are promising to be used for high resolution T1-weighted MR imaging and precise chemotherapy of tumors.

KEYWORDS Exceedingly small magnetic iron oxide nanoparticles (ES-MIONs), T1-weighted MR imaging, doxorubicin, RGD peptide, PEG shedding, theranostics.

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Currently, contrast agents for magnetic resonance imaging (MRI) are clinically used in 40-50% of all MR examinations.1-3 The MRI contrast agents include T1-weighted contrast agents (i.e. positive contrast agents), and T2-weighted contrast agents (i.e. negative contrast agents).4 The T1-weighted contrast agents decrease the protons’ longitudinal relaxation times inducing brighter MRI images, and the T2-weighted contrast agents shorten the protons’ transverse relaxation times resulting in darker MRI images. Because of the positive contrast, gadolinium (Gd) chelates as T1-weighted contrast agents dominate the current market of MRI contrast agents, which include Magnevist® (Gd-DTPA), Eovist® (Gd-EOB-DTPA), Omniscan® (Gd-DTPA-BMA), Dotarem® (Gd-DTOA), ProHance® (Gd-DO3A-HP), and so on.5 However, Gd causes nephrotoxicity because it may form complexes with ligands in vivo.6 Due to the better biocompatibility compared with the Gd-chelates, magnetic iron oxide nanoparticles (MIONs) have attracted increasing attention as MRI contrast agents.7,8 The MIONs with a large size (> 10 nm) are used as T2-weighted contrast agents because of their large transversal relaxivity (r2) and r2/r1 (r1 is longitudinal relaxivity).9 However, the development and production of MION-based T2-weighted contrast agents such as Feridex, Resovist, Combidex, Supravist, Clariscan, and Gastromark have largely ceased,10 presumably due to the fact that the dark images resulted from the T2-weighted contrast agents confuse those of some pathogenic conditions (e.g. hemorrhage, calcification and metal deposits), and the large magnetic moments of the T2-weighted contrast agents induce susceptibility artifact that destroys the background around disease regions and generates unclear images.11,12 The recently emerged exceedingly small MIONs (ES-MIONs) are promising to overcome the disadvantages of the Gd-chelate based T1-weighted contrast agents and MION based T2-weighted contrast agents because they have good biocompatibility and can be utilized as T1-weighted contrast agents. Up to now, the ES-MIONs have been synthesized by thermal decomposition method,10,13-17 solvothermal method,18 polyol method,19 reduction-precipitation method,20,21 and co-precipitation

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method.22,23 The ES-MIONs synthesized by these methods are all smaller than 5 nm, which is considered as an upper limit to be used as T1-weighted contrast agents. However, the best particle size below 5 nm for T1 imaging has not been defined because the relationship between the r1 (or r2/r1) and the particle size of ES-MIONs below 5 nm is still unknown. The key point to clarify the relationship is the synthesis of ES-MIONs with various particle sizes below 5 nm. Gao et al. reported the synthesis of manganese-doped iron oxide nanoparticles with controllable sizes. However, the particle sizes are all larger than 5 nm (5, 7, 9, 12 nm).24 Kolesnichenko et al. proposed synthesis of iron oxide nanoparticles with variable sizes of 3.2, 4.8 and 7.5 nm, with only two sizes below 5 nm.25 Hyeon et al. synthesized uniform iron oxide nanoparticles with different sizes for high-resolution T1 MR imaging, but there were also only two sizes below 5 nm (3 and 2.2 nm).13 To the best of our knowledge, there is still no systematic study of the relationship between the r1 (or r2/r1) and the size of ES-MIONs below 5 nm. In this study, we developed a co-precipitation synthesis method with precise size control of ES-MIONs below 5 nm (i.e. 1.9, 2.6, 3.3, 3.6, 4.2, 4.8 and 4.9 nm), and found that 3.6 nm is the optimal particle size for ES-MIONs to be utilized as T1-weighted MRI contrast agent. In addition, the targeting ligands may non-specifically bind to some non-targets on normal healthy cells, which results in non-specific uptake of nanoparticles, and reduces the specificity of active targeting of nanoparticles to tumors.26 To lower the non-specific uptake, we constructed a drug delivery system with precise targeting to tumors based on the 3.6 nm ES-MIONs for T1-weighted MR imaging and chemotherapy. Typically, the ES-MIONs were synthesized via an improved co-precipitation method using poly(acrylic acid) (PAA) as a stabilizer. Dimeric RGD peptide (RGD2) was then conjugated onto the ES-MIONs via the formation of amide bonds. The poly(ethylene glycol) methyl ether (mPEG) was grafted onto the ES-MIONs via an acid-labile β-thiopropionate linker. The anti-cancer drug doxorubicin hydrochloride (DOX) was finally loaded onto the RGD2 and mPEG conjugated ES-MION (ES-MION@RGD2@mPEG) via hydrogen bonds, ionic bonds and/or coordination bonds

to

construct

the

DOX

loaded

ES-MION@RGD2@mPEG 4


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(DOX@ES-MION@RGD2@mPEG)

(Figure

S1).

When

DOX@ES-MION@RGD2@mPEG

nanoparticles circulate in the blood stream (pH 7.4), the hydrophilic polymer mPEG inhibits RGD2 from coupling to healthy cells as the integrin ligand is hidden in the mPEG stealth. However, under the mild acidic tumor condition, the acid-labile β-thiopropionate linker is broken, which results in mPEG shedding from the nanoparticles. Thus, the hidden RGD2 is exposed and coupled to αvβ3 expressing cancer cells (Figure 1).

RESULTS AND DISCUSSION Precise Size Control of ES-MIONs Up to now, the optimal size of ES-MIONs below 5 nm for T1-weighted MRI is unkown.13,24,25 In this study, we developed a synthesis method with precise size control of ES-MIONs based on a co-precipitation method. The synthesis conditions and characterization results of the ES-MIONs are summarized in Table S1 and Table S2. PAA (2.0 ~ 5.0 mg/mL) was used as a stabilizer during the synthesis process, resulting in a plethora of carboxylate groups on the surface of the obtained ES-MION, which could be used for the following modifications with RGD2 and mPEG (Figure S1). From the TEM images and the size distribution measured from the TEM images (Figure S2), no significant difference was found between the ES-MION1-4 samples synthesized with various PAA concentrations. The iron concentrations of the obtained ES-MION solutions were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES), and the iron recovery was calculated from the molar ratio of iron in the obtained ES-MIONs to that in the feeding material. Due to the high iron recovery, the PAA concentration was fixed at 4.0 mg/mL for the subsequent syntheses. The dosages of iron precursors (FeCl3 and FeSO4) and ammonium hydroxide (NH3.H2O) were further tuned to control the ES-MION particle sizes (Table S1, and Table S2). The TEM images and the size distributions measured from the TEM images (Figure S3) showed successful synthesis of ES-MIONs with 7 different sizes below 5 nm (i.e. 1.9, 2.6, 3.3, 3.6, 4.2, 4.8 and 4.9 nm). The iron 5


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recoveries of ES-MION1-7 were all more than 80%, but those of ES-MION8-10 were less (Table S2), as excessive iron precursors were used to synthesize larger sized ES-MION8-10.

Optimal Size of ES-MIONs as T1 Contrast Agent In order to find the optimal sized ES-MIONs as T1 contrast agent, we measured the r1 and r2 values of the seven different-sized ES-MIONs on a 7T MRI scanner. To eliminate the influence of batch differences, we synthesized 3 batches for each sized ES-MIONs, and measured the T1 and T2 relaxation rates vs. Fe concentration (Figure S4 and Figure S5). The slopes of the linear lines were used as the r1 or r2 values. The average r1, r2, and r2/r1 values were summarized in Table S1 and Table S2. Figure 2a showed the average r1 and r2/r1 values vs. particle size (< 5 nm). With increasing particle size from 1.9 to 3.6 nm, the r1 value increased and the r2/r1 ratio decreased gradually. However, with the increase of particle size from 3.6 to 4.9 nm, the r1 value decreased and the r2/r1 ratio increased. Because a higher r1 value and lower r2/r1 ratio can result in better T1 imaging efficiency,27-29 3.6 nm was thus considered as the optimal size for ES-MIONs as T1 contrast agent. Although the size deviation is somewhat big and the dimensional difference between samples is actually small (especially 4.8 nm and 4.9 nm of ES-MIONs). The Fe precursor and NH3·H2O concentrations (shown in Table S1, S2) are the key to control the ES-MION sizes, and the differences of the Fe precursor and NH3·H2O concentrations for synthesis of different ES-MIONs are quite different (e.g. 1.6 and 1.0 mL of Fe precursor for synthesis of 4.8 nm and 4.9 nm of ES-MIONs, 17 and 13 mL of NH3·H2O for the synthesis of 4.8 nm and 4.9 nm of ES-MIONs). Therefore, the reproducibility of the ES-MIONs with specific average sizes is not a concern. In Figure 2a, the ES-MIONs with each specific average size were synthesized from 3 different batches under the same conditions. The good repeatability can be verified by the small error bars and the small P values (P < 0.02) as shown in Figure 2a.

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We further confirmed this by the T1-weighted MR images of ES-MIONs with different particle sizes and iron concentrations (Figure 2b). The MR images of ES-MION3 were the brightest due to the highest r1 and lowest r2/r1 among all 7 samples tested. The relative intensities of the ES-MION images measured by ImageJ and compared with pure water were shown in Figure 2c. Several factors can influence the r1 value of iron oxide nanoparticles. On the one hand, larger particles possess higher saturation magnetization (Ms), which results in stronger interaction between Fe and closely diffusing water molecules without interacting with Fe (the outer sphere), leading to larger r1 values (i.e. outer-sphere model). On the other hand, the specific surface area of larger nanoparticles is smaller, which results in less naked Fe on the nanoparticle surface that interacts directly with the hydrogen nuclei of water molecules (the inner sphere), causing a smaller r1 value (i.e. inner-sphere model). Therefore, the r1 value is not necessarily linearly correlated with particle size (Figure 2a). Similar theoretical models (the above-mentioned inner-sphere and outer-sphere models) also work on the influence of nanoparticle size on r2 value. However, the influence of nanoparticle size and magnetization is much bigger than that of the specific surface area (i.e. the outer-sphere model is more dominant) based on the following equation.30,31 ଵ ்మ

=

(ଶହ଺గ మ ఊమ /ସ଴ହ)௏ ∗ ெೞమ ௥మ ஽(ଵା௅/௥)

(1)

In equation (1), Ms is the saturation magnetization and r is the radius of magnetic core. Therefore, the larger nanoparticle size usually results in a larger r2 value (Table S1, Table S2).

Characterization of ES-MIONs The r1 and r2 values of the ES-MION3 (3.6 nm) were also measured at magnetic fields of 0.5 T (a MicoMR analyzing system) and 1.5 T (a clinical MRI scanner system) (Figure S6) and summarized in Table S1. With the decrease of magnetic field, the r1 value increased by 279% from 7.0 T to 1.5 T, and 44% from 1.5 T to 0.5 T. However, the influence of the magnetic field on r2 value is much 7


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smaller because ES-MION3 is exceedingly small (3.6 nm), resulting in a very small saturation magnetization (Ms) value of 9.5 emu/g (Figure 3). Similar findings were also observed in other reported ultrasmall nanoparticles.32 In addition, with the decrease of magnetic field, the r2/r1 decreased by 75% from 7.0 T to 1.5 T, and 27% from 1.5 T to 0.5 T. Therefore, lower magnetic field has better T1 imaging efficiency, which is consistent with other reported nanoparticles.32,33 Measured by a 1.5 T clinical MRI system, the r1 value of our ES-MION3 was up to 8.80 mM-1 s-1, and the r2/r1 ratio was only 2.6. Therefore, our ES-MION3 is a promising T1-weighted MRI contrast agent. Figure S7 showed the UV-vis spectra of the ES-MIONs with an average particle size of 4.2, 3.6, 3.3, 2.6 and 1.9 nm at 1.0 mM of iron concentration. Overlap of the absorption curves demonstrated that the particle size of ES-MIONs had no influence on the UV-vis absorption. Figure S8 showed the FT-IR spectra of the purified ES-MION3, 5 and 8. The similar FT-IR spectra manifest similar compositions of the ES-MIONs. The peaks at 3050, 2810 and 1400 cm-1 respectively correspond to the stretching vibration and bending vibration of –CH2– on the backbone of the PAA. The peaks at 3140, 1710 and 1100 cm-1 respectively correspond to the stretching vibrations of O-H, C=O, and C-O in the carboxyl groups,34,35 suggesting that the PAA were coated on the surface of ES-MIONs. Figure S9a showed the high resolution TEM (HR-TEM) image of ES-MION3. The interplanar distances of 0.25 and 0.30 nm are equivalent to the (311) and (220) lattice planes. The energy dispersive X-ray spectrum (EDS) of ES-MION3 (Figure S9b) demonstrated that the ES-MION3 were composed of iron and oxide. The copper peaks were from the copper grid used for TEM. Figure S9c-e showed the X-ray diffraction (XRD) patterns of the ES-MION3, ES-MION5, and ES-MION8, respectively. Three characteristic peaks (2θ ~ 35.5, 50.5, and 62.5°), marked by their indices [(311), (422), and (440)], were observed. All diffraction peaks including the positions and relative intensities matched well with those from references for magnetite. This result revealed that our ES-MIONs were pure magnetite.36,37 Although our ES-MIONs were very small, the XRD peaks were relatively sharp. Although it is generally true that crystal size is inversely proportional to the 8


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FWHM (full width at half maximum) of XRD peak, many factors, such as internal stresses, particle size, dislocation substructure, and stacking faults, can influence the broadening of XRD peaks.38 Figure S10 showed the XPS spectra of the ES-MION3, ES-MION5, and ES-MION8 corresponding to the Fe2p region. The levels of Fe2p3/2 and Fe2p1/2 indicated that the ES-MIONs were magnetite (Fe3O4) instead of maghemite (γ-Fe2O3), whose Fe2p1/2 level should be 724.0 eV.39,40

Preparation and Characterization of DOX@ES-MION@RGD2@mPEG To lower the non-specific uptake of nanoparticles by normal healthy cells, we constructed a drug delivery system with precise targetability to tumors based on the 3.6 nm of ES-MION3 for T1-weighted MR imaging and chemotherapy. As shown in Figure S1, integrin targeting ligand RGD2 was conjugated onto the ES-MION3 via formation of amide bonds. The mPEG was grafted onto the ES-MION3 via an acid-labile β-thiopropionate linker. The anti-cancer drug DOX was finally loaded onto the ES-MION3 via hydrogen bonds, ionic bonds and/or coordination bonds to construct the composite nanoparticle DOX@ES-MION3@RGD2@mPEG. Table

S3

showed

the

synthesis

conditions

and

characterization

results

of

the

ES-MION3@RGD2@FITC-PEG. The FITC-PEG content was determined by measuring the FITC fluorescence, which was as high as 41.6% due to the exceedingly small particle size and very large specific surface area of the ES-MIONs, which could be controlled by the feeding amount of FITC-PEG. The RGD2 conjugation efficiency was the similar to that of FITC-PEG (Table S3). The mPEG content, RGD2 content, and DOX loading content were calculated from the mass percentages of the conjugated mPEG, conjugated RGD2, and loaded DOX to the nanoparticle DOX@ES-MION3@RGD2@mPEG (Table S4). The DOX loading contents were 32.3 ± 1.0, 36.3 ± 1.6, 39.9 ± 2.1 and 43.6 ± 2.0% for DOX@ES-MION3@RGD2@FITC-PEG1-4, respectively. These DOX loading contents of our nanoparticles were much higher than most of the reported

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nanoparticles.41,42 The high DOX loading content can be ascribed to the large specific surface area of the nanoparticles. The particle size, water dispersity and magnetism of the DOX@ES-MION3@RGD2@mPEG3 were compared with ES-MION3

(Figure

3).

The

TEM images

indicated that the

DOX@ES-MION3@RGD2@mPEG3 was significantly larger than the ES-MION3 (Figure 3a, b) due to the high conjugation content of mPEG (12.1%) and high loading content of DOX (39.9 ± 2.1%). The core/shell structure of DOX@ES-MION3@RGD2@mPEG3 was not observed from the TEM images because the DOX and mPEG shell had a similar darkness to that of the ES-MION core. The hydrodynamic diameters of ES-MION3 and DOX@ES-MION3@RGD2@mPEG3 determined by dynamic light scattering (DLS) were 5.4 and 13.1 nm, respectively, which further confirmed the much larger size of DOX@ES-MION3@RGD2@mPEG3. 13.1 nm is much larger than the threshold requirement for renal clearance (~ 6 nm) to reduce the renal clearance,43 allows prolonged circulation of the nanoparticles, and enhances the targeting efficiency of the nanoparticles to tumors. In addition, both the TEM images (Figure 3a, b) and DLS size distributions (Figure 3c) demonstrated the good water dispersity of our DOX@ES-MION3@RGD2@mPEG3 nanoparticles. Figure 3d showed the field-dependent magnetization curves (H - M) of ES-MION3, ES-MION5, ES-MION8 and DOX@ES-MION3@RGD2@mPEG3 at 300 K. The saturation magnetization (Ms) values of ES-MION3 and DOX@ES-MION3@RGD2@mPEG3 were determined to be 9.5 and 12.6 emu/g.

The

slightly

larger

Ms

value

suggests

slight

aggregation

of

DOX@ES-MION3@RGD2@mPEG3 because aggregation of the iron oxide nanoparticles could increase the Ms value.44 The slight aggregation could be ascribed to high loading content of DOX (39.9 ± 2.1%). The low Ms values (e.g. 9.5 emu/g for ES-MION3) suppressed by the small particle size (e.g. 3.6 nm for ES-MION3) lead to small r2 values (e.g. 22.7 mM-1 s-1 at 1.5 T for ES-MION3), which benefit the T1-weighted MR imaging.20,21

Polymer Shedding and Drug Release Behaviors 10


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In order to lower the non-specific uptake of nanoparticles by normal healthy cells, we previously conjugated pH-sensitive polymers onto the surface of nanoparticles to hide the targeting ligands in blood stream (pH ~ 7.4) and expose them via the polymer shrinkage at tumor sites.45 However, the difference of the nanoparticle targeting efficiency to tumors with or without conjugation of pH-sensitive polymers was not significant, which may be ascribed to the covering of the targeting ligands by the shrunk polymers. To overcome this problem, we changed the strategy of ligand exposure at tumor sites from polymer shrinkage to polymer shedding in this study. The shedding behavior of the polymer FITC-PEG from the ES-MION3@RGD2@FITC-PEG3 nanoparticles (i.e. hydrolysis of the acid-labile β-thiopropionate linker) at pH 5.5, 6.0, 6.5 and 7.4 was shown in Figure S11. It’s obvious that stronger acidic condition led to faster polymer shedding. 81.9 ± 3.3 %, 65.6 ± 4.5 %, 52.6 ± 3.8 % and 23.7 ± 3.5 % of FITC-PEG were respectively shed from the nanoparticles after 72 h of incubation at pH 5.5, 6.0, 6.5 and 7.4. The pH sensitivity of our β-thiopropionate linker was comparable to the previously reported results.46,47 Our study suggests that the polymer shedding in the mild acid tumor environment should be much faster than that in the blood stream (pH ~ 7.4), causing on-demand exposure or hiding of the RGD2 to lower the non-specific uptake of the nanoparticles in the normal organs/tissues and realize integrin specific targeting of the nanoparticles in the tumor region. This is a key design that minimizes the nonspecific accumulation of nanoparticles to non-tumor tissues. The polymer mPEG is grafted onto the ES-MIONs via a well-known acid-labile β-thiopropionate linker, and the polymer shedding mechanism is based on the hydrolysis of the acid-labile β-thiopropionate linker. It is reported that this hydrolysis is driven by the generation of a partial positive charge on the ester carbonyl carbon because of the inductive effect of sulfur atom.46 The hydrolysis behavior of the acid-labile β-thiopropionate linker (Figure S11), and the zeta potential changes of DOX@ES-MION3@RGD2@mPEG3 after incubation in PBS with different pH values (Figure S12) double confirmed the polymer shedding mechanism.

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Our ES-MIONs and DOX@ES-MION3@RGD2@mPEG3 are both very stable in water because they were synthesized in aqueous phase method and stabilized with PAA. The stability of our DOX@ES-MION3@RGD2@mPEG3 was confirmed by the hydrodynamic diameter (dh) changes during storage up for to 30 days (Figure S12 d). The pH-dependent DOX release from DOX@ES-MION3@RGD2@mPEG3 was shown in Figure S13, with 82.0 ± 5.6, 58.0 ± 5.1, 37.9 ± 4.7 and 27.2 ± 4.1% of loaded DOX released from the nanoparticles at pH 5.5, 6.0, 6.5 and 7.4 in 72 h. Therefore, most of the loaded DOX would not be released in the circulation (pH ~ 7.4), but could be effectively released in the late endosome (pH ~ 5.5) after the nanoparticles were endocytosed by the cancer cells (Figure 1). This is another key point that helps reduce the side effect of DOX to healthy cells. In addition, DOX release behavior from DOX@ES-MION3@RGD2@mPEG3 in PBS (pH = 7.4) with 10 mg/mL of BSA (Figure S13 d) indicated that BSA molecules almost had no influence on the DOX release.

Precise Targeting To realize the precise targeting of nanoparticles to cancer cells, we conjugated hydrophilic polymer mPEG onto the surface of nanoparticles to hide the targeting ligand RGD2 in blood stream (pH ~ 7.4) and expose it after polymer shedding in the mildly acid tumor environment. Here, we investigated the feasibility of the precise targeting through cellular uptake of the nanoparticles after incubation with PBS (pH 6.5 and 7.4) for 72 h at 37 °C to mimic the normal physiological and tumor conditions. Figure S14 and Figure S15 showed the laser scanning confocal microscopy (LSCM) images of U-87 MG cells (integrin αvβ3 positive)48-50 and MCF-7 cells (integrin αvβ3 negative) incubated with DOX@ES-MION3@RGD2 or DOX@ES-MION3@RGD2@mPEG3 that was pre-incubated with PBS (pH = 7.4 or 6.5) for 72 h at 37 °C. The cells untreated with nanoparticles were used as the control. Figure 4 showed the merged LSCM images of the nanoparticle-internalized MCF-7 and U-87 MG cells. It was found that a large number of DOX@ES-MION3@RGD2@mPEG3 nanoparticles were internalized in U-87 MG cells (even in 12


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the nucleus) after incubation at pH 6.5, which was similar to that of the DOX@ES-MION3@RGD2 nanoparticles. However, very few DOX@ES-MION3@RGD2@mPEG3 nanoparticles were taken up by U-87 MG cells after incubation at pH 7.4. This is due to the fact that the mPEG can be shed from the nanoparticles to expose the RGD2 at pH 6.5, but not at pH 7.4. In addition, none of the nanoparticles were not effectively taken up by MCF-7 cells due to the low expression of integrin αvβ3 (Figure 4). These results demonstrate that our strategy of precise targeting via exposure or hiding of the targeting ligand RGD2 on demand is feasible. Figure S16 showed flow cytometry analysis of the nanoparticle-internalized MCF-7 and U-87 MG cells. Significant difference of relative fluorescence intensity between the MCF-7 and U-87 MG cells was observed for the DOX@ES-MION3@RGD2@mPEG3 nanoparticles incubated at pH 6.5, but not at pH 7.4, further confirming mPEG shedding at acidic pH to facilitate RGD2 binding.

MR Imaging and Chemotherapy on Cells In this study, we used hydrophilic polymer mPEG to hide the targeting ligand RGD2 at neutral pH to minimize interactions between the RGD2 and the non-specific receptors on the normal healthy cells, but expose the RGD2 at mildly acid tumor site to allow specific integrin binding. Overly dense PEGylation may result in incomplete mPEG shedding thus limited specific interaction between the RGD moiety and integrin αvβ3 on the surface of cancer cells. On the other hand, low density PEGylation would expose RGD2 on the surface of nanoparticles under normal physiological conditions, which can be recognized by the non-specific receptors expressed on normal healthy cells and thus induce non-specific uptake of the nanoparticles by the healthy cells. Therefore, the mEPG content plays an important role to realize the precise targeting. Here, we optimized the mEPG conjugation content according to the chemotherapeutic efficacies on cells (Figure 5). Figure 5a showed the cell viabilities of the nanoparticles on U-87 MG cells. The DOX@ES-MION3@RGD2@mPEG1-4 nanoparticles were incubated at pH 7.4 or 6.5 for 72 h before

incubation

with

cells to allow the

mPEG

shedding. Both

ES-MION3

and 13


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ES-MION3@RGD2@mPEG3 were almost non-toxic. The free DOX was more toxic than all of the DOX-loaded nanoparticles because it is a small molecule that is very easy to enter the nucleus.51,52 In addition, the cell viabilities of DOX@ES-MION3@RGD2@mPEG1-4 nanoparticles were DOX concentration dependent, and those incubated at pH 6.5 before incubation with cells were more toxic than those incubated at pH 7.4, presumably due to mPEG shedding at pH 6.5. The difference of

the

cell

viability

between

pH

7.4

and

6.5

was

the

largest

for

DOX@ES-MION3@RGD2@mPEG3 as compared with DOX@ES-MION3@RGD2@mPEG1, 2 and 4 (Figure 5b), which suggests that 12.1 % is the best mEPG conjugation content. Moreover, the nanoparticles showed very limited cytotoxicity to MCF-7 cells even at 10 µg/mL of DOX (Figure 5c) because of the low expression of αvβ3 integrin on MCF-7 cells. Compared with ES-MION3, DOX@ES-MION3@RGD2@mPEG3 has a similar r1 and slightly higher r2 (Figure S17a, b) due to the slightly larger particle size of the RGD and PEG modified ES-MION3 (Figure 3a-c). The T1-weighted MR images of U-87 MG cells with various contrast agents were shown in Figure S17c. The DMEM medium without any contrast agent was used as a control.

It

is

obvious

that

cells

incubated

with

DOX@ES-MION3@RGD2

and

DOX@ES-MION3@RGD2@mPEG3 at pH 6.5 PBS were of similar brightness, which were slightly

darker

than

those

of

Magnevist,

but

much

brighter

than

those

of

DOX@ES-MION3@RGD2@mPEG3 incubated at pH 7.4. The relative intensities of the MR images compared with the control were shown in Figure S17d.

In vivo MR Imaging and Cancer Chemotherapy To evaluate the MRI imaging efficiency of our DOX@ES-MION3@RGD2@mPEG3 nanoparticles in vivo, U-87 MG tumor model was established on nude mice and the contrast agent DOX@ES-MION3@RGD2@mPEG3, ES-MION3, or Magnevist was injected via tail vein. As shown in Figure 6a-c, the T1 contrast in the tumor sites was highest at 1, 4, or 12 h post-injection for Magnevist, ES-MION3, or DOX@ES-MION3@RGD2@mPEG3, respectively. The signal changes 14


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in tumors at different time points after contrast administration (Figure 6d-f) were quantified using ∆SNR calculated from the following equations: SNR = SImean / SDnoise

(2)

▵SNR = (SNRpost - SNRpre) / SNRpre × 100 %

(3)

The quantification results were consistent with the above-mentioned MRI images. The highest tumor ∆SNR after injection of DOX@ES-MION3@RGD2@mPEG3 nanoparticles is 203.4 ± 15.1% (at 12 h post-injection), which is much higher than that of Magnevist (55.9 ± 13.5% at 1 h post-injection), and ES-MION3 (63.4 ± 17.8% at 4 h post-injection). In addition, most of the highest tumor or tissue (e.g. liver) ∆SNR after injection of reported nanoparticles is smaller than 80%.16,32,53 The high MRI imaging efficiency of our DOX@ES-MION3@RGD2@mPEG3 nanoparticles for tumors may be ascribed to their high accumulation in the tumors. To examine the in vivo biodistribution of our DOX@ES-MION3@RGD2@mPEG3 nanoparticles, the in vivo T1-weighted MR images of U-87 MG tumor-bearing nude mice were obtained (Figure 7a, b), and the ∆SNR were calculated for tumor, liver, and spleen (Figure 7c, d). The highest ∆SNR values were 173.8 ± 17, 81.9 ± 28, and 78.2 ± 30% for tumor, liver and spleen, respectively. It is worth noting that the accumulation of DOX@ES-MION3@RGD2@mPEG3 nanoparticles in the tumor was higher than those in the liver and spleen. We further confirmed this result by measuring the Fe level in the U-87 MG tumor-bearing nude mice pre- or post-injection of DOX@ES-MION3@RGD2@mPEG3 by ICP (Figure 7e). The high targeting efficiency to tumors can be ascribed to the appropriate hydrodynamic diameter (13.1 nm) and precise targeting strategy. We also evaluated the renal excretion of DOX@ES-MION3@RGD2@mPEG3 (dh = 13.1 nm) and compared that with ES-MION3 (dh = 5.4 nm) (Figure S18). The T1-weighted MR images indicate that ES-MION3 could be seen in the bladder in 2 h, but DOX@ES-MION3@RGD2@mPEG3 was virtually not cleared through renal route due to its large dh. That’s also one of the reasons why our DOX@ES-MION3@RGD2@mPEG3 had a high targeting efficiency to tumors (Figure 7e). 15


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The

in

vivo

therapeutic

performance

of

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DOX@ES-MION3@RGD2@mPEG3

and

DOX@ES-MION3@RGD2 was evaluated using U-87 MG tumor mice compared with free DOX and PBS. The DOX dosage is 10 mg equiv. per kg body weight intravenously injected on day 0 and day 4. As shown in Figure 8a, the tumors in the PBS group grew exponentially, and one mouse had to be euthanized at day 16 post-injection due to the excessive tumor size (> 2000 mm3). The tumor growth of free DOX group was initially delayed but relapsed over time, with one mouse euthanized at day 30 post-injection. However, the tumor growths in both DOX@ES-MION3@RGD2@mPEG3 and DOX@ES-MION3@RGD2 groups were effectively suppressed and all the mice were eventually

tumor

free.

Although

DOX@ES-MION3@RGD2@mPEG3

group

the was

mean

relative

always

smaller

tumor than

volume that

of

of the

DOX@ES-MION3@RGD2 group since day 8 post treatment, the difference was not significant (P > 0.05). The excellent therapeutic performance of DOX@ES-MION3@RGD2 without PEG shedding was ascribed to the relatively small hydrodynamic particle size (~ 10 nm), which allows effective tumor accumulation via EPR effect. Figure S19 showed the photos of U-87 MG tumor-bearing nude mice on day 14 since the treatment, Figure 8b showed the survival of treated mice. In addition, the relative body weights of DOX@ES-MION3@RGD2@mPEG3 group and DOX@ES-MION3@RGD2 group were much higher than those of free DOX group (Figure 8c), which indicates that the side effect of DOX is significantly reduced via delivery by our nanoparticles. These results demonstrate that our nanoparticles are promising to be used for the treatment of human glioblastoma. The good therapeutic performance of our nanoparticles may be ascribed to the high accumulation in the tumor region due to EPR effect (Figure 7c-e), high uptake by U-87 MG cells with overexpression of integrin αvβ3 (Figure 4, Figure S16), and fast release of DOX in late endosome (pH ~ 5.5) (Figure 1, Figure S13).

16


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In addition, although RGD peptides recognize not only tumor cells that express αvβ3 integrin but also inflamed/angiogenic endothelium with increased level of integrin receptors,54 the dimeric peptide RGD2 used in this study was hidden in the mPEG, and would not be able to recognize integrin until the nanoparticles successfully extravasated and diffused in the slightly acidic tumor interstitium where the PEG was cleaved and RGD2 was exposed to bind integrin expressing tumor cells.

CONCLUSIONS In this study, we developed a co-precipitation synthesis method with precise size control of ES-MIONs below 5 nm, clarified the relationship between the r1 (or r2/r1) and the particle size, and drew a conclusion that 3.6 nm is the best particle size for ES-MIONs to be utilized as T1-weighted MRI contrast agent. The results of MRI, UV-vis, FT-IR, TEM, HR-TEM, EDS, DLS, XRD, XPS, and magnetization curves verified the compositions, structures, and physicochemical properties of ES-MIONs. Furthermore, to lower the non-specific uptake of nanoparticles by normal healthy cells, we constructed a drug delivery system DOX@ES-MION@RGD2@mPEG T1-weighted MR imaging and chemotherapy of integrin-expressing tumors. The hydrolysis of the acid-labile β-thiopropionate linker leads to PEG polymer shedding at tumor site and exposure of the ligand RGD2 to realize integrin specific binding. Treatment of integrin positive U87MG tumor mice with DOX@ES-MION3@RGD2@mPEG3 led to partial or even complete regression of tumors due to combined passive and active tumor targeting and pH responsive release of doxorubicin molecules.

EXPERIMENTAL METHODS Materials and Reagents. Poly(acrylic acid) (PAA, Mw = 1800), iron (III) chloride (FeCl3, ≥ 97%), gadolinium (III) nitrate hexahydrate (Gd(NO3)3·6H2O, 99.9%), triethylamine (TEA, ≥ 99%), cysteamine (CA, ≥ 98%), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC, ≥ 97%), acryloyl chloride (AC, ≥ 97%), poly(ethylene glycol) methyl ether (mPEG, Mw 5000), doxorubicin 17


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hydrochloride (DOX), phalloidin-FITC, and Hoechst 33258 were purchased from Sigma-Aldrich (USA). Iron (II) sulfate heptahydrate (FeSO4·7H2O) was purchased from Acros organics. FITC PEG hydroxyl (FITC-PEG-OH, Mw 5000) was purchased from Nanocs Inc. (Boston, USA). Glu-{Cyclo[Arg-Gly-Asp-(D-Phe)-Lys]}2 (i.e. RGD dimer, or RGD2, 97.92%, Mw = 1318.51) was purchased from C S Bio Co. (CA, USA 94025). Synthesis of ES-MIONs Smaller than 4 nm. 20 mL of PAA (Mw = 1800) solution (0.4 ~ 5.0 mg/mL) was first purged with nitrogen (≥ 50 min) for removal of oxygen. The polymer solution was then heated to reflux (100 oC). After that, 0.4 mL of iron precursor solution (50 ~ 500 mM FeCl3 plus 25 ~ 500 mM FeSO4) was quickly injected into the heated polymer solution, followed by addition of 6.0 mL of ammonia solution (2.8 ~ 28 %). The reaction was kept at 100 oC under magnetic stirring. After 1.0 h, the solutions were cooled down to room temperature. The obtained ES-MION1-7 were dialyzed (Mw cut-off 6-8 kDa) for 5 days in ultrapure water, which was changed every day. The dialyzed ES-MION1-7 were then concentrated by centrifugal ultrafiltration (Millipore, molecular size cutoff of 10 kDa). The Fe concentrations of the ES-MION1-7 solutions were determined by inductively coupled plasma optical emission spectrometry (ICP-OES; Agilent 5100). The recovery of the ES-MION1-7 was calculated from the molar ratio of Fe in the obtained ES-MIONs to that in the feeding materials. Synthesis of ES-MIONs Larger than 4 nm. 20 mL of PAA (Mw = 1800) solution (4 mg/mL) was first purged with nitrogen (≥ 50 min) for removal of oxygen. The polymer solution was then heated to reflux (100 oC) under magnetic stirring. After that, 0.4 mL of iron precursor solution (1.0 M FeCl3 plus 0.5 M FeSO4) was quickly injected into the heated polymer solution, followed by addition of 9.0 mL of ammonia solution (28%). The reaction was continued 1.0 h to obtain ES-MION8; or after 15 min of reaction, 0.6 mL of the iron precursor solution and 4.0 mL of the ammonia solution were injected followed by further 45 min of reaction to obtain ES-MION9; or after both 15 min and 30 min of reaction, 0.6 mL of the iron precursor solution and 4.0 mL of the ammonia solution were injected twice followed by further 30 min of reaction to obtain 18


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ES-MION10. The obtained ES-MION8-10 solutions were cooled down to room temperature, and then dialyzed (Mw cut-off 12-14 kDa) for 5 days in ultrapure water that was changed every day. The dialyzed ES-MION8-10 were then concentrated by centrifugal ultrafiltration (Millipore, molecular size cutoff of 10 kDa). The Fe concentrations of the ES-MION solutions were measured by ICP-OES. The recovery of the ES-MION8-10 were calculated from the molar ratio of Fe in the obtained ES-MIONs to that in the feeding materials. Synthesis of FITC-PEG-AC-CA. The FITC-PEG-OH (40 mg, 8 µmol) was dissolved in anhydrous tetrahydrofuran (THF, 2.0 mL) and then purged with nitrogen (≥ 50 min) to remove oxygen under ice water bath. After that, TEA (100 µL, 0.717 mmol) and acryloyl chloride (AC) (100 µL, 1.23 mmol), were added successively. The solution was then stirred at room temperature. After 16 h, 5.0 mL of cysteamine (CA) dissolved in N,N-dimethylformamide (DMF) (30 mg/mL, 1.94 mmol) was added, and the mixture was stirred for 24 h at room temperature. The obtained polymers were washed 3 times using PBS (pH = 7.4) by centrifugal ultrafiltration (Millipore, molecular size cutoff of 3.0 kDa), and finally dissolved in 4.0 mL of PBS. The concentration of the obtained FITC-PEG-AC-CA solution was determined to be 7.8 mg/mL by fluorescence spectrophotometer (F-7000, HITACHI, Japan). The recovery of FITC-PEG-OH was calculated to be 78%. Synthesis of mPEG-AC-CA. The mPEG (1.0 g, 0.20 mmol) was dissolved in THF (13 mL) and then purged with nitrogen (≥ 50 min) to remove oxygen under ice water bath. After that, TEA (460 µL, 3.3 mmol) and acryloyl chloride (AC) (200 µL, 2.46 mmol) were added successively. The solution was then stirred at room temperature. After 16 h, 10 mL of cysteamine (CA) dissolved in DMF (30 mg/mL, 3.88 mmol) was added. The solution was then stirred for 24 h at room temperature. The obtained polymers were washed 3 times using PBS (pH = 7.4) by centrifugal ultrafiltration (Millipore, molecular size cutoff of 3.0 kDa), and finally dissolved in 100 mL of PBS. The concentration of the obtained mPEG-AC-CA solution could be considered as 7.8 mg/mL 19


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because the recovery of FITC-PEG-OH and mPEG should be very close due to the similar reaction conditions. Synthesis of ES-MION3@RGD2@FITC-PEG or ES-MION3@RGD2@mPEG. The RGD2 and FITC-PEG-AC-CA (or mPEG-AC-CA) were conjugated onto the surface of ES-MIONs via the reaction between –COOH and –NH2 in the presence of EDC. Typically, 10 µL of EDC (56.5 µmol) and 100 µL of RGD2 (5.0 mg/mL, 3.8 mM) were added into 8.0 mL of the ES-MION3 solution (Fe concentration of 5.0 mM, ice cold) under magnetic stirring. After 4 h, 1.00, 0.50, 0.25 or 0.10 mL of the obtained FITC-PEG-AC-CA or mPEG-AC-CA (7.8 mg/mL, 1.56 mM) plus 0, 0.50, 0.75 or 0.90 mL of H2O were added into the mixtures. The reaction was kept at room temperature for 16 h under

magnetic

stirring.

The

obtained

ES-MION3@RGD2@FITC-PEG1-4

or

ES-MION3@RGD2@mPEG1-4 were washed 3 times using PBS by centrifugal ultrafiltration (Millipore, molecular size cutoff of 10 kDa) to remove unreacted EDC, RGD2 and FITC-PEG-AC-CA (or mPEG-AC-CA), and finally dissolved in 8.0 mL of PBS (pH 7.4) or saline. In the obtained ES-MION3@RGD2@mPEG1-4 solutions, the Fe concentrations were measured by ICP-OES. In addition, the conjugation contents of FITC-PEG, which were considered as equal to those of mPEG due to the same reaction conditions, were determined by fluorescence spectrophotometer. Hydrolysis of the Acid-labile β-Thiopropionate Linker. The hydrolysis of acid-labile β-thiopropionate

linker

(i.e.

shedding

of

FITC-PEG

from

the

surface

of

ES-MION3@RGD2@FITC-PEG3) was monitored at pH 5.5, 6.0, 6.5 and 7.4 via fluorescence determination of FITC-PEG. Typically, 2.0 mL of ES-MION3@RGD2@FITC-PEG3 in saline (CFe = 4.6 mM, CFITC-PEG = 331 µg/mL) was added in 10 mL of PBS with pH value of 5.5, 6.0, 6.5 or 7.4. The solutions with various pH values were kept in a shaking incubator (Excella E24, New Brunswick Scientific) at 37 °C. At predetermined time intervals, 1.0 mL of the solutions with different pH values were taken and subjected to centrifugal ultrafiltration (Millipore, molecular size cutoff of 10 kDa). The supernatants were then determined by fluorescence spectrophotometer and 20


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the fluorescence intensity (Ex = 495 nm, Em = 520 nm) was converted into the CFITC-PEG via a calibration curve constructed with standard FITC-PEG solutions. The hydrolysis behavior of the acid-labile β-thiopropionate linker at different pH values was monitored via plot of the shedded FITC-PEG content (i.e. the mass percentage of the shedded FITC-PEG to the total amount of FITC-PEG on the surface of nanoparticles) as a function of incubation time. Synthesis of DOX@ES-MION3@RGD2@mPEG. The DOX was loaded onto the surface of ES-MION3@RGD2@mPEG1-4 via the ionic bond, hydrogen bond and coordination bond as shown in Figure S1. Typically, 4.0 mL of ES-MION3@RGD2@mPEG1-4 (CFe = 4.0 mM) in PBS (pH 7.4) were mixed with 2.0 mL of DOX (2.0 mM, 1.16 mg/mL) under magnetic stirring at room temperature. After 24 h, the obtained DOX@ES-MION3@RGD2@mPEG1-4 solutions were washed using saline by centrifugal ultrafiltration (Millipore, molecular size cutoff of 10 kDa) to remove unloaded DOX. The finally obtained DOX@ES-MION3@RGD2@mPEG1-4 were dissolved in 4.0 mL of PBS (pH 7.4). The unloaded DOX in the supernatants was measured by UV-vis spectrophotometer and the absorbance at 233 nm was converted to the CDOX via a calibration curve constructed with standard solutions of DOX. The DOX loading content was calculated

from

the

mass

percentage

of

the

loaded

DOX

to

the

nanoparticle

DOX@ES-MION3@RGD2@mPEG. Release Behavior of DOX from DOX@ES-MION3@RGD2@mPEG3. The release behavior of DOX from DOX@ES-MION3@RGD2@mPEG3 was determined by UV-vis spectrophotometer at pH 5.5, 6.0, 6.5 or 7.4. The same experiments on ES-MION3@RGD2@mPEG3 were used as the control. Typically, 2.0 mL of DOX@ES-MION3@RGD2@mPEG3 (CFe = 4.6 mM, CDOX = 321 µg/mL) or ES-MION3@RGD2@mPEG3 (CFe = 4.6 mM) was added in 10 mL of PBS (pH = 5.5, 6.0, 6.5 or 7.4) without BSA, or PBS (pH = 7.4) with 10 mg/mL of BSA. The solutions with various pH values were kept in a shaking incubator (Excella E24, New Brunswick Scientific) at 37 °C. At predetermined time intervals, 1.0 mL of the solutions with different pH values were taken and subjected to centrifugal ultrafiltration (Millipore, molecular size cutoff of 10 kDa). The supernatants 21


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were then measured by UV-vis spectrophotometer and the absorbance at 233 nm was converted to the CDOX via a calibration curve constructed with standard solutions of DOX. The DOX release behavior at various pH values was monitored by plotting the cumulatively released DOX content as a function of incubation time.

ASSOCIATED CONTENT The authors declare no competing financial interest. Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental methods: protocols for cell and animal experiments. Table S1-4: synthesis conditions and characterization results. Figure S1: schematic representation for the chemical reactions and synthesis process. Figure S2, 3: TEM images and size distributions. Figure S4-6: r1 or r2 measurements with a magnetic field of 7.0, 1.5, or 0.5 T. Figure S7-10: UV-vis spectra, FT-IR spectra, HR-TEM image, EDS, XRD, and XPS spectra. Figure S11: hydrolysis behavior of the acid-labile

β-thiopropionate

linker.

Figure

S12.

Zeta

potential

changes

of

DOX@ES-MION3@RGD2@mPEG3 after incubation in PBS with different pH values. Figure S13: release behaviors of DOX. Figure S14, 15: the LSCM images. Figure S16: flow cytometry analysis. Figure S17: MR imaging on cells. Figure S18: T1-weighted MR images of U-87 MG tumor-bearing nude mice for analysis of bladder showing evidence of renal excretion. Figure S19: photos of tumor-bearing nude mice on the 14th day post-treatment (PDF).

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] 22


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Author Contributions #

Z.S. and T.C. contributed equally to this work.

ACKNOWLEDGMENTS This work is supported in part by Youth Innovation Promotion Association of Chinese Academy of Sciences (2016269) (Z.S.), the Intramural Research Program (IRP), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH) (Grant No. ZIA EB000073), the Public Welfare Technology Application Research Project of Zhejiang Province (2017C33129), the National Key Research & Development Program (2016YFC1400600), National Natural Science Foundation of China (Grant Nos. 51761145021, 61571278 and 21305148), Bureau of Science and Technology of Ningbo Municipality City (Grant Nos. 2015B11002), and NSFC-Guangdong Province Joint Project on National Supercomputer Centre in Guangzhou (NSCC-GZ) (A.W.).

REFERENCES (1) Shen, Z.; Wu, A.; Chen, X. Iron Oxide Nanoparticle Based Contrast Agents for Magnetic Resonance Imaging. Mol. Pharm. 2017, 14, 1352-1364. (2) Jia, Z.; Song, L.; Zang, F.; Song, J.; Zhang, W.; Yan, C.; Xie, J.; Ma, Z.; Ma, M.; Teng, G.; Gu, N.; Zhang, Y. Active-Target T1-Weighted MR Imaging of Tiny Hepatic Tumor Via RGD Modified Ultra-Small Fe3O4 Nanoprobes. Theranostics 2016, 6, 1780-1791. (3) Wang, Y.; Chen, J.; Yang, B.; Qiao, H.; Gao, L.; Su, T.; Ma, S.; Zhang, X.; Li, X.; Liu, G.; Cao, J.; Chen, X.; Chen, Y.; Cao, F. In vivo MR and Fluorescence Dual-Modality Imaging of Atherosclerosis Characteristics in Mice Using Profilin-1 Targeted Magnetic Nanoparticles. Theranostics 2016, 6, 272-286. (4) Gao, Z.; Ma, T.; Zhao, E.; Docter, D.; Yang, W.; Stauber, R. H.; Gao, M. Small is Smarter: Nano MRI Contrast Agents – Advantages and Recent Achievements. Small 2016, 12, 556-576. (5) Yan, G. P.; Robinson, L.; Hogg, P. Magnetic Resonance Imaging Contrast Agents: Overview and Perspectives. Radiography 2007, 13, e5-e19. 23


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(27) Kaittanis, C.; Shaffer, T. M.; Ogirala, A.; Santra, S.; Perez, J. M.; Chiosis, G.; Li, Y.; Josephson, L.; Grimm, J. Environment-Responsive Nanophores for Therapy and Treatment Monitoring Via Molecular MRI Quenching. Nat. Commun. 2014, 5, 3384. (28) Mikhaylov, G.; Mikac, U.; Magaeva, A. A.; Itin, V. I.; Naiden, E. P.; Psakhye, I.; Babes, L.; Reinheckel, T.; Peters, C.; Zeiser, R.; Bogyo, M.; Turk, V.; Psakhye, S. G.; Turk, B.; Vasiljeva, O. Ferri-Liposomes As an MRI-Visible Drug-Delivery System for Targeting Tumours and Their Microenvironment. Nat. Nanotechnol. 2011, 6, 594-602. (29) Kislukhin, A. A.; Xu, H.; Adams, S. R.; Narsinh, K. H.; Tsien, R. Y.; Ahrens, E. T. Paramagnetic Fluorinated Nanoemulsions for Sensitive Cellular Fluorine-19 Magnetic Resonance Imaging. Nat. Mater. 2016, 15, 662-668. (30) Zhou, Z.; Zhao, Z.; Zhang, H.; Wang, Z.; Chen, X.; Wang, R.; Chen, Z.; Gao, J. Interplay between Longitudinal and Transverse Contrasts in Fe3O4 Nanoplates with (111) Exposed Surfaces. ACS Nano 2014, 8, 7976-7985. (31) De Leon-Rodriguez, L. M.; Martins, A. F.; Pinho, M. C.; Rofsky, N. M.; Sherry, A. D. Basic MR Relaxation Mechanisms and Contrast Agent Design. J. Magn. Reson. Imaging 2015, 42, 545-565. (32) Zhou, Z.; Wu, C.; Liu, H.; Zhu, X.; Zhao, Z.; Wang, L.; Xu, Y.; Ai, H.; Gao, J. Surface and Interfacial Engineering of Iron Oxide Nanoplates for Highly Efficient Magnetic Resonance Angiography. ACS Nano 2015, 9, 3012-3022. (33) Hyon Bin Na, H. B.; In Chan Song, I. C.; Taeghwan Hyeon, T. Inorganic Nanoparticles for MRI Contrast Agents. Adv. Mater. 2009, 21, 2133-2148. (34) Sheng Eng, A. Y.; Sofer, Z.; Sedmidubsky, D.; Pumera, M. Synthesis of Carboxylated-Graphenes by the Kolbe-Schmitt Process. ACS Nano 2017, 11, 1789-1797. (35) Kang, T. W.; Jeon, S. J.; Kim, H. I.; Park, J. H.; Yim, D. B.; Lee, H. R.; Ju, J. M.; Kim, M. J.; Kim, J. H. Optical Detection of Enzymatic Activity and Inhibitors on Non-Covalently Functionalized Fluorescent Graphene Oxide. ACS Nano 2016, 10, 5346-5353. (36) Cheong, S.; Ferguson, P.; Feindel, K. W.; Hermans, I. F.; Callaghan, P. T.; Meyer, C.; Slocombe, A.; Su, C. H.; Cheng, F. Y.; Yeh, C. S.; Ingham, B.; Toney, M. F.; Tilley, R. D. Simple Synthesis and Functionalization of Iron Nanoparticles for Magnetic Resonance Imaging. Angew. Chem. Int. Ed. 2011, 50, 4206-4209.

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Figure 1. Design of DOX@ES-MION@RGD2@mPEG composite nanoparticles and the principle of reducing non-specific uptake by normal healthy cells with non-specific receptors. When the DOX@ES-MION@RGD2@mPEG nanoparticles are circulating in the blood stream (pH ~ 7.4), the hydrophilic polymer mPEG can prevent RGD2 from binding to the healthy cells with non-specific receptors because RGD2 is hidden in the mPEG. However, in the tumor microenvironment, the acid-labile β-thiopropionate linker is broken, which results in mPEG shedding from the nanoparticles. The hidden RGD2 ligands are then exposed and able to bind to cancer cells with αvβ3 expressed on the surface.

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Figure 2. MRI efficiencies of the ES-MIONs. (a) The r1 value and r2/r1 ratio of ES-MIONs as a function of the particle size (< 5 nm) (mean ± SD, n = 3). * P < 0.02. (b) T1-weighted MR images of ES-MION3, 5, 8 at various Fe concentrations (0.031 ~ 1.000 mM) (TE = 20 ms, TR = 500 ms). Magnetic field = 7.0 T. (c) Relative intensity of the MR images for ES-MION3, 5, 8 at various Fe concentrations as shown in (b), measured by the ImageJ and compared with the pure water (CFe = 0).

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Figure 3. TEM images of ES-MION3 (a) and DOX@ES-MION3@RGD2@mPEG3 (b) showing the

well-dispersed

nanoparticles.

(c)

Size

distribution

of

ES-MION3

and

DOX@ES-MION3@RGD2@mPEG3 measured by DLS. (d) Field-dependent magnetization curves (H - M) of ES-MION3, ES-MION5, ES-MION8, and DOX@ES-MION3@RGD2@mPEG3 at 300 K.

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Figure

4.

The

LSCM

images

of

U-87

MG

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or

MCF-7

cells

incubated

with

DOX@ES-MION3@RGD2 or DOX@ES-MION3@RGD2@mPEG3 dialyzed against PBS (pH = 7.4 or 6.5) for 72 h at 37 °C. The cells untreated with nanoparticles were used as the control. The cytoskeleton was stained with phalloidin-FITC (green) and the nucleus was stained with Hoechst (blue). The DOX-loaded nanoparticles were red.

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Figure 5. (a) U-87 MG cell viabilities treated with different nanoparticles (mean ± SD, n = 5). DOX@ES-MION3@RGD2@mPEG1-4 nanoparticles were incubated at pH 7.4 or 6.5 for 72 h before incubation with cells. (b) Plot of the difference value of the cell viability for DOX@ES-MION3@RGD2@mPEG1-4 between pH 7.4 and 6.5 as a function of the DOX concentration. (c) MCF-7 cell viabilities treated with different nanoparticles (mean ± SD, n = 5). Free DOX was used as a control.

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Figure 6. T1-weighted MR images of U-87 MG tumor-bearing nude mice (slice orient: axial) (a-c) and quantitative analysis of the tumors (d-f) after intravenous injection of Magnevist (CGd = 5.0 mg / kg), ES-MION3 (CFe = 5.0 mg / kg), or DOX@ES-MION3@RGD2@mPEG3 (CFe = 5.0 mg / kg).

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Figure 7. T1-weighted MR images of U-87 MG tumor-bearing nude mice (slice orient: coronal) (a, b) and quantitative analysis of tumor, liver, and spleen (c, d) after intravenous injection of DOX@ES-MION3@RGD2@mPEG3 (CFe = 5.0 mg/kg). (e) Fe level in different organs/tissues in the U-87 MG tumor-bearing nude mice at 12 h post-injection or pre-injection of DOX@ES-MION3@RGD2@mPEG3. 35


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Figure

8.

Anti-tumor

efficacies

of

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DOX@ES-MION3@RGD2

and

DOX@ES-MION3@RGD2@mPEG3 in U-87 MG tumor mice (mean ± SD, n = 5). PBS and free DOX were used as controls. (a) Tumor volume changes of mice treated with PBS, free DOX, DOX@ES-MION3@RGD2, or DOX@ES-MION3@RGD2@mPEG3. (b) Mouse survival in the different treatment groups. (c) Body weight changes of mice in the different treatment groups.

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Graphic Abstract A co-precipitation method with precise size control was developed for ES-MIONs. The relationship between the r1 (or r2/r1) and the particle size of ES-MIONs demonstrate that 3.6 nm is the best particle size for ES-MIONs to be used as T1-weighted MRI contrast agent and for drug delivery as cancer theranostics.

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Multifunctional Theranostic Nanoparticles Based on Exceedingly

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