SiO2 Nanoparticles with

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Rational design of GO modified Fe3O4/SiO2 nanoparticles with combined rhenium-188 and gambogic acid for magnetic target therapy Yuxiang Yang, Yicheng Liu, Chao Cheng, Haowei Shi, Huan Yang, Hongming Yuan, and Chaoying Ni ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07589 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 12, 2017

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ACS Applied Materials & Interfaces

Rational design of GO modified Fe3O4/SiO2 nanoparticles with combined rhenium-188 and gambogic acid for magnetic target therapy Yuxiang Yanga,b*, Yicheng Liu,a Chao Cheng, c Haowei Shi,a Huan Yang, a and Hongming Yuan, d Chaoying Nib* a

School of Chemistry and Molecular Engineering, East China University of Science &

Technology, Shanghai 200237, China. b

Department of Materials Science and Engineering, University of Delaware, DE 19716, USA

c

Department of Nuclear Medicine, Changhai Hospital, The Second Military Medical

University, Shanghai 200237, China. d

State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, Jilin University,

Changchun 130012, China. Tel: +1 (302) 831-6359; E-mail: [email protected], [email protected] KEYWORDS: europium oxychloride dopant, graphene oxide modified, multifunctional, radionuclide imaging, magnetic target bio-distribution, PEI-GO convergence effect ABSTRACT: Peanut-like magnetic-fluorescent Fe3 O 4/SiO2 nanoparticles, with an effective dynamic diameter of 180 nm, were synthesized in this study via EuO+ doping and coupling of two Fe3 O4 cores reassembly through solvothermal process. Spherical pure Fe3 O 4/SiO2 nanoparticles with an effective dynamic diameter of 230 nm were also prepared for comparison. We designed graphene oxide modified core-shell Fe 3 O4/SiO2 nanoparticles as nano-carrier for loading gambogic acid following labeling with radioisotope rhenium-188. We also performed gambogic acid loading and releasing on

ACS Paragon Plus Environment


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gambogic acid-loaded magnetic nanoparticles, in vivo biodistribution as well as magnetic drug targeting therapy experiments. Results indicated that the gambogic acid-loaded magnetic nanoparticles demonstrate a clear pH-dependent drug release behavior, having higher release rate in acidic environments. The in vivo biodistribution of the magnetic nanoparticles has morphologic dependence and the peanut-like nanoparticles (PN-Fe3 O4) tend to accumulate more in the spleen, lung and liver than the spherical nanoparticles (S-Fe 3 O4). The targeted therapy showed higher efficacy of PN-Fe 3 O4 in inhibiting tumor cell growth than the non-targeted therapy. The PEI grafting of PN-Fe 3 O4 with amide bond was also designed to find an effective active targeting anti-tumor agent considering the fact that PEI-GO conjugate has higher gambogic acid load efficiency and the convergence effect. 1. INTRODUCTION Chemotherapy and radio-therapy are the most commonly used strategies for clinical treatment of cancer but significant negative side effects from radiation and chemotherapeutic agents cause great limitation in the treatment of cancers. Such side effects include gastrointestinal symptoms, radiation thyroiditis, cisplatin-induced nephrotoxicity and oculo-visual side effects.1-2 The targeted drug delivery systems that transport the anti-cancer drugs to target lesion sites have potential to overcome the drug’s side effects. With emergence and development of nanotechnology, a large number of drug nano-carriers have been fabricated3-4 and among them, magnetic nanoparticles have shown great potential due to their applications in magnetic resonance imaging as contrast enhancement agents, magnetic drug targeting and magnetic hyperthermia in cancer treatment.5-8 The in vivo behavior of the nano-carriers is complex and generally the nanoparticles


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are rapidly captured by the reticular endothelial system (RES) in vivo. Research has shown that the surface charge9-11 and particles’ size12-13 can affect the in vivo biodistribution of the nanaoparticles. The nanoparticles in tumor tissues are mainly affected by their enhanced permeability and retention (EPR) effect. This theory postulates that certain size and shape of the particles tend to accumulate in the tumor tissues much more than in normal tissues, because the tumor’s newly formed blood vessels are usually abnormal in form and architecture. Many strategies have been applied for the preparation of magnetic nanoparticles with different morphologies, including template-induction, magnetic field induction and controlled experiment parameters14-17. There are however few reports about how doped rare-earth acyl ions can assist in controlling the morphology of the magnetic nanoparticles. Graphene oxide (GO) is a novel one-atom-thick two-dimensional graphitic carbon system that promises tremendous potential applications in targeted drug delivery, due to its two-dimensional nanostructure and adjustable surface chemistry. Xiaoying Yang et al found that GO can be used as a nanocarrier which allows for controlled loading and release of doxorubicin hydrochloride with high

capacity

under

controlled

pH

conditions.18

Hungwei

Yang

et

al

prepared

gadolinium-functionalized GO that can be loaded with micro RNA and epirubicin.19 The GO has the potential capacity to load many bioactive molecules via simple π-π stacking with a relatively high capacity due to its abundant surface groups. Gambogic acid (GA), with molecular formula C38H44O8, is the natural compound extracted from gamboge and has recently been established as a potent anticancer agent that can inhibit the growth of a wide variety of tumor cells, including breast cancer, gastric cancer, pancreatic cancer cells, hepatocellular carcinoma and pulmonary carcinoma20. Despite the potency of GA both in



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vitro and in vivo, it has been reported to have poor water-solubility and high molecular weight, so it is poorly absorbed in humans and therefore unlikely to have a significant effect in vivo, which will limit their transition from research to clinical applications21. A typical peanut-like magnetic Fe3O4@SiO2-GO based drug carrier system could solve the above challenges by improving water solubility of Fe3O4@SiO2-GO nanoparticles, increasing their cellular uptake and potentially minimizing toxicity to adjacent normal cells. This nanoformulation, characterized by high drug-loading and controlled release efficiency, could maximize the drug delivery efficacy to the tumor area and distinguish specific cancer cells22. As reported23, the important role of radionuclide Re-188 is based on its decay characteristics which are suitable for emission computed tomography imaging, tumor diagnosis and radio-therapy in nuclear medicine. The Re-188 could be used to label GO with high yield, showing high stability in vitro and in vivo and rapid uptake in lungs, liver and spleen24. The GA was therefore chosen as chemotherapy agent model while the Re-188 was chosen as a radio-therapy agent model in this work. The multimode Re-188 radiolabeled magnetic nanoparticles and loaded with GA will achieve the goal of minimizing the side effects while maximizing the therapeutic efficacy, due to the fact that both of GA and 188Re-labelled radiopharmaceuticals are effective for regional target therapy and the peanut-like nano magnetic particles are more easily captured in vivo by tumor tissue. Besides, 188Re-labelled nanoparticles can be applied not only in radionuclide imaging and CT imaging contrast agents but also in internal radiotherapy of tumours. Moreover, PEI-GO conjugate has higher gambogic acid load efficiency and convergence effect to tumours. Integration of these advantages on multi-functionalizing nanoparticles has promising potential in a wide variety of


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biomedical fields. Fe3O4 nanoparticles with two different morphologies were synthesized in this research by doping them with Eu element via solvothermal method. Silica coating and GO modification were adopted to enhance their stability, making the particles to be capable of functionalizing with different molecules. Firstly, the GO modified nanoparticles were labelled with Re-188 nuclide to study the multimode and mode coupled effects of Re-188 radiolabeled GA-loaded peanut-like magnetic-fluorescent Fe3O4/SiO2 nanoparticles. The multifunctional nanoparticles were then injected into the tumour-bearing nude mouse to study their treatment effect. Their magnetic target effect on the biodistribution was also studied. Finally, the therapeutic effect was observed by the single photon emission computed tomography (SPECT).

2. MATERIAL AND METHODS Synthesis of Fe3O4 nanoparticles and europium doped Fe3O4 nanoparticles Pure Fe3O4 nanoparticles (S-Fe3O4) were prepared according to a modified solvothermal method. Typical synthesis of Fe3O4 was carried out through the solvothermal system using 0.7 g FeCl3 as iron source in the present of 20 ml EG, 1.8 g NaAc and 0.5 ml DEG. The mixture was stirred vigorously till it became homogeneous. The mixture was sealed in a 50 mL PTFE-lined stainless steel autoclave and heated at 200 °C for 12 h followed by slow cooling to room temperature. The resulting product was thoroughly washed with absolute ethanol to remove all residual reagents, followed by separation by magnetic field. The europium doped Fe3O4 nanoparticles (PN-Fe3O4) were prepared through the same strategy by an addition of 0.025 g EuOCl into the mixture.



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Silica-coating and amino-modification of Fe3O4 nanoparticles Silica coated magnetic core-shell nanoparticles were synthesized via Stöber method. Typically, 50 mg doped nanoparticles were dispersed in the mixture of deionized water (20 ml) and ethanol (80 ml). Concentrated ammonia aqueous solution (2.0 mL, 28 wt.%) and tetraethyl orthosilicate (TEOS, 1 ml) were then added into the mixture. After mechanical stirring at 30°C for 12 h, the magnetic nanocomposites were separated and washed with ethanol and water, followed by drying in vacuum at 60°C for 12 h. The dried nanocomposites were re-suspended in the dried toluene (50 ml), followed by addition of 3-aminopropyl triethoxysilane (APTES, 1 ml). The mixture was then heated to reflux for 12 h. The amino-modified magnetic nanocomposites (Fe3O4@SiO2-NH2) were then separated and washed with ethanol and water, followed by drying for 12 h. The silica-coated S-Fe3O4 and PN-Fe3O4 were named as S-Fe3O4-SiO2 and PN-Fe3O4-SiO2 respectively. Preparation of Fe3O4@SiO2-GO 200 mg GO was treated with ultrasound wave in 60 ml of water for GO exfoliation. GO was prepared

from

natural

graphite

by

modified

Hummer’s

method25.

20

mg

of

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 16 mg of N-hydroxysuccinnimide (NHS) were then added into the mixture and the mixture was then stirred to form homogenous suspension. 200 mg of amino-modified magnetic nanoparticles were then added and the reaction was carried out at 80°C for 1 h under mechanical stirring. The GO-modified (Fe3O4@SiO2-GO) nanocomposites were separated under the magnetic field and washed. The GO-modified S-Fe3O4-SiO2 and PN-Fe3O4-SiO2 were then named as S-Fe3O4-GO and PN-Fe3O4-GO respectively. Samples characterization


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The morphology and size of nanoparticles was examined by JEM-2010F field emission transmission electron microscope (200 kV) and structural information was observed by JEM-3010 LaB6 transmission electron microscope with XEDS (HRTEM) operating at 300 kV. The magnetic study was performed with a Quantun Design MPMS-7. X-ray photoelectron spectroscopy (XPS) spectra were recorded on a Kratos AXIS ULTRA DLD X-ray photoelectron spectrometer using an Al (Mono) X-ray as the excitation source. Gambogic acid loading and release 1.5mg, 2.5mg, 5mg and 10mg of GA were respectively added into 30 mL of 10 mM phosphate-buffered saline (PBS, pH 7.4) with 20 mg of PN-Fe3O4-GO, treated under ultrasound for 30 min, and dispersed using an ultrasonic horn for 4 h. Then, the mixture in vessel was placed in a temperature controlled shaking incubator (model: TQZ-312) with the speed of 150 rpm and kept at 37 °C for 24 h. Finally, it was magnetically separated and washed with deionized water for three times. The supernatant liquid and washed solution were collected to measure the amount of the free GA, while the GA loaded nanoparticles were kept for next release determination. The free GA content in the supernatant was quantified using SHIMADZU UV-VIS Spectrophotometers UV-2600 at 360 nm. The GA loading efficiency and drug loading content were calculated using the following equation: Loading efficiency = Wtotal drug−Wdrug in supernatant/Wtotal drug* 100

The release of the GA was carried out by adding the above obtained GA loaded nanoparticles into 25 mL of 10 mM fresh PBS (pH=5.7), which was then separated at regular intervals. The free GA content in PBS solution was determined by recording absorbance at 360 nm. After that, another 25 mL of 10 mM fresh PBS (pH=5.7) was added into GA loaded nanoparticles to perform



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release experiment following free GA content determination. The accumulative release rate was total GA release amount divided by GA loading amount. The GA loaded S-Fe3O4-GO, PN-Fe3O4-GO and PN-Fe3O4-GO-PEI were named as S-Fe3O4-GO-GA, PN-Fe3O4-GO-GA and PN-Fe3O4-PEI-GA respectively. Polyethyleneimine (PEI) grafting GO and its GA loading efficiency and release ratio Excellent nanocarriers for effective delivery of chemical drugs were prepared by polyethyleneimine (PEI) grafting on the PN-Fe3O4@SiO2-GO. Briefly, a mixture of 20 mg Fe3O4@SiO2-GO, 20 mL of de-ionized water, 5 mg EDC, 4 mg NHS and 0,1,2,3,4 and 5 ml(0.1 mg/ml) of PEI were subjected to 24-hr shaken reaction to generate PN-Fe3O4@SiO2-GO-PEI (PN-Fe3O4-GO-PEI) for loading of GA (PN-Fe3O4-PEI-GA) respectively. The measurements on loading efficiency and release ratio were carried out by addition of 2.5 mg of GA into 30 mL of 10 mM PBS (pH 7.4). 20 mg of PN-Fe3O4-GO-PEI nanoparticles were added in the same step described in above. Preparation of Re-188-labeled Fe3O4@SiO2-GO and Fe3O4@SiO2-GO-PEI loaded with GA The 188ReO4- was reduced using stannous chloride. typically; 8 mg of SnCl2, 0.1 ml of 0.5 mol/L sodium gluconate solution, 0.4 ml of PBS (pH=5.5) and 5 mg GA loaded nanoparticles were mixed to form homogeneous solution and subjected to ultrasonic treatment for 15 min. 1 ml of 5mCi/ml

188

ReO4--leacheate was then added and the mixture was heated to 70°C for 2 h. The

obtained nanoparticles were separated by magnetic field and washed with deionized water, and radioactivity of the labeled nanoparticles was then detected using isotope activity meter. The Re188 labelled S-Fe3O4-GO-GA, PN-Fe3O4-GO-GA and PN-Fe3O4-PEI-GA were then named as S-Fe3O4-GA-Re, PN-Fe3O4-GA-Re and PN-Fe3O4-PEI-GA-Re respectively.


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In vivo biodistribution study in the normal mice Female healthy mice were subjected to injection with Re-188 labeled S-Fe3O4-GO and PN-Fe3O4-GO nanoparticles via tail vein (0.1 ml of 0.1mCi per mouse, at a dose of 8 mg/kg). The mice were then sacrificed by carbon dioxide inhalation to collect major organs for measuring the radioactivity by the gamma counter. Treatment of VX-2 tumor-bearing nude mice VX-2 tumor-bearing nude mice were prepared by transplanting tumor cells (with tumor diameter of about 0.1 cm) under the skin of both forelimbs. When the size of the tumors grew to 0.5-1 cm, four groups were set according to injection reagents, each group containing three mice as follows: Re-188 labeled D-GA and Re-188 labeled P-GA (0.1 ml of 0.15mCi per tumor, at 16 mg GA/kg body mass dose);

188

ReO4- leacheate (0.1 ml 0.15mCi per tumor) and control group had no

treatment. SPECT-CT images were then taken for the mice from Re-188 labeled D-GA, Re-188 labeled P-GA and 188ReO4-leacheate groups after magnetic targeting of one side tumor for 1 h. CT images were taken every 24 h from the treated mice to evaluate the volume of tumors. 3. RUSULTS AND DISCUSSION Synthesis of spherical and peanut-like magnetic nanoparticles Control of nanomaterial’s morphology is generally achieved via template directing. This was the first time, as far as we know, that europium oxychloride (EuOCl) was used as doping agent for controlling the size and shape of Fe3O4 nanoparticles. The EuOCl was synthesized according to reference26 and its XRD pattern is shown in Fig. S1 and the Bragg peaks, inter-planar spacing “d” with different Miller indices for EuOCl crystal are referenced in Table S1. The TEM images in Fig. 1A～D exhibit the effect of EuOCl on the size and shape of the Fe3O4 nanoparticles, and the



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HRTEM images in Fig. 1E and F show structural information of the doped Fe3O4 (PN-Fe3O4) nanoparticles. The pure Fe3O4 (S-Fe3O4) nanoparticles have an average size of 220 nm but the doped Fe3O4 (PN-Fe3O4) nanoparticles show a morphology of coupled double spheres with a uniform distribution. The length and width of the double-spherical nanoparticles fall within the range of 140～202 nm and 75～125 nm respectively. Almost all of the spherical nanoparticles shown in Fig. 1B were mutually coupled to become PN-Fe3O4. The doped Fe3O4/SiO2 (PN-Fe3O4-SiO2) nanoparticles also have a peanut-like shape and exhibit core-shell structure (Fig. 1D), with size ranging from 120 nm to 170 nm for each spherical particle. The pure Fe3O4/SiO2 (S-Fe3O4-SiO2) showed uniform spherical particles of core-shell morphology (Fig. 1C). The laser scattering particle analyzer was also used to determine their average particle sizes. Results (Fig. S2) show that the dynamic effective diameter of the PN-Fe3O4-SiO2 was 180 nm and that of the S-Fe3O4-SiO2 was 230 nm. The XRD patterns in Fig. S3 show that both S-Fe3O4 and PN-Fe3O4 have characteristic cubic crystal structures for the Fe3O4 (JCPDS 019-0629). HRTEM image for the PN-Fe3O4 in Fig. 1E provides more detailed structural information, as it can obviously be seen that the doped nanoparticles are assembled with small nanocrystals. The interlayer distance of these small nanocrystals has arranged crystal lattice with an interplanar spacing of 0.256 nm, in good agreement with (311) lattice planes. 0.309 nm is assigned to the (220) lattice planes of PN-Fe3O4 nanocrystals. The lattice constant for the PN-Fe3O4 nanoparticles (0.8368 nm) was reduced as compared to that of S-Fe3O4 (0.8396 nm). Detailed calculation of the lattice constant is given in Table S2. The smaller lattice constant for the doped particles leads to a smaller cell volume, which


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may have contributed to the small size of the doped particles.27 In correspondence to Table S1, the 0.678 nm for the interplanar spacing shown in Fig. 1E agrees well with the (001) lattice planes for the EuOCl crystals, indicating that the EuOCl has been successfully doped in the Fe3O4 nanoparticles. With increasing magnification of Fig. 1D, the image from the PN-Fe3O4-SiO2 recorded in Fig. 1F exhibits well defined core-shell and peanut-like structure. The coupled double spherical nanoparticles have the lengths and widths of about 150～180 nm and 90～120 nm respectively, and the silica shell is about 9～13 nm in width. HRTEM image for PN-Fe3O4-SiO2 inserted in the top and bottom right-hand corner of Fig. 1F shows that the interplanar spacing of 0.660 nm and 0.251 nm agree with (001) and (311) lattice planes of EuOCl and PN-Fe3O4 respectively, demonstrating that the core structure of doped Fe3O4 has not changed after Silica coating. Magnetic measurements were carried out at room temperature. The M-H curves (Fig. S4) show that the saturation magnetization for the PN-Fe3O4-SiO2 and S-Fe3O4-SiO2 is 43.7 emu/g and 75.4 emu/g, respectively, indicating that the EuO+-doping reduces saturation magnetization of the particles. This is mainly due to the fact that the PN-Fe3O4-SiO2 has smaller size than the S-Fe3O4-SiO2, so PN-Fe3O4-SiO2 shows apparent typical super magnetic behaviors. Besides, “frozen orbital” approximation makes the Eu3+ less Bohr magnetron than the Fe3+28, thus leading to decreased saturation magnetization in the PN-Fe3O4 nanoparticles. ZFC-FC curves for the S-Fe3O4 and PN-Fe3O4 are measured in the range of 2K - 300K. As can be seen from ZFC curves in Fig.S5A and S5B, the TB temperatures for the S-Fe3O4 and PN-Fe3O4 are 200 k and 136 K respectively, probably because the PN-Fe3O4 has a longer relaxation time and smaller energy barriers with temperature range reaching TB29, which may be related to the smaller size of the



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PN-Fe3O4. In our nanoparticle fabrication, ethylene glycol served both as solvent and reducing agent, NaAc for electrostatic stabilization to prevent particle agglomeration, and DEG as surfactant. When using EuOCl as doping source, the europium ion (EuO+) was doped into the octahedral site vacancies in the surface layer of the Fe3O4 core in the solution. Oxygen coordinated with Fe3+ in the Fe3O4 core, with assistance from protonation of DEG, forming the intermediate shown in Fig. 2A. This was due to lone-pair electrons in the oxygen atom of the EuO+ and empty d orbital at the iron center from another Fe3O4 core. This means that one doping EuO+ ion played a role in coupling the two Fe3O4 core in the solution with the assistance of ethylene glycol. To verify the formation of intermediate compound, the peanuts-like Fe3O4 nanoparticles were synthesized by solid phase synthesis at room temperature, using the added DEG as initiator. Briefly, 0.7 g of FeCl3, 0.025 g of EuOCl and 2 ml of DEG were mixed and grinded in an agate mortar for 8 h to form gel, followed then by heating in a tube type electric-resistance furnace at 120 °C under ambient argon gas. The obtained brown compound was analyzed by the electrospray ionization mass spectrometry (ESIMS) and results are shown in Fig. S6. The peak at m/z=488.3 is assigned to molecular ion from the compound, while that at m/z=474.97 is assigned to {(EuO)Fe2(OCH2CH2OCH2CH2O) (OCH2CH2OCH2O)}+. The peaks at m/z= 450.38, 437.19 are assigned to {(EuO)Fe2(OCH2CH2OCH2O)2+4H+} and {(EuO)Fe2(OCH2CH2O-CH2O)(OCH2O -CH2O)+4H+}

respectively,

{(EuO)Fe2(OCH2CH2O)2+6H+}.

while

the

one

at

m/z=406.3

is

The

other

peak

at

m/z=362.33

was

assigned assigned

to to

{(EuO)Fe2O(OCH2CH2O)}+, while the main peak at m/z=318.3 is assigned to {(EuO)Fe2O2}+. These characteristic peaks are all in good agreement with the supposed structure of the


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intermediate compound, demonstrating that the EuO+ ion assisted solvothermal method and forced the two Fe3O4 cores to couple and re-assemble into the peanuts-like Fe3O4 particles, as shown in the Fig. 2B. To demonstrate participation of europium in the self-assembly of the peanuts-like Fe3O4, the EDAX and ICP-AES analyses were carried out. The EDAX-ZAF quantitative analysis show that the Eu Wt% and Eu/Fe atom ratio on the surface of nanoparticles are 6.69% and 1:28.4 respectively (shown in Fig. S7). The ICP-AES quantitative analysis shows that the Eu Wt% and Eu/Fe atom ratios for the whole nanoparticles are 5.70% and 1:44.8 respectively. These findings suggest that the Eu Wt% on the surface of the nanoparticles is higher than that of the bulk nanoparticles, thus the EuO+ ions mainly stay on the surface of the particles to assist the coupling of the Fe3O4 cores. Fig. S8 shows the excitation and emission spectra from the PN-Fe3O4 at room temperature. The excitation spectrum, monitored at 610 nm, displays a wide absorption peak at 320 nm which is assigned to the f-f transition of the Eu3+ ions. Upon excitation of the Eu3+ ions at 320nm, the emission spectrum exhibits four groups of emission lineswhich are ascribed to 5D0-7FJ (J = 1 – 4 ) transitions of the Eu3+ ions, respectively. The main peak at 612 nm corresponds to 5D0-7F2 electron dipole transition of the Eu3+, and the peaks at 595 nm, 653 nm and 699 nm correspond to 5D0-7F1 magnetic dipole transition,

5

D0-7F3 transition and

5

D0-7F4 transition, respectively. This

demonstrates that the PN-Fe3O4 possesses good fluorescence after doping with EuOCl.

Strategy for the synthesis of doped Fe3O4/SiO2-GO, Fe3O4/SiO2-GO-PEI nanoparticles and the combination with GA and Re-188



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The synthesis of Fe3O4@SiO2-GO nanoparticles is described as follows; the S-Fe3O4 and PN-Fe3O4 were synthesized first, and then coated with silica according to our previous work.30 The amino group was then modified on the silica surface via hydrolysis of APTES. The average molar ratio of amino group on the silica surface was determined to be about 45 mmol/mg nanoparticles based on color reaction between amino group and ninhydrin at 570 nm.24 The stripped GO nanosheet could easily bind with amino groups on the silica surface to generate the Fe3O4@SiO2-GO nanoparticles in the presence of EDC and NHS. This was due to the presence of plenty reactive functional carboxyl (COOH) groups. Cationic polymers such as PEI are widely used for gene and siRNA delivery. GO has been found to be an effective nanocarrier for delivery of multiple anticancer drugs. An integration of GO and cationic polymers is therefore expected to facilitate the loading of anticancer drugs via electrostatic adsorption and aromatic anticancer drugs via stacking onto GO sheets.31 PEI grafted Fe3O4@SiO2-GO was therefore prepared by covalent linking of PEI to GO through the formation of an amide bond using EDC chemistry, as illustrated in Figure 3a. The chemical conjugation of PEI to GO was confirmed by Raman spectroscopic and X-ray photoelectron spectroscopy (XPS). The chemical conjugation of GO to Fe3O4@SiO2 and PEI-Grafted Fe3O4@SiO2-GO were confirmed by Raman spectroscopy. As shown in Fig. 3b, both of the

S-Fe3O4-GO and

PN-Fe3O4-GO exhibit two peaks at about 1362.6 cm-1 and 1592.1 cm-1, characteristic of the GO bands. But the PN-Fe3O4-SiO2 has no obvious band in the Raman spectrum. This indicates that the GO had been successfully modified on the surface. The PN-Fe3O4-GO sample shows two peaks with stronger intensity than those for S-Fe3O4-GO, indicating a strong linkage of GO on the PN-Fe3O4-GO sample. When PEI grafting of GO occurs on the surface of the Fe3O4@SiO2-GO,


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the intensity of 1362.6 cm-1 and 1592.1 cm-1 decreases obviously, indicating GO reacts with PEI. In order to study possible chemical interactions between PEI and PN-Fe3O4-GO, X-ray photoelectron

spectroscopy (XPS)

measurements

were

taken

for

PN-Fe3O4-GO

and

PN-Fe3O4-GO-PEI (Fig. 4). The XPS spectra of PN-Fe3O4-GO and PN-Fe3O4-GO-PEI in Fig. 4a show the characteristic peaks, such as C(1s), N(1s), and O(1s) at 287.5 eV, 400.2 eV and 531.5 eV respectively. Apparently, the peak of N1s in PN-Fe3O4-GO-PEI is stronger than that of PN-Fe3O4-GO, and more specifically, the ratio of C1s/N1s in the spectrum of PN-Fe3O4-GO is 95.29/4.71, while the ratio of C1s/N1s in the spectrum of PN-Fe3O4-GO-PEI is 93.98/6.02, and an obvious increase of N1s content indicates that polyethyleneimine (PEI) is successfully grafted onto PN-Fe3O4-GO. Fig. 4b is the C1s spectrum of PN-Fe3O4-GO and PN-Fe3O4-GO-PEI. The peak at 284.85 eV belongs to the peaks of the C non-oxygen bond (C-C, C = C, C-H), but the peak at 287.65 eV is attributed to the peak of the C oxygen-containing bond (C-O, C-O-C, O=C-O). It is clear that the C-oxygen bond in PN-Fe3O4-GO-PEI is very weak, indicating that PEI is partially reduced by GO. The fine spectra of C1s of PN-Fe3O4-GO and PN-Fe3O4-GO-PEI are shown in Fig. 4c and d, respectively. The C1s peak can be further divided into four peaks attributable to carbon atoms with different functional groups, including C=C (284.7 eV), C–O (285.6 eV), C–O–C (286.9 eV) and O-C–O (288.5 eV) bonds, respectively. Compared with the C=C peak, the peak of the PEI modified oxide is much weaker, indicating that GO was successfully reduced. However, there are still some residual oxygen groups in PN-Fe3O4-GO-PEI, indicating that PEI was partially reduced, and the above analysis indicates that PEI has been successfully grafted onto PN-Fe3O4-GO.



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GA loading efficiency and GA release of doped Fe3O4/SiO2-GO, Fe3O4/SiO2-GO-PEI nanoparticles The GO modified magnetic nanoparticles highly loaded with GA and the PN-Fe3O4-GO were further tested. The PN-Fe3O4-GO changed color from dark to gray after being loaded with the pale-yellow drug GA. UV-vis spectrum of the resulting PN-Fe3O4-GO-GA revealed obvious broad peak wavelength within range of 378～514 nm (Fig. 5 A), but the PN-Fe3O4-GO had no absorption, suggesting the loading of GA onto the PN-Fe3O4-GO. In comparison with the characteristic absorption peak for free GA at 360 nm, the absorption of PN-Fe3O4-GO-GA showed the bathochromic effect, which was probably due to the surface effect of the GO. The absorbance at 360 nm was therefore used to determine the loading, and the readout at 360 nm was compared with standard curve generated from the free GA at different concentrations, to calculate the unloaded GA concentration (Fig. 5B). The loading efficiency on the PN-Fe3O4-GO and S-Fe3O4-GO surface gradually reached 86.00% and 78.85% respectively, by addition of 1.5 mg, 2.5 mg, 5 mg and 10 mg of GA into 30 mL of 10 mM phosphate-buffered saline (pH 7.4) (PBS) with 20 mg nanoparticles (Table 1). The high loading efficiency can be attributed to simple π-π stacking. As shown in Table 1, the GA loading efficiency of PN-Fe3O4-GO increased with increases GA loading amount on PN-Fe3O4-GO. When the GA loading amount is changed from 1.5 mg to 5 mg, the GA loading efficiency is kept within the range of 45.87 to 53.37 mg/g, and the GA release ratio is also kept to about 70.5%.This is due to the fact that the layer-by-layer stacking of GA on PN-Fe3O4-GO leads to little change of free GA in the original solution. The trends of GA release with release time are shown in Fig. 6A. When duration of GA


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release occurs within 20 h, the GA release increases rapidly no matter what GA loading amount is on PN-Fe3O4-GO. The GA release increases faster when the GA loading was 1.5 mg. When duration of release extends from 20 h to 80 h, the GA release rises gently to about 70.5% for 1.5 mg, 2.5 mg and 5.0 mg GA loading respectively. By contrast, the GA release only reaches 44.54% for 10 mg GA loading. The high GA loading efficiency on the PN-Fe3O4-GO prohibits the diffusion of GA molecules, leading to a slow-release of GA. 2.5 mg GA was therefore considered to be the optimum loading amount per 20 mg of PN-Fe3O4-GO-PEI in the next study on the effects of PEI on GA release, in order to reduce the cost of expensive gambogic acid. A comparison of PN-Fe3O4-GO with S-Fe3O4-GO loading efficiency is shown in Table 1, which shows that the PN-Fe3O4-GO has higher GA loading efficiency and release rate as expected due to their peanut-like structure. Fig. 6B shows that the GA loading efficiency increases with increased PEI for the PEI grafted Fe3O4@SiO2-GO. Because covalent linking of PEI to GO can form an amide bond using EDC chemistry,31 the GA has both 6-hydroxy and 30-carboxy groups, and the generated amide bond could form the hydrogen bonding with the two groups of GA for immobilizing GA. So, when the addition of PEI increases, the strength of hydrogen bonding between GA and PN-Fe3O4-GO-PEI also increases, leading to an increased GA loading efficiency. When the PEI reaches 4-6 mL, the curves become relatively flatter. Because PEI functionalization increases the positive charge on the surface of GO and contributes to the electrostatic repulsion32, the GA loading efficiency could not increase with the PEI amount over 6 ml in this study. Fig. 7A shows the GA release rate increases rapidly within the first 10 h, and the release rate then reaches equilibrium after 20 h. The increased PEI amount from 0 to 5 mL induces the



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decrease of equilibrium releasing rate from 68.2% to 35.4%, which is directly related to the hydrogen bonding between GA and PN-Fe3O4-GO-PEI. The release data indicate that the 2.5 mg GA loaded PN-Fe3O4-GO-PEI could be the target delivered to the lesion sites and maintained for more than 20 hours, and the GA could be released efficiently for about 43.00 %. A comparison of Fig. 6A and 7A suggests that either GA loading or PEI grafting amount could not facilitate GA release of nanoparticles. Though an increase of GA loading amount finally generates a slow increase of total release amount, a bit more GA may exhibit cell toxicity via several mechanisms. The PEI-GO conjugate meanwhile shows much lower cytotoxicity than PEI itself31, implying that the PN-Fe3O4-GO-PEI has significant low cytotoxicity. However, higher PEI grafting amount could induce higher GA loading efficiency, which significantly lowers the cytotoxicity of the PN-Fe3O4-GO-PEI/GA and substantially enhances its chemotherapy efficacy31. As shown in Fig. 6B and Fig. 7B, though 2.5 mg GA loaded PN-Fe3O4-GO-PEI decreases accumulative release rate, it still has both high GA-loading efficiency (67.9 mg/g) and total release amount (28.73 mg/g) when the PEI volume was 3 mL, indicating the optimum PEI volume． The XPS were further carried out to quantitatively analyze the contents of carbon and nitrogen in GA, PN-Fe3O4-GO-PEI and GA loaded PN-Fe3O4-GO-PEI sample. As shown in Fig. 8, GA has only a strong binding energy peak of C1s at 284.8 eV but it has no N1s peak, indicating that the GA has no nitrogen incorporation. GA loaded PN-Fe3O4-GO-PEI sample has a stronger C1s peak at 284.8 eV and relatively high N1s peak at 400.2 eV than those of PN-Fe3O4-GO-PEI sample. The positive charge of the PN-Fe3O4-GO-PEI nanoparticles is important for the association with the negatively charged GA and subsequent load efficiently.33 It was also found that the increased carbon content and decreased nitrogen content were


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related with element composition in the GA (C38H44O8). The GA loaded PN-Fe3O4-GO-PEI has a mole ratio of C1s:N1s at 95.29:4.71, which has higher carbon content and lower nitrogen content than that of the PN-Fe3O4-GO-PEI (the mole ratio of C1s:N1s at 93.98:6.02). The finding confirms that the PN-Fe3O4-GO-PEI can load GA effectively, in good agreement with the previous discussion. The effect of pH on doped Fe3O4/SiO2-GO, Fe3O4/SiO2-GO-PEI nanoparticles loaded with GA The loading of GA on PN-Fe3O4-GO and PN-Fe3O4-GO-PEI is shown in Fig. 9. As expected, the loading efficiency of GA on PN-Fe3O4-GO-PEI is higher than PN-Fe3O4-GO at the same pH (5.7 or 7.4). For example, the loading efficiency of GA on PN-Fe3O4-GO-PEI is 48.86%, higher than that of PN-Fe3O4-GO (36.64%) in PBS (pH = 7.4). It is obvious that PN-Fe3O4-GO-PEI has higher loading efficiency of GA in PBS at pH =7.4 than that at pH = 5.7. The pH-dependence of the loading may be due to the different degree of hydrogen bonding interaction between the GA and the PEI-GO conjugate in PN-Fe3O4-GO-PEI nanoparticles under different pH conditions. The hydrophilic face of GA molecules includes the carboxylate ion and several oxygen atoms at pH value of 7.4,34 but at lower pH, the GA molecules only have positively charged carboxyl functional groups, and the amide bond of PEI-GO conjugate is liable to associate with positive hydrogen ions and it weakens the hydrogen bond between the drug and the PN-Fe3O4-GO-PEI nanoparticles significantly, leading to the hydrogen bonds being easily broken, and so less drug loading efficiency. Studies have shown that tumor tissues exhibit a lower extracellular pH (pH 5.7-7.2) than normal tissue and plasma (pH 7.4) due to anoxic environment, and the pH is further reduced in the



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intracellular compartment, such as lysase body (pH 4-5) and endosome (pH 5-6). This difference can be used to control the release of anticancer drug designs to reduce side effects on cytotoxicity and promote drug efficacy. The drug release properties of GA-loaded magnetic nanoparticles in PBS solutions at pH 5.7 and pH 7.4 were investigated respectively. As shown in Fig. 9, PN-Fe3O4-GO-GA and PN-Fe3O4-PEI-GA all show a clear pH-dependent drug release behavior. The release rate of PN-Fe3O4-GO-GA and PN-Fe3O4-PEI-GA drug is relatively slow at pH 7.4, and slows down after 6 hours with an initial release of about 15% and 9% respectively. Their cumulative release reaches 24.2% and 11.5% for PN-Fe3O4-GO-GA and PN-Fe3O4-PEI-GA respectively after 50 hours. In contrast, the release of the two nanoparticles in PBS solutions at pH 5.7 was much faster and also about 67% and 42.5% of the drug are released in the PBS solution at pH 5.7 for PN-Fe3O4-GO-GA and PN-Fe3O4-PEI-GA drug respectively after 50 hours. Drugs and nanoparticles carry a positive charge at a lower pH, which provides the necessary exclusion between them. These factors are the main drivers of drug release in acidic environments. De-labelling rate of Re-188 from PN-Fe3O4-GO-GA and PN-Fe3O4-PEI-GA Due to the special structure of the GO,35, 36 the low valence Re-188 ion can be labelled on the surface of the GO modified nanoparticles when the Na188ReO4was reduced by SnCl2 in the mixed solution of sodium gluconate and phosphate buffer solution (PBS) at 5.5 pH.37 The amounts of Re-188 ion loaded onto the PN-Fe3O4-GO-GA, PN-Fe3O4-PEI-GA and S-Fe3O4-GO-GA were 0.2 mCi/mg, 0.2 mCi/mg and 0.18 mCi/mg, respectively. The stability of the labelled Re-188 is shown in Fig. 10. Part of the labelled Re-188 is released from the nanoparticles at the first 1h and the labelling rates for the S-Fe3O4-GO-GA, PN-Fe3O4-GO-GA and PN-Fe3O4-PEI-GA are about 17%,


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20% and 7.4% respectively, which then remains stable in the next 2 h. These findings demonstrate that the PN-Fe3O4-PEI-GA has strong coordination ability to low valence Re-188 ion due to the presence of generated amide bond.

Biodistribution of two shaped nanoparticles in mice with or without magnetic targeting To investigate the biodistribution of two nanoparticles in mice, the PN-Fe3O4-GA-Re and S-Fe3O4-GA-Re were injected into the mice via caudal vein and the nanoparticles were then detected by the SPECT and γ-counter. The mice were divided into three groups according to the dissected time after injection; 1 h, 2 h and 3 h. Among them, the 1 h group was chosen to take the SPECT images to qualitatively evaluate the distribution before dissection. The SPECT images in Fig. 11 show that both of two nanoparticle groups are mainly occupied in the tissues and fewer of them could cross the blood-brain barrier. As shown in Fig. 11, the majority of the S-Fe3O4-GA-Re occupies the excretory system only detected in the A region. These findings mean that the majority of the S-Fe3O4-GA-Re has been rapidly removed from circulation and accumulates in the bladder. Strong signal could be detected in both A and B regions in the PN-Fe3O4-GA-Re case, demonstrating that the PN-Fe3O4-GA-Re is more easily captured by the macrophages in the RES and therefore the PN-Fe3O4-GA-Re mainly occupies the RES. The tissues shown in Fig. 11C were weighted and counted by a counter for radioisotope and the percentage of radioactivity administered dose per gram of tissue ID/g % was used to express the isotope retention. The biodistributions of the two nanoparticles at 1 h, 2 h and 3 h after injection are shown in Fig. 11 D, E, F. Their distribution in the non-excretory tissues (bone, brain, heart, intestine, lungs, muscles, and spleen) more



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effectively highlighted the differences in the delivery properties of the S-Fe3O4-GA-Re vs PN-Fe3O4-GA-Re. The PN-Fe3O4-GA-Re nanoparticles were found to accumulate more than 3-fold higher than the S-Fe3O4-GA-Re in the lungs, liver and spleen, the possible reason being that the PN-Fe3O4-GA-Re has smaller particle size and peanuts-like morphology with aspect ratio of about 2. While the PN-Fe3O4-GA-Re circulates in vivo with blood circulation, its long axis surface would tend to interact with the vascular wall or cytomembrane. This leads to a larger bonding force per particle compared with the sphere,38 resulting in higher concentration in these organs. Further, the PN-Fe3O4-GA-Re concentration in the lungs, liver and spleen remains at a high level after 2 h and 3 h, and only its concentration in the lungs slightly decreases after 2 h (Fig. 11G). In addition, the S-Fe3O4-GA-Re concentration in the lungs, liver and spleen is also maintained at a relatively stable level after 2 h and 3 h. Moreover, the PN-Fe3O4-GA-Re concentration in the blood increases after 2 h, which is related to the decrease of concentration in lungs. In contrast, the S-Fe3O4-GA-Re concentration in the blood shows a slight decrease, consistent with a previous study.39 The biodistribution of the nanoparticles shows that the majority of the particles are arrested in the first encounter with the RES via cauda vein injection. The nude mice bearing two VX2 tumors on both left and right forelimbs were chosen as nude model. For treatment of the tumor-bearing nude model, the multi-mode functionalized nanoparticles were directly injected into the tumor site. To demonstrate higher efficacy of this targeted therapy for inhibition of tumor cell growth, one side tumor was placed under an external pulsed magnetic field generated by medical pulse gradient magnetic field generator, as shown in Fig. 12A. The CT images from the nude model obviously show that the nude mice have two tumors on both sides (Fig.12B, C).


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Four groups of tumor-bearing nude models were set for injection of drugs: PN-Fe3O4-PEI-GA, PN-Fe3O4-GA-Re, S-Fe3O4-GA-Re, free Re-188, together with a group with no treatment (NT). The nude model with injected drugs was imaged with SPECT-CT after magnetic targeting at one side for 1h, as shown in Fig. 12D. The signal intensity was obviously strong in the tumor tissue in the nano-drug treated groups, indicating that the PN-Fe3O4-GA-Re and S-Fe3O4-GA-Re mainly accumulated in the tumor site, but for the free 188ReO4- leachate group, the signal could be detected in the whole body of the nude models. This indicated that the two nanoparticles groups, both PN-Fe3O4-GA-Re and S-Fe3O4-GA-Re, accumulated more efficiently into the tumor sites when compared with free 188

ReO4- leachate. A possible reason for this is that the EPR effect is from the magnetic

nanoparticles in the tumor vasculature. Besides, the signal intensity from the tumor in the magnetic targeted side (region Q and S) was stronger than that from the non-targeted side (region R and T) in the two groups of particles. The intensity ratio Q/R for PN-Fe3O4-GA-Re and S/T for S-Fe3O4-GA-Re is 1.37 and 1.19 respectively, demonstrating that the PN-Fe3O4-GA-Re had higher sensitivity in vivo magnetic targeted delivery performance than that from the S-Fe3O4-GA-Re. Moreover, the intensity ratio Q/S is 1.15, indicating that the PN-Fe3O4-GA-Re had high fluorescence performance. These results suggest that peanut-like magnetic-fluorescent PN-Fe3O4-GA-Re can be effectively used as targeted drug delivery systems to the in vivo sites of interest under an external magnetic field.

Effects of nano-shapes and PEI grafted on VX2 tumor therapy The nude models were fed to evaluate the treatment outcome of the three nano-drugs in the



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next three days including S-Fe3O4-GA-Re, PN-Fe3O4-GA-Re and PN-Fe3O4-PEI-GA-Re. The CT images were taken to assess the volume of the tumors every 24 h. The tumor volume was calculated by serially measuring tumor dimensions in three orthogonal directions (long axis: a; short axis: b; high axis: c) in the CT images. Tumor volume was calculated using the formula (4/3)πabc and plotted over time as represented in Fig. 13A. The tumor volume from the individual nude models in each group was normalized with respect to the initial tumor volume prior to treatment. The results show that the tumor volume from the treatment groups significantly decreases with feeding time while the tumor volumes from the No Treatment (NT) and Free Re-188 groups all increase instead. The tumor volume from the NT group and free Re-188 is 3 and 2 times of the initial volume in the third day respectively. However, the tumor volume for the PN-Fe3O4-GA-Re and S-Fe3O4-GA-Re decreases to about 3/4 and 9/10 of the initial tumor volume respectively. The higher accumulation of PN-Fe3O4-GA-Re in the tumor site damages the tumor tissue and reduces the tumor volume than the S-Fe3O4-GA-Re, indicating that the PN-Fe3O4-GA-Re is more easily in vivo captured by the tumor tissue and therefore the PN-Fe3O4-GA-Re mainly occupies the tumor tissue. Greater percentage of GA drug and Re-188 is released from the peanut-like PN-Fe3O4-GA-Re than from spherical S-Fe3O4-GA-Re, thus more tumor cells are killed by GA drug and Re-188, leading to the shrinking of tumor volume. From Fig. 13A, it can be seen that the tumor volume (about 84% for S-Fe3O4-GA-Re, and 63% for PN-Fe3O4-GA-Re) under an external magnetic field is lower than that (about 90% for S-Fe3O4-GA-Re, and 75% for PN-Fe3O4-GA-Re) without the magnetic targeting due to the targeted delivery performance from the magnetic nanoparticles. These findings suggest that an


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external magnetic field targeting can indeed enhance an enrichment of magnetic nanomaterials, leading to an increased therapeutic effect. The tumor volume for the PN-Fe3O4-PEI-GA-Re group appears smaller than half of the initial tumor volume, much lower than that for the S-Fe3O4-GA-Re and PN-Fe3O4-GA-Re under an external magnetic field, demonstrating that the PN-Fe3O4-PEI-GA-Re has better inhibiting effect to the growth of tumor. This is probably due to the fact that the PEI-GO conjugating with the amide bond has the convergence effect, by making the magnetic nanoparticles to preferably accumulate in the tumor sites. Fig. 13B shows SPECT image of PN-Fe3O4-PEI-GA-Re nanoparticles in VX2 tumor-bearing nude mice. The magnetic PN-Fe3O4-PEI-GA-Re nanoparticles concentrate on the tumor sites as time goes by, and few nanoparticles are observed at the periphery of normal cells, indicating that the PN-Fe3O4-PEI-GA-Re nanoparticles have good active targeting performance. These findings provide a platform and guidelines for the preparation of effective active targeting anti-tumor agent. Conclusion In conclusion, spherical and peanut-shaped Fe3O4 nanoparticles were synthesized via simple solvothermal and EuO+ ion assisted solvothermal methods respectively, where the doping EuO+ ion plays the role of a template in the coupling of two Fe3O4 cores. The GO modified spherical S-Fe3O4 (S-Fe3O4-GO) and peanut-like PN-Fe3O4 (PN-Fe3O4-GO) were then fabricated and could all be cleared via the excretory system. PN-Fe3O4-GO has strikingly higher gambogic acid (GA) loading efficiency and release rate than the S-Fe3O4-GO, due to the peanut-like structure. High GA loading efficiency on PN-Fe3O4-GO leads to low release rate of GA, hence 2.5 mg GA was



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considered to be the optimum quantity for GA release of about 70.5%. The GA-loaded magnetic nanoparticles show a clear higher release rate at pH 5.7 than that at pH 7.4 due to the positive charge carried on both drugs and nanoparticles at a lower pH. To find effective active targeting anti-tumor agent, PEI was grafted to the PN-Fe3O4-GO. The multi-mode functionalized S-Fe3O4-GA-Re, PN-Fe3O4-GA-Re and PN-Fe3O4-PEI-GA-Re were prepared by Re-188 ion radiolabeling the PN-Fe3O4-GO-GA, PN-Fe3O4-PEI-GA and S-Fe3O4-GO-GA respectively. Both of SPECT and γ-counter reveal that the peanut-shaped PN-Fe3O4-GA-Re has higher sensitivity in the in vivo magnetic targeted delivery performance and is more easily captured by the macrophages in the RES than the spherical S-Fe3O4-GA-Re. PEI-GO conjugate has a convergence effect and PN-Fe3O4-PEI-GA-Re exhibits a good active targeting performance. This study also establishes a new method for the preparation of active targeting anti-tumor agent. Supporting Information The two tables and XRD patterns of EuOCl, S-Fe3O4, PN-Fe3O4, PN-Fe3O4-SiO2 and the EDAX analysis are provided in the supporting information. The M-H and ZFC-FC curves, as well as excitation and emission spectra are also provided. In addition, ESIMS spectra reveal fragments of intermediate compound. This material is available free of charge via the internet at http://pubs.acs.org. Acknowledgements The authors are grateful to the financial support from National Natural Science Foundation of China (Grant No. 20971043, No. 20577101) and to Mr Xiang-nong Liu, at the Analysis & Testing Center, Yangzhou University for help in sample characterization.


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(18) Yang, X. Y.; Zhang, X. Y.; Ma, Y. F.; Huang, Y.; Wang, Y. S.; Chen, Y. S. Superparamagnetic Graphene Oxide–Fe3O4 Nanoparticles Hybrid for Controlled Targeted Drug Carriers. J. Mater. Chem. 2009, 19, 2710-2714. (19) Yang, H. W.; Huang, C. Y.; Lin, C. W.; Liu, H. L.; Huang, C. W.; Liao, S. S.; Ma, C. C. M. Gadolinium-Functionalized Nanographene Oxide for Combined Drug and MicroRNA Delivery and MagneticResonance Imaging. Biomaterials 2014, 35, 6534-6533. (20) Saeed, L. M.; Mahmood, M.; Xu Y.; Nima, Z.A.; Kannarpady, G. K.; Bratton, S. M.; Dervishi, E.; Casciano, D.; Crooks, P. A.; Radominska-Pandya, A.; Biris, A. S. Nanodelivery of Gambogic Acid by Functionalized Graphene Enhances Inhibition of Cell Proliferation and Induces G0/G1 Cell Cycle Arrest in Cervical, Ovarian, and Prostate Cancer Cells. RSC Adv. 2015, 5, 44022-44030. (21) Hothi, P.; Martins, T. J.; Chen, L. P.; Deleyrolle, L.; Yoon, J.; Reynolds ,B.; Foltz, G. High-Throughput Chemical Screens Identify Disulfiram as an Inhibitor of Human Glioblastoma Stem Cells. Oncotarget 2012, 3, 1124-1136. (22) Wang, Z. H.; Zhou, C. F.; Xia, J. F.; Via, B.; Xia, Y. Z.; Zhang, F.F.; Li, Y. H.; Xia, L. H. Fabrication and Characterization of a Triple Functionalization of Graphene Oxide with Fe3O4, Folic Acid and Doxorubicin as Dual-Targeted Drug Nanocarrier, Colloids Surf., B 2013, 106, 60-65. (23) Knapp Jr, F. F.; Beets, A. L.; Guhlke S.; Zamora, P. O.; Bender, H.; Palmedo, H.; Biersack,

H.

J.

Availability

Tungsten-188/Rhenium-188

of

Generator

Radiopharmaceuticals for Cancer Treatment.

Rhenium-188 for

From

Preparation

of

the

Alumina-Based

Rhenium-188-Labeled

Anticancer Res. 1997, 17, 1783-1795.



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(24) Zhang, X. Y.; Li, J.; Zhu, Y.; Qi, Y. J.; Zhu, Z. Y.; Li, W. X.; Huang, Q. Nanographene Oxide Labeling with 188Re. Nucl. Sci. Tech. 2011, 22, 99-104. (25) Tung, V. C.; Allen, M. J.; Yang, Y.; Kaner, R. B. High-Throughput Solution Processing of Large-Scale Grapheme. Nat. Nanotechnol. 2009, 4, 25-29. (26) Burns, J. B.; Peterson, J. R.; ; Haire, R. G. Standard Enthalpies of Formation for Europium, Gadolinium, and Lutetium Oxychlorides, Calculated from Measured Enthalpies of Solution. J. Alloys. Compd. 1998, 265, 146-152. (27) Lamber, R.; Wetjen, S.; Jaeger, N. I. Size Dependence of the Lattice Parameter of Small Palladium Particles. Phys. Rev. B 1995, 51, 10968-10971. (28) Huan, W. W.; Cheng, C.; Yang, Y. X.; Yuan, H. M.; Li, Y. X. A Study on The Magnetic and Photoluminescence Properties of Eun+ and Sm3+ Doped Fe3O4 Nanoparticles. J. Nanosci. Nanotechnol.2012, 12, 4621-4634. (29) Chang, M. T.; Chou, L. J.; Hsieh, C. H.; Chueh, Y. L.; Wang, Z. L.; Murakami, Y.; Shindo, D. Magnetic and Electrical Characterizations of Half-Metallic Fe3O4 Nanowires. Adv. Mater. 2007, 19, 2290-2294. (30) Shi, H. W.; Huang, Y.; Cheng, C.; Ji, G. Y.; Yang, Y. X.; Yuan, H. M. Preparation and Characterization of Chain-Like and Peanut-Like Fe3O4@SiO2 Core–Shell Structure. J. Nanosci. Nanotechnol. 2013, 13, 6953-6960. (31) Zhang, L.M.; Lu, Z. X.; Zhao, Q. H.; Huang, J.; Shen, H.; Zhang, Z. Enhanced Chemotherapy Efficacy by Sequential Delivery of siRNA and Anticancer Drugs Using PEI-Grafted Graphene Oxide. Small 2011, 7, 460-464.


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(32) Huang, Y. P.; Hung, C. M.; Hsu, Y. C.; Zhong, C. Y.; Wang, W. R.; Chang, C. C.; Lee, M. J. Suppression of Breast Cancer Cell Migration by Small Interfering RNA Delivered by Polyethylenimine-Functionalized Graphene Oxide. Nanoscale Res. Lett. 2016, 11, 1-8. (33) Zhou, X.; Laroche, F.; Lamers, G.; Torraca, V.; Voskamp, P.; Lu, T.; Chu, F. Q.; Spaink, H. P.; Abrahams, J. P.; Liu, Z. F. Ultra-Small Graphene Oxide Functionalized with Polyethylenimine (PEI) for Very Efficient Gene Delivery in Cell and Zebrafish Embryos. Nano Res. 2012, 5, 703–709. (34) Weakley, T. J.; Cai, S. X.; Zhang, H. Z.; Keana, J. F. Crystal Structure of The Pyridine Salt of Gambogic Acid, J. Chem. Crystallogr. 2001, 31, 501-505. (35) Tang, X. Z.; Cao, Z.; Zhang, H. B.; Liu, J.; Yu, Z. Z. Growth of Silver Nanocrystals on Graphene by Simultaneous Reduction of Graphene Oxide and Silver Ions with a Rapid and Efficient One-Step Approach. Chem. Commun. 2011, 47, 3084-3086. (36) Zhao, G. X.; Wen, T.; Chen, C. L.; Wang, X. K. Synthesis of Graphene-Based Nanomaterials and Their Application in Energy-Related and Environmental-Related Areas. RSC Adv. 2012, 2, 9286-9303. (37) Miao, Y. B.; Owen, N. K.; Whitener, D.; Gallazzi, F.; Hoffman, T. J.; Quinn, T. P. In Vivo Evaluation of 188Re-Labeled Alpha-Melanocyte Stimulating Hormone Peptide Analogs for Melanoma Therapy. Int. J. Cancer 2002, 10, 480-487. (38) Venkataraman, S.; Hedrick, J. L.; Ong, Z. Y.; Yang, C.; Ee, P. L. R.; Hammond, P. T.; Yang, Y. Y. The Effects of Polymeric Nanostructure Shape on Drug Delivery.

Adv. Drug

Deliver Rev. 2011, 63, 1228-1246. (39) Aggarwal, P.; Hall, J. B.; Mcleland, C. B.; Dobrovolskaia, M. A.; McNeil, S. E. Nanoparticle



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Interaction with Plasma Proteins as it Relates to Particle Biodistribution, Biocompatibility and Therapeutic Efficacy. Adv. Drug Deliver Rev. 2009, 61, 428-437.


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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


Figures captions: 1. Fig. 1 TEM of A), S-Fe3O4 B), PN-Fe3O4, C), S-Fe3O4-SiO2 and D), PN-Fe3O4-SiO2

E),

HRTEM of PN-Fe3O4, F) HRTEM of PN-Fe3O4-SiO2 2. Fig. 2 (A), Molecular structure of intermediate, (B), possible mechanism for formation of peanut-like morphology 3. Fig. 3 (a) Strategy for synthesis of PEI grafting Fe3O4@SiO2-GO nanoparticles, and (b)Raman spectrum of D-Fe3O4-SiO2, S-Fe3O4-GO, PN-Fe3O4-GO and PN-Fe3O4-GO-PEI 4. Fig. 4 X-ray Photoelectron Spectroscopic (XPS) analysis of the PN-Fe3O4-GO and PN-Fe3O4-GO-PEI sample 5. Fig. 5 Ultraviolet absorption spectrum of PN-Fe3O4-GO and GA 6. Fig. 6 GA release rate of PN-Fe3O4-GO (A) and Changes of GA loading efficiency with PEI (B) 7. Fig. 7 GA release rate of PN-Fe3O4-GO-PEI (A), and Changes of Accumulative Release rate and Total release amount (B) with different PEI volumes 8. Fig. 8 XPS spectra of Carbon and Nitrogen in PN-Fe3O4-GO-PEI and GA loaded PN-Fe3O4-GO-PEI sample 9. Fig. 9 Loading efficiency and release rate of Fe3O4@SiO2-GO and Fe3O4@SiO2-GO-PEI under different pH conditions (pH 5.7, or pH 7.4) 10. Fig. 10 De-labelling rate of Re-188 from PN-Fe3O4-GO-GA, PN-Fe3O4-PEI-GA and S-Fe3O4-GO-GA 11. Fig.11 Digital camera and SPECT images from mouse and their organs, (A), PN-Fe3O4-GA-Re and (B), S-Fe3O4-GA-Re, (C), photo from the organs. ID/g % from the organs at (D), 1 h, (E), 2 h, (F), 3 h and (G), ID/g % of blood at different time



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

12. Fig.12 Digital photo and CT images from the mouse (A), under magnetic field, (B), top view in CT images (C), sectional view. CT images, SPECT images and SPECT-CT images from mice after injection with PN-Fe3O4-GA-Re, S-Fe3O4-GA-Re and free Re-188(D). 13. Fig.13 Changes of tumor size in three days (A), tumors volume and (B), SPECT image of PN-Fe3O4-PEI-GA-Re nanoparticles in VX2 tumor-bearing nude mice with different time


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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


A

B

C

D

E

F

Fig. 1 TEM of A), S-Fe3O4 B), PN-Fe3O4, C), S-Fe3O4-SiO2 and D), PN-Fe3O4-SiO2 E), HRTEM of PN-Fe3O4, F) HRTEM of PN-Fe3O4-SiO2



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

CH2CH2OCH 2CH2 O

A

O Fe EuO Fe O

O CH2CH2 OCH2CH2

B

O O Fe O n

CH2CH2OCH2 CH 2 O O Fe

O

O Fe EuO Fe

O O

O

Fe

O

O Fe O O n

CH 2CH2OCH2CH2

Fig. 2 (A), Molecular structure of intermediate, (B), possible mechanism for formation of peanut-like morphology


Page 36 of 49

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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


a

HOOC

NH2

H2N

COOH

O

OH

NH2

COOH

OH

NH2

H2N

COOH

NH C

HOOC

NH

NH2

C COOH

O

PEI

O

b

CONH

NH C OH NH C

O

CONH

Fig. 3 (a) Strategy for synthesis of PEI grafting Fe3O4@SiO2-GO nanoparticles, and (b) Raman spectrum of PN-Fe3O4-SiO2, S-Fe3O4-GO, PN-Fe3O4-GO, PN-Fe3O4-GO-PEI



I E P O O G G - -4444 4444 OOOO OOOO e3333 e3333 F F - N N P P

O G 4444 OOOO e3333 F N P

8 7 2 0 8 2 2) 8V 2e 4( 8y 2g 6r 8e 2n 8E 8g n 2i 0d 9n 2i 2B 9 2 4 9 2 6 9 2 8 9 2

0 0 2 1 0 0 0 1 ) 0V e 0( 8y g r 0e 0n 6E g n i 0d 0i n 4B

0 0 2

0000

I E P O G 4444 OOOO e3333 F N P

dddd

O G 4444 OOOO e3333 F N P

cccc O C

O C C O C

C O C

) . u . a ( y t i s n e t n I

C = C

C = C

) . u . a ( y t i s n e t n I

s 1

s 1 N

ps 22 ii SS

) . u . a ( y t i s n e t n I

C

L L K O

I E P O G 4444 OOOO e3333 F N P

) . u . a ( y t i s n e t n I

s 1 O

s 1 C

bbbb

aaaa

O C = O

O C = O

5 9 2 ) v e ( y g 0r 9e 2n e g n i g n 5i 8B 2

0 8 2

5 9 2 ) V e ( y 0r g 9e 2n e g n i d 5n 8i 2B

0 8 2

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 38 of 49



Fig. 4 X-ray Photoelectron Spectroscopic (XPS) analysis of the PN-Fe3O4-GO and PN-Fe3O4-GO-PEI sample Fig. 4a: XPS full spectra, Fig. 4b: XPS C1s spectra. The red curves for each graphical representation correspond to the C1s signal as collected from each material. The curves (Fig. 4c: PN-Fe3O4-GO, and Fig. 4d: PN-Fe3O4-GO-PEI) underneath the C1s peaks, correspond to mathematical models of the each underlying moiety present in the samples (i.e., p–p shake up; C=O, C–OH, COOH, and C–C bonds).


Page 39 of 49

0.7

0.8 0.7

0.6

40µg/ml 20µg/ml 10µg/ml 8 µg/ml 6 µg/ml 4 µg/ml 2 µg/ml

0.5 0.4

y=0.0164x+0.0125 slope=0.0164 R2=0.99

0.5

Absorbance

0.6

Absorbance

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


0.3

0.4 0.3 0.2

0.2

0.1

0.1

0.0

0.0 300

350

400

450

500

Wavelength（ nm）

550

600

0

5

10

15

20

25

30

35

The concentration of GA（ µg/ml）

A

B

Fig. 5 Ultraviolet absorption spectrum of PN-Fe3O4-GO and GA Fig. 5A Ultraviolet absorption spectrum of PN-Fe3O4-GO loading with GA amount; Fig. 5B Standard curve of the free GA concentration response to absorbance at 360 nm


40

45


1.5mg 2.5mg 5.0mg 10.0mg

70

(A)

GA Loading Efficiency(mg/g)

80

GA Release Rate (%)

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

60 50 40 30 20

75

Page 40 of 49

(B)

70 65 60 55 50

10

45

0 0

20

40

60

Time(h)

80

0

1

2

3

4

5

6

PEI Volume(ml)

Fig. 6 GA release rate of PN-Fe3O4-GO (A) and Changes of GA loading efficiency with PEI (B)


0ml 1ml 2ml 3ml 4ml 5ml

60 50 40 30 20 10

70 65 60 55 50 45 40 35 30 25 20 15

33

(B)

32 31 30 29 28 27 26

Total Release Amount(mg/g)

(A)

70

GA release rate(%)

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


Accumulative Release Rate(%)

Page 41 of 49

25

0 0

10

20

30

40

50

60

70

Time(h)

0

1

2

3

4

5

PEI Volume(ml)

Fig. 7 GA release rate of PN-Fe3O4-GO-PEI (A), and Changes of Accumulative Release rate and Total release amount (B) with different PEI volumes



2000

18000

C1s Counts/S

12000

6000

N1s

GA

Counts/S

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 42 of 49

GA

1500

1000

C1s: N1S=95.29:4.71 PN-Fe3O4-PEI-GA

C1s: N1S=93.98:6.02

PN-Fe3O4-PEI-GA

500

PN-Fe3O4-GO-PEI

0 298 296 294 292 290 288 286 284 282 280 278

PN-Fe3O4-GO-PEI

412 410 408 406 404 402 400 398 396 394

Binding Energy

Binding Energy

Fig. 8 XPS spectra of Carbon and Nitrogen in PN-Fe3O4-GO-PEI and GA loaded PN-Fe3O4-GO-PEI sample


Page 43 of 49

PN-Fe3O4-GO-GA-5.7

90

PN-Fe3O4-GO-GA-7.4

80

0 3

GA released(%)

0 4 0 2 0 1

) % ( y c n e i c i f f e g n i d a o l

A A G G - I O E G P - 4444 4444 OOOO OOOO e3333e3333 F F - N N P P

0 5

PN-Fe3O4-PEI-GA-5.7

70

PN-Fe3O4-PEI-GA-7.4

60 50 40 30 20 10

4 . 7 = H p

7 . 5 = H p

0000

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


0 0

10

20

30

40 50 Time(h)

60

Fig. 9 Loading efficiency and release rate of Fe3O4@SiO2-GO and Fe3O4@SiO2-GO-PEI under different pH conditions (pH 5.7, or pH 7.4)



% e t a R g n i l l e b a l e D

0 0 e e 2 R R e 0 - R 8 A 1 A G 0 G A 6 I - G 1) 4444 E 0n 4444 OOOO P 4i 1 e3333 OOOO3333 4444 0m F e OOOO 2( - F 1 e3333 N 0T F P S 0 1 N 0 P 8 0 6 0 4 0 2 0000 0 2 2 0 8 6 4 2 0 8888 6666 4444 2 2 1 1 1 1 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

Fig. 10 De-labelling rate of Re-188 from PN-Fe3O4-GO-GA, PN-Fe3O4-PEI-GA and S-Fe3O4-GO-GA


Page 44 of 49

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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


Fig.11 Digital camera and SPECT images from mouse and their organs, (A), PN-Fe3O4-GA-Re and (B), S-Fe3O4-GA-Re, (C), photo from the organs. ID/g % from the organs at (D), 1 h, (E), 2 h, (F), 3 h and (G), ID/g % of blood at different time



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

Fig.12 Digital photo and CT images from the mouse (A), under magnetic field, (B), top view in CT images (C), sectional view. CT images, SPECT images and SPECT-CT images from mice after injection with PN-Fe3O4-GA-Re, S-Fe3O4-GA-Re and free Re-188 (D).


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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


B

Fig.13 Changes of tumor size in three days (A), tumors volume and (B), SPECT image of PN-Fe3O4-PEI-GA-Re nanoparticles in VX2 tumor-bearing nude mice with different time



Table captions: Table 1 Loading efficiency of GA on the PN-Fe3O4-GO and S-Fe3O4-GO

1.0 mg

1.5 mg

2.5 mg

5.0 mg

10 mg

46.28

45.87

49.79

53.37

86.00

Release rate %

68.72

70.51

69.86

71.75

44.54

Release amount

31.80

32.34

34.78

38.29

38.30

GA loading

45.01

43.11

45.40

50.70

78.85

Release rate

66.01

68.99

67.14

69.72

45.63

Release amount

29.71

29.74

30.48

35.35

35.98

Gambogic acid (GA)

GA loading

PN-Fe3O4-GO S-Fe3O4-GO

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 48 of 49

efficiency mg/g

efficiency mg/g


Page 49 of 49

Graphical abstract

c

H2N

HOOC

NH2

COOH

O

OH

NH2

COOH

OH

NH2

H2N

NH

NH2

PEI

O NH C

NH C

C

O

COOH

NH C

HOOC

CONH

50 loading efficiency(%)

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


COOH

D-GO-GA D-GO-PEI-GA

40 30 20 10

OH

O

0

CONH

pH=5.7

pH=7.4


SiO2 Nanoparticles with

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