Ultrasmall Super-paramagnetic Iron Oxide Nanoparticle for T2

Subscriber access provided by University of Rochester | River Campus & Miner Libraries

Article

Ultrasmall Super-paramagnetic Iron Oxide Nanoparticle for T2-weighted Magnetic Resonance Imaging Yaling Wang, Chao Xu, Ya-Nan Chang, Lina Zhao, Kai Zhang, Yuliang Zhao, Fuping Gao, and Xueyun Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10030 • Publication Date (Web): 08 Aug 2017 Downloaded from http://pubs.acs.org on August 8, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Ultrasmall Super-paramagnetic Iron Oxide Nanoparticle for T2-weighted Magnetic Resonance Imaging Yaling Wang,† Chao Xu,†,⊥ Yanan Chang,† Lina Zhao,† Kai Zhang,§Yuliang Zhao,† Fuping Gao† and Xueyun Gao*,‡ †

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High

Energy Physics, Chinese Academy of Sciences, Beijing, China. ‡

Department of chemistry and chemical engineering, Beijing University of Technology.

⊥

College of Chemistry and Material Science, Shandong Agricultural University, Tai‫׳‬an,

Shandong, China. §

Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of

Sciences, Beijing, China.

ABSTRACT The facile method to synthesize ultrasmall size super-magnetic iron oxide nanoparticle with good mono-dispersity, high relaxivity is desired for magnetic resonance imaging (MRI) technology. Herein, we developed a one-step method to direct the formation of super-paramagnetic iron oxide nanoparticle (uBSPIO) using albumin at mild condition. The

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 27

resulting uBSPIO possess ultrasmall size (4.78 ± 0.55 nm) and super high MR relaxivity (444.56 ± 8.82 mM-1·s-1). After grafted by the luteinizing hormone release hormone peptide (LHRH), the uBSPIO could targeted and accumulated in the tumor site. Finally, the uBSPIOs had good stability, did not induce cytotoxicity in vitro or major organ toxicity in vivo. The uBSPIOs are promising contrast agent for MR imaging.

KEYWORDS. BSA, Biomineralization, Ultrasmall Superparamagnetic Iron Oxide, T2-weighted Magnetic Resonance Imaging. 1. INTRODUCTION Recent years, versatile nanoparticles have been developed for biological applications. Among these nanomaterials, super-paramagnetic iron oxide nanoparticles (denoted as SPION) have attracted a lot of interest because of their very specific physicochemical properties, such as better sensitivity, low toxicity, and possibility to easily modify their surface for bio-application.1 SPION, hydrodynamic diameter ranging from 5 nm to 300 nm, have been rational designed for diagnostic and therapeutic applications.2 Such as, in vivo cell track and monitor after regenerative cytotherapy,3 targeted delivering drugs,4 distinguishing pathological tissues from healthy surrounding tissues in magnetic resonance imaging (MRI).5-7 MRI technology has several advantages including excellent anatomic detail, enhanced soft tissue contrast, and high spatial resolution (submillimeter).8 The noninvasive and painless diagnosis of disease via MRI can eliminate the risk of cancer caused by X-ray radiation.9 The only drawback of MRI is the relatively low sensitivity, which demands high quality SPION to ensure efficiently imaging effects. For in vivo MR tumor imaging, the SPION should have narrow size distribution, high relaxivity and specific tissue targeting ability, no matter via active


2

Page 3 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


or passive targeting routes.2, 10 Plenty of procedures based on physical, biological and chemical methods have been developed for synthesizing the small size SPION for T2-weighted MRI.11-13 Proteins have been used to synthesize or functionalize the SPION, to enhance the stability, hydrophility, and relaxivity of the SPION.14-18 Few natural or synthetic ferritins cages have been introduced in small size iron oxide nanoparticle construction for MRI.5,

18-19

For example,

Toshiro Kitagawa et al. had synthesized RGD peptide functionalized iron oxide nanoparticles inside the engineered human ferritin for in vivo vascular inflammation and angiogenesis MRI.18 Despite of the high cost of ferritin, complicated synthesize procedures, N2 atomosphere and high temperature were necessarily for the iron oxide nanoparticles synthesis,18-19 and these conditions hindered the extensively application of ferritin caged iron oxide nanoparticle for MRI. In contrast, Bovine serum albumin (BSA) is known as the most abundant representative serum protein; it is commercial availability and low cost. BSA-based bio-mineralization process many advantages such as good methodology reproducibility, biocompatibility and bio-stability.20 Thus BSA has been introduced in mineralizing the uniform-sized metal oxide nanoparticles at routine laboratory conditions, such as metal nanoclusters, quantum dots and gadolinium oxide nanoparticles.20-23 In addition, its abundant active groups on amino acid residues (-NH2, -COOH) facilitate functionalization on the protein scaffold, and the negative charged amino acids provide plenty of positions to complex and stable iron ions.24 Therefore, using BSA mineralized SPION to achieve the tumor targeted MRI is highly potential. As we know, the data about using BSA as a bio-template to direct the formation of Fe3O4 for T2-weighted tumor MRI have not been reported yet. Here, we developed a one pot procedure to synthesize the ultra-small Fe3O4 nanoparticle (denoted uBSPIO) using BSA as scaffold under mild conditions, followed by the conjugation of


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 27

luteinizing hormone releasing hormone peptide (LHRH) with albumin. The resulting uBSPIOs possess small core diameter (4.78 ± 0.55 nm), high saturation magnetization (84.32 emu/g) and robust transverse relaxivity (444.56 ± 8.82 mM-1·s-1). The LHRH grafted with uBSPIOs, enabled the LHRH-uBSPIOs to selectively accumulate and distribute in the tumor. Besides the good MRI contrast effect, the uBSPIOs did not cause cytotoxicity in vitro or major organ toxicity in vivo. 2. RESULTs AND DISCUSSION 2.1 Synthesis and characterization of uBSPIO A one step biomineralization method was developed to synthesize ultra-small size magnetite nanoparticles (uBSPIO) with BSA. Briefly, as shown in Scheme 1a, the aqueous solution mixture of ferric and ferrous salts was added into the BSA under stirring. Subsequently, the solution of NaOH was injected into the mixture to regulate the pH to above 12. The solution was kept in 37 °C water bath for 4 h. LHRH was covalently grafted on the uBSPIO for the tumor targeting imaging via T2-weighted MRI (Scheme 1b). The negatively charged residues directed Fe ions into the inner cavity of the protein and involved in the binding and stabling the iron ions during the nucleation process.24-25 The molecular dynamics simulation of the BSA structure suggested the abundant negatively charged residues (Glu and Asp) and caves exists in the BSA (see figure S1). This benefits the formation of Fe3O4 with BSA as scaffold.


4

Page 5 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. (a) the procedure of the BSA-templated uBSPIO synthesis and functionalization. (b) LHRH-uBSPIO as a LHRH receptor targeted contrast agent for tumor MR imaging. The shape, size of the crystal and the hydrodynamic diameter of the SPION influence the biological application.2 The resulting uBSPIO nanoparticle solution was dark brown in color (Figure 1a). X-ray powder diffraction (XRD) analysis (Figure 1b) of uBSPIO nanoparticles revealed that a single-phase cubic inverse spinel Fe3O4 nanostructure was formed, which corresponded to the JCPDS file 19-0629. According to the Scherrer formula, the mean crystallite diameter was estimated about 4.5 nm.26-27 The X-ray photoelectron spectroscopy (XPS) spectra of Fe 2p3/2 (711.1 eV) and Fe 2p1/2 (724.3 eV) could be ascribed to Fe3+ species in the uBSPIO nanoparticle (Figure 1c). The absence of the satellite peak at around 718 eV revealed the formation of Fe3O4 in our experiments.28 The TEM micrographs (Figure 1d) showed that the cores of the uBSPIO nanoparticles had good uniformity, and that almost all the nanoparticles were mono-dispersed and no obvious aggregation. The average core size of the uBSPIO


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 27

nanoparticle of 4.78 ± 0.55 nm was calculated by measuring 100 uBSPIO nanoparticles (Figure 1e), which was in accordance with the XRD data. As shown in Figure 1f, the dominant hydrodynamic diameter of the uBSPIO nanoparticle was about 5.4 ± 0.93 nm (98%), which further illustrated the good stability and uniformity of these nanoparticles.

Figure 1. (a) Digital photographs of the BSA and uBSPIO solutions. (b) The XRD pattern of the uBSPIO (black and red curves corresponded to the raw data and FFT smoothed data, respectively). (c) The XPS spectra of Fe 2p in the uBSPIO sample (black and red curves corresponded to the experimental data and fitted data, respectively). (d)Transmission electron microscope (TEM) image of uBSPIO. (e) The size distribution of the uBSPIO. (f) The


6

Page 7 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hydrodynamic diameter number weighted Nicomp distribution of the uBSPIO. (g) A HRTEM image of a representative region of the uBSPIO. (h) and (i) Images corresponding to the different crystalline images of the white square-marked Fe3O4 nanoparticles shown in (g). High-resolution TEM (HRTEM) could provide more detailed property of the uBSPIO nanoparticle such as the structural information. Figure 1g showed a representative TEM image of uBSPIO nanoparticles which further supported the crystalline nature. The lattice spacings were also calculated at approximately 4.85 Å and 2.97 Å (Figure 1h), which were corresponded to the separation between the [111] and [220] lattice planes, respectively. The 2.55 Å and 2.51 Å interlayer distances (Figure 1i) agreed well with the lattice spacing of [311] facet of the cubic phase of Fe3O4 (JCPDS file 19-0629). The small size, well mono-dispersity, single-phase cubic inverse spinel Fe3O4 structural characteristics, imply a good MR efficiency for uBSPIO. 2.2 The MR efficiency of uBSPIO. The two paramount importance properties, regarding the MRI application, of the imaging agent are superparamagnetic behavior and induced relaxation properties. To understand the MR efficiency, the magnetization and relaxation rate were measured. The lyophilized uBSPIO nanoparticles were used to test the hysteresis loop. Figure 2a showed the magnetization curve of the uBSPIO nanoparticle, in which negligible hysteresis was observed, suggested that the iron oxide nanoparticles obtained were superparamagnetic. The saturation magnetization of the Fe3O4 nanoparticles (uBSPIO nanoparticle) (MS = 84.32 emu/g) was much higher than that reported for the Fe3O4 nanoparticles of a comparable size,29 suggesting that uBSPIO is a promising candidate for MRI.30


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 27

Figure 2. The Super-paramagnetic behavior and in vitro MRI properties of uBSPIOs. (a) The hysteresis loops of the uBSPIO at 300 K. (b) T1- and T2-weighted MR phantom images of the uBSPIO with series of iron concentrations (Fe: 0.024, 0.048, 0.074, 0.097, 0.121, 0.146, 0.170, and 0.195 mM), obtained under a 7 T magnetic field. (c) The plot of relaxation rates (1/T1) and (1/T2) with the iron concentration of uBSPIO. Typically, the ultra-small-sized SPIONs contributed to the decrease in the brightness of the T2images, darkening effect of the image (negative contrast) can be observed.31 Thus, the phantom images and relaxation rate of uBSPIO were measured to evaluate the contrast efficiency of the uBSPIO. As shown in Figure 2b, T2-weighted MR phantom images of the uBSPIO were progressively darkened with increasing Fe concentration. In contrast, the T1-weighted images of the uBSPIO became slightly brighter with increasing Fe concentration. A linear relationship was found between the longitudinal (1/T1) and transverse (1/T2) relaxation rates and iron concentration for the uBSPIO nanoparticles (Figure 2c). It was concluded that, as expected, the


8

Page 9 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


uBSPIO nanoparticles had a low longitudinal (0.93 ± 0.04 mM-1·s-1) and high transverse (444.56 ± 8.82 mM-1·s-1) relaxivity (Figure 2c). In addition to the r1,2 relaxivities, the r2/r1 ratio needs to be considered. Because the signal intensity will be significantly attenuated even in T1-weighted MRI, if the T2-weighted image contrast effect is too strong. Therefore, the r2/r1 ratio of the effective T1-weighted image contrast agents is much smaller than that of the T2-weighted image contrast agents. For uBSPIO nanoparticles an r2/r1 ratio of 478 was obtained. In addition, the high magnetization saturation of the uBSPIO nanoparticles indicates these nanoparticles are more suitable for T2-weighted MRI.

Figure 3. The optical absorption of the uBSPIO in (a) 10% FBS solution with 24 h and (b)-(d) corresponding to the digital photos, absorption spectra and zeta potential of uBSPIO incubated with various pH PBS buffer (6, 6.5, 7, 7.5, 8, 8.5) for 24 h, respectively.


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 27

2.3 The in vitro stability and safety of uBSPIO. The stability of the uBSPIO nanoparticle is critical to its storage and usage in in vivo MRI, enabling the choice of the optimal interval between injection and imaging.32 The optical absorption, digital photo, zeta potential of uBSPIO nanoparticles were recorded in 10% FBS solution and PBS buffer with various pH. As can be seen in Figure 3a, no obvious changes were noticed in the optical absorption range from 220 nm to 800 nm, indicating that the uBSPIO nanoparticles can keep stable in the FBS medium for over 24 h. We also noticed that after 24 h incubation in PBS buffer with pH varied from 6 to 8.5, all uBSPIO colloidal solutions maintained the transparent light brown color, no precipitation was observed (Figure 3b). Correspondingly, no significant changes of optical absorption and zeta potential (absolute value >37.5 mV) of uBSPIO solution with pH varied from 6 to 8.5 were found (Figure 3c and 3d). The high zeta potential value indicated the good dispersion stability of the uBSPIO.2 The charge of surface and the interparticle steric repulsions introduced by BSA,1, 33-34 enabling the good stability of uBSPIO nanoparticles, and indicating uBSPIO are suitable for further biological application. 2.4 The safety of LHRH grafted uBSPIO. The LHRH peptide have been widely applied to functionalize the nanoparticles to target tumor tissue,35-36 because in several kinds of cancer, such as lung caner, breast cancer etc., its receptor always overexpressed. A peptide, LHRH, was covalently grafted on the uBSPIO to evaluate the safety and the contrast effect for MR tumor imaging. As shown in the figure S3, through the Prussian blue staining for iron assessment, Fe distributed in the A549 cell (LHRH receptor positive37-38) after LHRH-uBSPIO incubation 12 h, in contrast, there was no obvious distribution


10

Page 11 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for the uBSPIO incubation group. This proved the surface functionalization with LHRH could increase the intracellular efficiency of the uBSPIO.

Figure 4. The cytotoxic effects of LHRH-uBSPIOs on A549 cells. (a–g) The live and dead stained fluorescence images and (h) the viability of A549 cells after incubated with various concentration of LHRH-uBSPIOs (Fe concentrations: 0, 10, 25, 50, 100, 300, and 600 µg/mL) for 24 h. The green colour indicates the presence of live cells and red colour represents dead cells. (i) hemolysis analysis. Ultrapure water (+) were set as positive controls and PBS (-), negative controls. Inset: Digital photos for direct observation of hemolysis.


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 27

Before MRI in vivo, we also evaluated the safety of LHRH-uBSPIO. The cytotoxicity of the LHRH-uBSPIO regarding the A549 cell line were studied through live/dead stain and CCK-8 assays. As shown in Figure 4, very few dead cells (red color) were found in the Fe-containing groups, even when the Fe concentration reached 600 µg/mL (Figure 4a–g). This was further demonstrated using the viability assay (CCK-8). We found that in almost all experimental groups, the proportion of live cells was ˃95% (Figure 4h). When the Fe concentration reached 600 µg/mL, only 6% of A549 cells were dead. In addition, the influence of LHRH-uBSPIO nanoparticles of different Fe concentrations on the hemolytic behavior of red blood cells was studied. Even when the concentration of LHRH-uBSPIO nanoparticles reached 600 µg/mL, negligible hemolysis was detected, indicating good blood compatibility (Figure 4i) of these nanoparticles. Therefore, the low cytotoxicity and nice biocompatibility promised the application of LHRH-uBSPIO for MRI.


12

Page 13 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. T2-weighted in vivo MRI and corresponding signal loss percentage of tumor after tail vein injection of LHRH-uBSPIOs. (a) Coloured MR images from a mouse at 0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 h post LHRH-uBSPIO injection (coronal plane, 7 T micro-MRI). T: tumor; (b) The corresponding statistic histogram involving the MRI signal loss percentage of tumor obtained at each time point. Three areas (1 mm × 1 mm) were selected to measure the T2 value and calculate the mean signal loss percentage and error bar. To assess the negative contrast effect of the uBSPIO nanoparticles, the dynamic distribution in tumor was tracked by means of T2-weighted MRI. The saline (control group) and the LHRHuBSPIO (experimental group) were intravenously injected into the mice through the tail vein. From the colored MR images (Figure 5a) and the corresponding histogram of signal loss percentage (Figure 5b), the LHRH-uBSPIO nanoparticles began to accumulate in the tumor at 0.5 h time point; obvious darkening was observed at 1 h post LHRH-uBSPIO nanoparticle injection (8.03% signal loss). And a plateau was observed after 1.5 h post-injection; around 15% signal loss were maintained for almost 3 hours. This gave researcher plenty of time to evaluate the pathological information of tumor. To further observe the intra-tumor distribution of LHRHuBSPIO nanoparticles, we sacrificed the saline and LHRH-uBSPIO-injected mice after the MRI assays; frozen sections of the tumor were stained with Prussian blue to observe the Fe distribution. Fe nanoparticles were found in the tumor (Figure S4). Majority of the small-sized LHRH-uBSPIO nanoparticles located near the blood vessels, and almost no mass aggregation was observed.


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 27

Figure 6. Photomicrographs showing hematoxylin and eosin (H&E)-stained histological tissue sections of major organ from mice receiving (a) saline (control), (b) & (c) uBSPIO and LHRHuBSPIO saline solution injection for 7 days, respectively. Furthermore, hematoxylin and eosin staining was used to investigate the histological variations of the mice 7 days after uBSPIO nanoparticles administration (Figure 6). No histological difference of the major organs was found between the uBSPIO, LHRH-uBSPIO and the control groups, indicating that no tissue damage occurred as the result of the uBSPIO nanoparticles injection. These preliminary results show that the uBSPIO had good biocompatibility in short term at the tested Fe concentration. 3. CONCLUSIONS

In summary, we have rapidly fabricated ultrasmall iron oxide nanoparticles (mean core size: 4.78 ± 0.55 nm) using the BSA-templated mild bio-mineralization method. The resulting Fe3O4 possessed superparamagnetism, good mono-dispersity, stability and had very high transverse relaxivity. The uBSPIO nanoparticles exhibited good in vivo T2-weighted MRI ability, and the


14

Page 15 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


LHRH grafted uBSPIO can actively targeted the tumor for imaging. The uBSPIO nanoparticles have good biocompatibility and may be suitable for the diagnosis of tumor and other kinds of tissue abnormalities using MRI. 4. EXPERIMENTAL METHODS

4.1 Molecular Simulations of Negatively Charged Residues Distributed in BSA. To understand the mechanism of uBSPIO nanoparticle formation in BSA, we performed molecular simulation to study the negatively charged residues distributed in BSA. We relaxed the crystal structure of BSA (PDB ID: 3V03) in solvation by means of a molecular dynamics calculation for 15 ns using the NAMD2.9 package. The residues Asp and Glu were highlighted in red and blue throughout the relaxed BSA. These visualizations were rendered by visual molecular dynamics. 4.2 Materials. The ferrous and ferric salts (FeCl2·4H2O and FeCl3·6H2O), BSA and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich. Calcein-AM, propidium iodide (PI) and the Cell Counting Kit-8 were purchased from Dojindo Laboratories, Japan. A Perls Stain Kit was obtained from Solarbio Life Science (Cat#G1390: Beijing Solarbio Science & Technology Co. Ltd, China). The cell culture medium and wash buffer were RPMI-1640, 0.25% trypsin-EDTA (1×); FBS and PBS buffer were obtained from Hyclone. The A549 cell line was purchased from the Cancer Institute and Hospital, Chinese Academy of Medical Sciences. The optical cutting temperature compound was obtained from Sakura Finetek USA, Inc. 4.3 Synthesis of uBSPIO. The freshly prepared iron salt mixture aqueous solutions (the stoichiometric ratio of Fe3+ to Fe2+ was 1:2; the final volume was 0.8 mL) was added to the BSA (20 mg/mL; 10 mL) solution under vigorous stirring at 37°C; the reaction was stopped at 4 h post 1 mL of NaOH (1 M) was injected into the mixture solution. The dialysis tube (MWCO: 30 KDa,


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 27

(500–4000) µL Midi D-tube, Merck) was used to remove free BSA and other ions. The purified uBSPIO nanoparticles were used to perform the following assays. 4.4 The Surface Modification of uBSPIO with LHRH peptide. The LHRH peptide (Pyroglutamic acid-HWSYGLRPG-AHx, AHx represent the 6-aminocaproic acid, purity: >95%) was obtained from Wu Han Moon Biosciences Co, Ltd. In the typical synthesis, 29.41 mg LHRH was dissolved in the 1 mL MES buffer solution (pH=6), then 4.35 mg EDC and 2.61 mg NHS were added into the LHRH MES solution, then reacted for 45 min, shield from light at room temperature. Subsequently, the previously prepared uBSPIO (5.9 mL) were introduced into the activated LHRH solution, and rapidly adjusted pH of the mixture solution to 7.5 and the reaction were continued for 3.5 h. At the end of the conjugation, LHRH-uBSPIO were purified use the dialysis tube to remove free LHRH peptide and other ions for further use. The hydrodynamic diameter of LHRH-uBSPIO increased to ~9.9 nm after surface modification. And the zeta potential is about -40 mV in water. 4.5 Characterization of uBSPIO and LHRH-uBSPIO nanoparticles. The detailed characterization experiments proceeded similarly to our previous work.22 For HRTEM, the uBSPIO nanoparticle was dispersed and dried on the ultrathin carbon-coated copper grids before TEM studies. The HRTEM image was obtained using a JEM-2100 TEM (Japan). The hydro-diameter and zeta potential of uBSPIO and LHRH-uBSPIO were measured at 25°C using a dynamic light scattering particle sizer (NICOMP 380/ZLS [PSS]). XRD data were recorded by X-ray powder diffractometer (Rigaku D/max-2500, Japan). The XPS measurements were performed on an ESCALab220i-XL spectrometer (VG Thermo, USA). Optical absorption spectra of the BSA, uBSPIO and LHRH-uBSPIO nanoparticles were measured on a spectrophotometer (Shimadzu UV-1800, Japan).


16

Page 17 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4.6 The in vitro relaxivity of the uBSPIO. 0.5 mL of the uBSPIO nanoparticle (0.024, 0.048, 0.074, 0.097, 0.121, 0.146, 0.170, and 0.195 mM of the Fe element) solution were placed in 0.5mL centrifuge tubes, the corresponding phantom images and the mean T1 or T2 signal intensity values were acquired using a 7 T BioSpec70/20USR Micro-MRI Scanner (Bruker) at room temperature. 4.7 Stability of uBSPIO. The stability of uBSPIO nanoparticles were investigated by incubating them in 10% FBS for 0, 1, 2, 4, 12, and 24 h and in PBS solution at various pH (6, 6.5, 7, 7.5, 8, and 8.5) for 24 h at room temperature. The appearance of the precipitate was assessed by visual inspection, then the corresponding UV-vis absorption and zeta potential of uBSPIO nanoparticles were measured after incubation. 4.8 In Vitro A549 Cell targeting Evaluation of LHRH-uBSPIO. To observe the targeting ability of uBSPIO after surface functionalization, 5 × 10 4 A549 cells were seeded in the glassbottom dish in a 1640 medium. After pre-culture for 12 h, the culture medium in the dishes were removed. The freshly prepared LHRH-uBSPIO and uBSPIO (Fe: 2 mM) were added to the dish. After incubation for 12 h, cells were washed twice, then fixed 15 min with 4% paraformaldehyde. The intracellular distribution of Fe nanoparticles was studied by Prussian blue staining method. Cells were stained followed the instructions of the Perls Stain Kit. Finally, the colored images of cell were performed at an inverted fluorescence microscope (Olympus X73, Japan,10× objective lens). 4.9 Live/Dead Staining of A549 cell. For the live and dead stain, a density of 5×104 A549 cells were seeded and cultured in 6-wells plates for 12 h; then they were incubated with LHRHuBSPIO nanoparticle-containing culture medium (Fe concentration: 0, 10, 25, 50, 100, 300, and 600 µg/mL) for 24 h. Then A549 cell were washed and stained with the stain solution of Calcein-


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 27

AM (2 µmol/L) and PI (4 µmol/L) for 10 min. Subsequently, the cell fluorescence images were acquired. 4.10 In Vitro Cell Viability Evaluation of LHRH-uBSPIO. The cell viability assay were performed using Counting Kit-8 (CCK-8). A series of concentrations of LHRH-uBSPIO with Fe (0, 10, 25, 50, 100, 300, and 600 µg/mL) were incubated with experimental cell groups. There were three parallel samples for each condition. After culturing the samples for 24 h, each group of cells were washed and incubated with 10% (v/v) CCK-8 solution for 2 h. Then the absorbance of each group at 450 nm was recorded to calculated the cell viabilities. 4.11 Hemolysis assay. 1 mL blood sample was collected from mice, then diluted to 10 mL with PBS. To perform hemolysis assay, 0.5 mL of diluted red blood cell suspension solution was mixed with 0.5 mL (i) PBS solution of LHRH-uBSPIO nanoparticles (Fe concentration: 0 to 600 µg/mL); (ii) 0.5 mL PBS; and (iii) deionized water, respectively. After 2 h incubation, samples were centrifuged at 10050 rpm/min for 3 min. The absorbance at 570 nm of the supernatants was measured to calculate the hemolysis percentage. 4.12 Histology Examinations. All animal experiments were performed in compliance with the local ethics committee. Female BABL/c athymic nude mice (nu/nu, 4 weeks of age; body weight, approximately 20 g) used for in vivo experiments were obtained from Beijing HFK Bioscience Co., Ltd. Every five mice were feeding in one cage, with water and food available ad libitum according to Institutional Animal Care and Use Committee approval. The in vivo toxicity of the saline, uBSPIO and LHRH-uBSPIO nanoparticles to mice were evaluated by observing histological variations in the major organs of the mice at 7 days post injection. The obtained organs were stained with H&E for examination.


18

Page 19 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4.13 Xenografted Tumor Model Establishing. To establish the subcutaneous xenografted tumor model, a mixture of 1 × 107 A549 cell suspension (50 µL) and matrix gel (50 µL) was subcutaneously inoculated into the right leg of the mice. In vivo assays were performed when the tumor volume reached 200–500 mm3. 4.14 In Vivo MR tumor imaging and biodistribution studies. For in vivo MRI, the 0.2 mL saline-containing LHRH-uBSPIO nanoparticles (4.5 mg/mL, Fe concentration) were intravenous injected into the control and experimental mice via tail vein, respectively. The whole body was exposed to a 7 T BioSpec70/20USR Micro-MRI Scanner (Bruker), T2-weighted MR images were acquired at different time points (Preinjection, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 h). The mice were anesthetized using isoflurane gas during the entire MRI scan. To quantitatively observe the distribution of the uBSPIO nanoparticles, the tumor T2 values were measured at each time points using the map_MSME sequence, TR: 3000 ms, and TE: 6.5–104 ms. All the experimental sequences were the standard sequences, provided by the BioSpec70/20USR scanner (Bruker). 4.15 Prussian Blue Staining of Tumor after MRI. To directly observe the intra-tumor distribution of the LHRH-uBSPIO nanoparticles (Fe element), saline containing the nanoparticles was injected into mice after in vivo MRI assay; saline injected mice were set as control. They were sacrificed and the tumors collected after MRI experiments. The resected tissue was kept at -80 °C. The frozen tissues were sectioned at 10 µm, and then stained using the Perls Stain Kit (Prussian blue reaction) following the standard protocol. And the Fe distribution images were taken using an inverted fluorescence microscope (Olympus X73, Japan). The in vivo experiments were performed similarly to our previous work.22


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 27

ASSOCIATED CONTENT Supporting Information. Information on the theoretical study about the BSA, the DLS data of LHRH-uBSPIO, the Fe distribution of LHRH-uBSPIO in A549 cells and tumor of are available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Tel: 86-10-88236709 Fax: 86-10-88236456. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the National Key Basic Research Program of China (2013CB932703), National Natural Science Foundation of China (21390414, 11535015, 21675157, 21425522, 31300827, 81472851, U1632110), Beijing science and Technology Commission special project for frontier technology in life sciences (Z171100000417008) and National Key Research and Development Program of China (2016YFA0400900). We also thank Dr. Yuqing Wang at the National Center for Nanoscience and Technology of China, for helping with Micro MRI data collection and image processing.


20

Page 21 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1)

Laurent, S.; Henoumont, C.; Stanicki, D.; Boutry, S.; Lipani, E.; Belaid, S.; Muller, R. N.;

Vander Elst, L., Superparamagnetic Iron Oxide Nanoparticles. In MRI Contrast Agents, Springer: 2017, pp 55-109. (2)

Mahmoudi, M.; Sant, S.; Wang, B.; Laurent, S.; Sen, T., Superparamagnetic iron oxide

nanoparticles (SPIONs): development, surface modification and applications in chemotherapy. Adv. Drug Deliver. Rev. 2011, 63, 24-46. (3)

Liao, N.; Wu, M.; Pan, F.; Lin, J.; Li, Z.; Zhang, D.; Wang, Y.; Zheng, Y.; Peng, J.; Liu,

X.; Liu, J., Poly (dopamine) coated superparamagnetic iron oxide nanocluster for noninvasive labeling, tracking, and targeted delivery of adipose tissue-derived stem cells. Sci. Rep. 2016, 6, 18746. (4)

Asadi, H.; Khoee, S.; Deckers, R., Polymer-grafted superparamagnetic iron oxide

nanoparticles as a potential stable system for magnetic resonance imaging and doxorubicin delivery. RSC Adv. 2016, 6, 83963-83972. (5)

Zhao, Y.; Liang, M.; Li, X.; Fan, K.; Xiao, J.; Li, Y.; Shi, H.; Wang, F.; Choi, H. S.;

Cheng, D.; Yan, X., Bioengineered Magnetoferritin Nanoprobes for Single-Dose NuclearMagnetic Resonance Tumor Imaging. ACS Nano 2016, 10, 4184-4191. (6)

Rui, Y.; Liang, B.; Hu, F.; Xu, J.; Peng, Y.-F.; Yin, P.; Duan, Y.; Zhang, C.; Gu, H., Ultra-

large-scale Production of Ultrasmall Superparamagnetic Iron Oxide Nanoparticles for T1weighted MRI. RSC Adv. 2016, 6, 22575-22585.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(7)

Page 22 of 27

Hachani, R.; Lowdell, M.; Birchall, M.; Hervault, A.; Mertz, D.; Begin-Colin, S.; Thanh,

N. T. K., Polyol synthesis, functionalisation, and biocompatibility studies of superparamagnetic iron oxide nanoparticles as potential MRI contrast agents. Nanoscale 2016, 8, 3278-3287. (8)

Sharifi, S.; Seyednejad, H.; Laurent, S.; Atyabi, F.; Saei, A. A.; Mahmoudi, M.,

Superparamagnetic iron oxide nanoparticles for in vivo molecular and cellular imaging. Contrast Media Mol. Imaging 2015, 10, 329-355. (9)

Laurent, S.; Henoumont, C.; Stanicki, D.; Boutry, S.; Lipani, E.; Belaid, S.; Muller, R. N.;

Vander Elst, L., Imaging Probes, In MRI contrast agents: from molecules to particles, Springer: 2016, pp 13-21. (10) Lee, N.; Hyeon, T., Designed synthesis of uniformly sized iron oxide nanoparticles for efficient magnetic resonance imaging contrast agents. Chem. Soc. Rev. 2012, 41, 2575-2589. (11) Gao, J.; Gu, H.; Xu, B., Multifunctional Magnetic Nanoparticles: Design, Synthesis, and Biomedical Applications. Acc. Chem. Res. 2009, 42, 1097-1107. (12) Gupta, A. K.; Gupta, M., Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 2005, 26, 3995-4021. (13) Huang, S.-H.; Juang, R.-S., Biochemical and biomedical applications of multifunctional magnetic nanoparticles: a review. J. Nanopart. Res. 2011, 13, 4411-4430. (14) Huang, J.; Wang, L.; Lin, R.; Wang, A. Y.; Yang, L.; Kuang, M.; Qian, W.; Mao, H., Casein-coated iron oxide nanoparticles for high MRI contrast enhancement and efficient cell targeting. ACS Appl. Mater. Interfaces 2013, 5, 4632-4639.


22

Page 23 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(15) Xie, J.; Wang, J.; Niu, G.; Huang, J.; Chen, K.; Li, X.; Chen, X., Human serum albumin coated iron oxide nanoparticles for efficient cell labeling. Chem. Commun. 2010, 46, 433-435. (16) Ma, X.; Gong, A.; Chen, B.; Zheng, J.; Chen, T.; Shen, Z.; Wu, A., Exploring a new SPION-based MRI contrast agent with excellent water-dispersibility, high specificity to cancer cells and strong MR imaging efficacy. Colloid. Surface. B 2015, 126, 44-49. (17) Shen, Z.; Wu, H.; Yang, S.; Ma, X.; Li, Z.; Tan, M.; Wu, A., A novel Trojan-horse targeting strategy to reduce the non-specific uptake of nanocarriers by non-cancerous cells. Biomaterials 2015, 70, 1-11. (18) Kitagawa, T.; Kosuge, H.; Uchida, M.; Iida, Y.; Dalman, R. L.; Douglas, T.; McConnell, M. V., RGD targeting of human ferritin iron oxide nanoparticles enhances in vivo MRI of vascular inflammation and angiogenesis in experimental carotid disease and abdominal aortic aneurysm. J. Magn. Reson. Imaging 2017, 45, 1144-1153. (19) Uchida, M.; Terashima, M.; Cunningham, C. H.; Suzuki, Y.; Willits, D. A.; Willis, A. F.; Yang, P. C.; Tsao, P. S.; McConnell, M. V.; Young, M. J., A human ferritin iron oxide nano‐ composite magnetic resonance contrast agent. Magnet. Reson. Med. 2008, 60, 1073-1081. (20) Cui, Y.; Yang, J.; Zhou, Q.; Liang, P.; Wang, Y.; Gao, X.; Wang, Y., Renal Clearable Ag Nanodots for in Vivo CT Imaging and Photothermal Therapy. ACS Appl. Mater. Interfaces 2017, 9, 5900-5906. (21) Wang, Y.; Xu, C.; Zhai, J.; Gao, F.; Liu, R.; Gao, L.; Zhao, Y.; Chai, Z.; Gao, X., LabelFree Au Cluster Used for in Vivo 2D and 3D Computed Tomography of Murine Kidneys. Anal. Chem. 2015, 87, 343-345.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 27

(22) Xu, C.; Wang, Y.; Zhang, C.; Jia, Y.; Luo, Y.; Gao, X., AuGd Integrated Nanoprobe for Optical/MRI/CT Triple-modal In Vivo Tumor Imaging. Nanoscale 2017, 9, 4620-4628. (23) Zhang, B.; Jin, H.; Li, Y.; Chen, B.; Liu, S.; Shi, D., Bioinspired synthesis of gadoliniumbased hybrid nanoparticles as MRI blood pool contrast agents with high relaxivity. J. Mater. Chem. 2012, 22, 14494-14501. (24) Ceci, P.; Ilari, A.; Falvo, E.; Bellapadrona, G.; Fornara, M.; Morea, V., Biomimetic Materials Synthesis from Ferritin-Related, Cage-Shaped Proteins. INTECH Open Access Publisher: 2012; Chapter 6, pp 113-136. (25) Andrews, S. C., The Ferritin-like superfamily: Evolution of the biological iron storeman from a rubrerythrin-like ancestor. Biochim. Biophys. Acta 2010, 1800, 691-705. (26) Scherrer, P., Bestimmung der inneren Struktur und der Größe von Kolloidteilchen mittels Röntgenstrahlen. In Kolloidchemie Ein Lehrbuch, Springer: 1912, pp 387-409. (27) Monshi, A.; Foroughi, M. R.; Monshi, M. R., Modified Scherrer equation to estimate more accurately nano-crystallite size using XRD. World J. Nano Sci. Eng. 2012, 2, 154-160. (28) Lu, W.; Ling, M.; Jia, M.; Huang, P.; Li, C.; Yan, B., Facile synthesis and characterization of polyethylenimine-coated Fe3O4 superparamagnetic nanoparticles for cancer cell separation. Mol. Med. Rep. 2014, 9, 1080-1084. (29) Pereira, C.; Pereira, A. M.; Rocha, M.; Freire, C.; Geraldes, C. F. G. C., Architectured design of superparamagnetic Fe3O4 nanoparticles for application as MRI contrast agents: mastering size and magnetism for enhanced relaxivity. J. Mater. Chem. B 2015, 3, 6261-6273.


24

Page 25 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(30) Arosio, P.; Baldi, G.; Chiellini, F.; Corti, M.; Dessy, A.; Galinetto, P.; Gazzarri, M.; Grandi, M. S.; Innocenti, C.; Lascialfari, A.; Lorenzi, G.; Orsini, F.; Piras, A. M.; Ravagli, C.; Sangregorio, C., Magnetism and spin dynamics of novel encapsulated iron oxide superparamagnetic nanoparticles. Dalton Trans. 2013, 42, 10282-10291. (31) Palma, S. I.; Carvalho, A.; Silva, J.; Martins, P.; Marciello, M.; Fernandes, A. R.; Del Puerto Morales, M.; Roque, A. C., Covalent coupling of gum arabic onto superparamagnetic iron oxide nanoparticles for MRI cell labeling: physicochemical and in vitro characterization. Contrast Media Mol. Imaging 2015, 10, 320-328. (32) Jin, R.; Lin, B.; Li, D.; Ai, H., Superparamagnetic iron oxide nanoparticles for MR imaging and therapy: design considerations and clinical applications. Curr. Opin. Pharmacol. 2014, 18, 18-27. (33) Xie, J.; Chen, K.; Huang, J.; Lee, S.; Wang, J.; Gao, J.; Li, X.; Chen, X., PET/NIRF/MRI triple functional iron oxide nanoparticles. Biomaterials 2010, 31, 3016-3022. (34) Tong, S.; Hou, S.; Zheng, Z.; Zhou, J.; Bao, G., Coating optimization of superparamagnetic iron oxide nanoparticles for high T2 relaxivity. Nano Lett. 2010, 10, 46074613. (35) Gao, F.; Cai, P.; Yang, W.; Xue, J.; Gao, L.; Liu, R.; Wang, Y.; Zhao, Y.; He, X.; Zhao, L.; Huang, G.; Wu, F.; Zhao, Y.; Chai, Z.; Gao, X., Ultrasmall [64Cu] Cu Nanoclusters for Targeting Orthotopic Lung Tumors Using Accurate Positron Emission Tomography Imaging. ACS Nano 2015, 9, 4976-4986.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 27

(36) Dharap, S. S.; Wang, Y.; Chandna, P.; Khandare, J. J.; Qiu, B.; Gunaseelan, S.; Sinko, P. J.; Stein, S.; Farmanfarmaian, A.; Minko, T., Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide. Proc. Natl. Acad. Sci. USA 2005, 102, 12962-12967. (37) Gao, F.; Li, L.; Fu, C.; Nie, L.; Chen, D.; Tang, F., LHRH-PE40 Fusion Protein Tethered Silica Nanorattles for Imaging-Guided Tumor-Specific Drug Delivery and Bimodal Therapy. Adv. Mater. 2013, 25, 5508-5513. (38) Deng, X.; Klussmann, S.; Wu, G.-M.; Akkerman, D.; Zhu, Y.-Q.; Liu, Y.; Chen, H.; Zhu, P.; Yu, B.-Z.; Zhang, G.-L., Effect of LHRH-PE40 on target cells via LHRH receptors. J. Drug Target. 2008, 16, 379-388.


26

Page 27 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC graph


27

Ultrasmall Super-paramagnetic Iron Oxide Nanoparticle for T2

Recommend Documents