In Vivo Study of Spiky Fe3O4@Au Nanoparticles with Different Branch

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In Vivo Study of Spiky FeO@Au Nanoparticles with Different Branch Lengths: Biodistribution, Clearance, and Biocompatibility in Mice Hongjian Zhou, Sangjin Oh, Ji Eun Kim, Fengming Zou, Dae Youn Hwang, and Jaebeom Lee ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00505 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

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ACS Applied Bio Materials

In Vivo Study of Spiky Fe3O4@Au Nanoparticles with Different Branch Lengths: Biodistribution, Clearance, and Biocompatibility in Mice

Hongjian Zhoua,†, Sangjin Ohb, Ji Eun Kimc,†, Fengming Zoud,†, Dae Youn Hwangc and Jaebeom Leee,*

a

Key Laboratory of Materials Physics, Centre for Environmental and Energy

Nanomaterials, Anhui Key Laboratory of Nanomaterials and Nanotechnology, Institute of Solid State Physics, Chinese Academy of Sciences, Hefei 230031, P. R. China b

Department of Cogno-Mechatronics Engineering, Pusan National University, Busan

46241, Republic of Korea c

Department of Biomaterials Science, College of Natural Resources and Life Science

/ Life and Industry Convergence Research Institute, Pusan National University, Miryang 50463, Republic of Korea d High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui 230031,

China e Department

of Chemistry, Chungnam National University, Daejeon, 34134, Republic

of Korea

Abstract Magnetoplasmonic nanoparticles (Fe3O4@Au NPs) have been proven to be effective theranostic agents in genetic transmission, drug targeted delivery system, as well as in photothermal treatment. Herein, two spiky magnetoplasmonic NPs with different branch lengths and numbers (short- and long-branched spiky Fe3O4@Au NPs) were 1

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specifically designed to determine theirs in vivo behaviors. The biocompatibility, biodistribution, and clearance of spiky Fe3O4@Au NPs were examined in mice. Organ distributions showed that intravenously administered spiky Fe3O4@Au NPs cumulated mainly in liver and spleen, and particle shape significantly affected their in vivo behaviors. The higher tendency in bioaccumulation of short-branched rather than longbranched spiky Fe3O4@Au NPs was observed in the spleen because of long-branched spiky Fe3O4@Au NPs with a high aspect ratio were internalized more slowly than shortbranched spiky Fe3O4@Au NPs. Serum biochemistry and transmission electron microscopy of ultra-histological structures indicated that spiky Fe3O4@Au NPs did not exhibit distinct toxicity in vivo and posed no potential risk of causing liver and kidney dysfunction. These findings lay the foundation for the design of future theragnostic agents.

Keywords: magnetoplasmonic nanoparticles; branched nanostructure; biodistribution; shape; nanotoxicity; shaping nanomedicine

1. Introduction The particle size, geometry, surface functional groups, and nanostructure of nanoparticles all significantly regulate theirs in vitro and vivo behaviors for various biomedical applications 1-6. Among them, particle geometry significantly affects the in vivo fate of nanoparticles 2,4,7. Although spherical nanoparticles have been successfully utilized in various biomedical applications because of their ease of fabrication, many 2


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recent findings have suggested that the control of particle geometry (rod, disk, ellipsoid, hemispheres, and so forth) can enormously improve the efficacy of targeted particulate carriers

8-10.

Over the last decade, researchers have developed various ways of

controlling the geometry of different materials, providing an opportunity to explore the impact of nanoparticle geometry on their in vivo fate 11-13. Compared with nanospheres, non-spherical nanoparticles are able to form a multitude of polyvalent interactions with cell membranes

1,4,14,15.

It has been verified

that the geometry of nanoparticles in circulation affects their ability to evade immune uptake and escape from the blood flow to bind with their target 16. This is essential for targeting, especially in the case of vascular targets, since geometrically enhanced targeting can effectively neutralize hemodynamic forces and extricate the nanoparticles from the endothelium

14,15.

For example, Smith et al.

16

found that prolate ellipsoids

have higher adhesive rate and slower rate of internalization than spheral nanoparticles. Ghandehari et al.1 also demonstrated that the uptake of gold nanorods with a high aspect ratio (size: 10 nm × 45 nm; aspect ratio: 4.5) was lower than that of gold nanospheres in the liver and spleen. Of greater note, Maysinger et al.17 reported that urchin-like gold nanoparticles induced an inflammatory response in microglial cells, which increased interleukin-1α production compare with globose and clubbed nanoparticles. These distinctions demonstrated different effect on immune system depending on the geometry of nanoparticles. Thus, “shaping” nanomedicine can provide new opportunities to address important and unmet clinical needs. Magnetoplasmonic nanoparticles integrate two nanocrystal domains (iron oxide 3



and gold) into a single architecture (i.e., Fe3O4@Au core-shell NPs) through solid-state interfaces. The single architecture features greater surface chemistry tunability and improved optical and superparamagnetic properties compared with those of the individual components, which promoted their applications in miscellaneous biomedical applications, e.g. biosensing, bioimaging, photothermal treatment, genetic transmission and drug targeted delivery system 18-23. We previously reported spiky iron oxide-gold core-shell nanoparticles (Fe3O4@Au NPs) with well-defined shapes and distinctive topological structures following a self-assembly method 24. Subsequently, our research group studied the cytotoxicity of spiky Fe3O4@Au NPs to tumor cells and the effects of spiky Fe3O4@Au NP shapes on gene

expression profiling related to cell

proliferation, cell cycle, cell differentiation, etc. 25. This research provided fundamental information on the impact of geometry of nanoparticles for cellular immune responses and cyto/genotoxicity. However, it is necessary to acquire a deeper level of understanding into their in vivo behaviors in order to expand their applications in modern medicine. Illuminating the effect of particle geometry on their in vivo behaviors could thoroughly aid us in understanding the toxicological profile of these nanomaterials, which is important for their transformation into clinical practice. In this paper, we systematically evaluated the in vivo behavior including distribution, clearance and biocompatibility of spiky Fe3O4@Au NPs with different branch lengths and numbers (short- and long-branched) in mice following intravenous injection. To assess the biodistribution and clearance of spiky Fe3O4@Au NPs, the content of gold (Au) in blood, urine, feces, as well as tissue organs were measured via 4


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inductively coupled plasma optical emission spectroscopy (ICP-OES). Moreover, the biocompatibility of spiky Fe3O4@Au NPs was evaluated through the analysis of serum biochemistry and ultra-histological structure.

2. Experimental details 2.1 Preparation of spiky Fe3O4@Au core-shell NPs with different branch lengths The preparation of spiky Fe3O4@Au NPs were employed HEPES as a reducing agent and stabilizer to reduce HAuCl4 solution, as reported previously 26,27. The HEPES (100 mmol/L) aqueous solution was produced via deionized water in advance, and then 1 M NaOH was using to adjusted its pH to 7.4 at 25 °C. To produce longbranched spiky Fe3O4@Au NPs, 500 μL of 0.136 μM Fe3O4 solution was mixed with 10 mL of HEPES (100 mM) and 0.25 mL of HAuCl4 (20 mM) aqueous solution. The mixed solution was maintaining in 25 oC water bath without shaking. After 30 min, the solution color was transferred from grayish brown to green and subsequently changed to greenyellow color. The short-branched spiky Fe3O4@Au NPs were obtained as the same procedure as long-branched spiky Fe3O4@Au NPs except for using 10 mM of HEPES aqueous solution in the reaction system. 2.3 Biodistribution of spiky Fe3O4@Au NPs in vivo Briefly, thirty mice were arbitrarily divided into three groups (n = 10 mice): saline solution treatment (control group), short-branched spiky Fe3O4@Au NP treatment (short group), and long-branched spiky Fe3O4@Au NP treatment (long group). After 5



anesthesia with a ketamine/xylazine mixture (40:5 mg/kg, i.p.), mice in the short group and the long group were injected with short-branched spiky Fe3O4@Au NPs and longbranched spiky Fe3O4@Au NPs, respectively. The NP suspensions (in 1× PBS) were injected intravenously at a dose of 30 g/kg. Mice in the control group acquired the same volume of 1× PBS by the same technique. At one day or 7 days after NP injection, all ICR mice were euthanized using CO2 gas, and seven types of tissues (heart, liver, lung, spleen, kidney, abdomen fat, and brain), blood, and urine samples were gained and stored in Eppendorf tubes at −80 °C until assayed. Urine samples were gathered on cellophane to avoid fecal contamination. For in vivo toxicity studies, the body weight of each mouse was monitored for seven days after NP or PBS administration. To measure NP concentration, blood, urine, faces, and tissue samples were wholly lysed in aqua regia, and subsequently the lysed solution was centrifuged at 1,000 × g for 5 min. Following evaporation, the resulting precipitates were redispersed in 10 mL of 0.05 M HCl. The gold (Au) content in the lysed solution was analyzed by ICP-OES (ICP-6300, Thermo Fisher Scientific, USA) with radio frequency power at 1150 W and nebulizer gas flow of argon at 0.5 L/min. 2.4 TEM of the hepatic, splenic, and renal tissues TEM measurements were employed to investigate the uptake and subcellular localization of spiky Fe3O4@Au NPs in the liver, spleen, and kidney tissues. Briefly, tissue samples were collected from animals seven days after NP administration and were instantaneously fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate 6


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buffer overnight. 2% osmium tetroxide were used to fix these specimens following graded alcohol solutions to dehydrate. Finally, Ultrathin sections (100 nm) were embedded in Epon and mounted on copper grids for TEM analysis (HITACHI, H-7600, Japan) at 60 kV, uranyl acetate and lead citrate as contrast. The samples in the control group were prepared following the same procedures. 2.5 Statistical analysis All results were displayed as the mean ± standard deviation (SD) and statistically analysed by GraphPad 6.02 software (San Diego, CA, USA) by t-test. The level of significance in all analysis was accepted at P value ˂ 0.05.

3. Results and discussion 3.1 Characterization of the Fe3O4@Au NPs We synthesized spiky Fe3O4@Au NPs with different branch lengths to evaluate theirs in vivo behavior in mice

28-31.

Citrate-modified Fe3O4 NPs as seeds, the spiky

Fe3O4@Au core-shell NPs were grown from a simple reaction system where served as a reducing agent and stabilizer. Different concentrations of HEPES were used to tailor the number and length of branches in spiky Fe3O4@Au NPs. The physicochemical characteristics of spiky Fe3O4@Au NPs are outlined in Table S1, and their typical transmission electronic microscopic (TEM) images are exhibited in Figure 1. It was observed that the as-synthesized short-branched spiky Fe3O4@Au NPs were nearspherical, possessing a full core with cutty, anomalo, and bulletheaded branches. The 7



average diameter of the short-branched spiky Fe3O4@Au NPs was 51.4 nm, and each branch was cone-shaped with a length of 10 nm. The number of branches was approximately 10–15 per NP via counting in TEM images. With the increase in HEPES concentration up to 100 mM, the number of branches decreased to 4-6 per NP and the branch changed to rod shape at a length of 25–33 nm. The dimensions of the longbranched spiky Fe3O4@Au NPs averaged 48.2 nm. Both types of spiky Fe3O4@Au NPs were highly monodispersed in size, as measured by TEM. Both types showed negative zeta potential values, which were thought to arise from HEPES as the surfactant on spiky Fe3O4@Au NPs during the preparation process (Table S1). The surface plasmon resonance (SPR) frequency of spiky Fe3O4@Au NPs was significantly different depending on their branch lengths and numbers 32. From the UV-vis spectra of spiky Fe3O4@Au NPs (Figure 1C), it was clearly observed that the short-branched spiky Fe3O4@Au NPs had a prominent SPR peak at 575 nm, which correlated to approximately 51.4 nm size with short gold tips. In contrast, long-branched spiky Fe3O4@Au NPs showed two distinct SPR peaks, transverse and longitudinal, at 540 nm and 745 nm, respectively, which rendered similar optical properties as those of gold nanorods. Owing to their specific optical properties, the spiky Fe3O4@Au NPs have potential to be effective theranostic agentia for genetic transmission, drug targeted delivery system, as well as in photothermal treatment 33,34.

8


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Figure 1. Typical TEM images of (A) short- and (B) long-branched spiky Fe3O4@Au NPs. (C) UV-vis spectra of spiky Fe3O4@Au NPs.

3.2 Biodistribution of spiky Fe3O4@Au NPs in mice The physicochemical properties of nanoparticles determine their in vivo fate and transportation across biological barriers after intravenous administration. In this work, the biodistributions of two spiky Fe3O4@Au NPs with similar sizes (51.4 nm for shortbranched spiky NPs and 48.2 nm for long-branched spiky NPs), but different geometries, were evaluated in mice. To determine whether the morphology of spiky Fe3O4@Au NPs affects their biocompatibility in vivo, the blood, urine, feces, as well as several tissue organs (heart, kidneys, lungs, liver, spleen, and abdominal fat) were collected on the first day after intravenous administration, and gold content was analyzed by ICP-OES. The biodistribution of spiky Fe3O4@Au NPs normalized to organ weight on the first day after intravenous administration is shown in Figure 2. The distributions of short- and long-branched spiky Fe3O4@Au NPs were expressed as μg/g of tissue based on the average gold content in the major representative organs/tissues taken from five individual mice. It can be observed in Figure 2 that the liver and spleen were the preferential sites for accumulation of both short- and long-branched spiky Fe3O4@Au NPs. Interestingly, the Au content in the spleen was significantly different 9



for short- and long-branched spiky Fe3O4@Au NPs, at 7.18 μg/g and 4.88 μg/g, respectively. This revealed that the short-branched spiky Fe3O4@Au NPs might be disposed to accumulate in the spleen compared with long-branched spiky Fe3O4@Au NPs. However, Au was scarcely detected in the heart, kidney, lung, and abdominal fat of spiky NP-injected animals. Au was also not found in the brain in mice treated with either short- or long-branched spiky Fe3O4@Au NPs on the first day after intravenous administration, indicating that both types of spiky Fe3O4@Au NPs were unable to pass through the hemato encephalic barrier in current experiment. Meanwhile, in our similar experiments, the Au NPs with 20 nm size penetrated the hemato encephalic barrier and accumulated in different organs (Figure S2 in the Supporting Information). The recovery percent of gold for short- and long-branched spiky Fe3O4@Au NPs was individually accounted for 61.1% and 68.3% of the original injected dose, which could because of the accumulation of spiky Fe3O4@Au NPs in other unanalyzed tissue and organs (such as skin, skeleton, and soft tissue). This result was consistent with those in other reports whereby a high percentage of injected Au NPs remained in the carcass 35. According to literatures, nanoparticles are usually eliminated by macrophages in the reticuloendothelial system (RES), spleen and bone marrow, as well as Kupffer cells in the liver

36.

Also, nanoparticles larger than 6 nm cannot be filtered by glomerular

filtration and urinary excretion

37.

Therefore, the conspicuous cumulation in liver

revealed that the hepatobiliary system might be the principal pathway to eliminate two types of spiky Fe3O4@Au NPs through mononuclear phagocytes in the RES 36, which was by the general consensus on the fate of nanoparticles in vivo 38. 10


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To verfiy the reason of low gold recovery percent for short- and long-branched spiky Fe3O4@Au NPs on the first day after intravenous administration, the amount of gold present in feces was measured via ICP-OES. Figure 2 shows that the accumulative gold content in feces for short- and long-branched spiky Fe3O4@Au NP was approximately 1.98 μg/g and 2.06 μg/g, respectively.

Figure 2. Biodistribution of spiky Fe3O4@Au NPs with different branch lengths (shortand long-branched) in the blood, urine, feces, and representative organs of mice on the first day after intravenous administration. 3.3 Uptake and subcellular localization of spiky Fe3O4@Au NPs in liver and spleen We further evaluated the cumulation of short- and long-branched spiky Fe3O4@Au NPs in liver and spleen on the first (short-term) and seventh day (long-term) after intravenous administration. Figure 3 demonstrates that both types of spiky Fe3O4@Au NPs were cumulated in the liver and spleen. The spiky Fe3O4@Au NPs were rapidly cleared from blood after intravenous administration, and then mainly cumulated in the liver and spleen due to their elimination via the RES 4,36,38. In the liver and spleen, the changes in cumulation over time were somewhat different (Figure 3). In the liver, Au content gradually decreased from 8.83 μg/g to 5.17 μg/g in mice injected 11



with short-branched spiky Fe3O4@Au NPs during seven days and from 9.13 μg/g to 5.92 μg/g in those injected with long-branched spiky Fe3O4@Au NPs. In contrast, the spleen displayed a gradual increase in Au content during seven days for short-branched spiky Fe3O4@Au NPs (7.18 μg/g to 10.16 μg/g) and long-branched spiky NPs (4.88 μg/g to 7.45 μg/g). A proposed clearance mechanism of spiky Fe3O4@Au NPs by the spleen was that the injected spiky Fe3O4@Au NPs may be transported through venous circulation or diffuse into the lymph nodes and are then trapped in the spleen 39. The increased uptake of spiky Fe3O4@Au NPs in the spleen during seven days in our experiment was consistent with the proposed mechanism. There was a tendency for higher bioaccumulation of short-branched than long-branched spiky Fe3O4@Au NPs in the spleen. This may predict that short-branched spiky Fe3O4@Au NPs have a higher risk of long-term toxicity compared with long-branched spiky Fe3O4@Au NPs. In the current study, comparing the uptake of the two spiky Fe3O4@Au NPs with different branch lengths, a contact angle parameter was formulated, which was quantitatively related to particle internalization velocity. The long-branched spiky Fe3O4@Au NPs, with a high aspect ratio, aligned their macroaxis in parallel to the cell membrane. Therefore, they were internalized more slowly than short-branched spiky Fe3O4@Au NPs 4.

12


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Figure 3. Short-term (one day) and long-term (seven days) uptake of short- and longbranched spiky Fe3O4@Au NPs in (A) hepatic and (B) splenic tissues

To further determine the ultrastructural location of the spiky Fe3O4@Au NPs, TEM images of liver, spleen, and kidney were acquired on the seventh day after intravenous administration. Contrast to the control group, it has no ultra-histological abnormality in the liver, spleen, or kidney in any of the spiky Fe3O4@Au NP-treated mice (Figure 4). Unlike the control group, monodispersed or agglomerated short- and long-branched spiky Fe3O4@Au NPs were trapped in Kupffer cells in the liver and macrophages in the spleen for all treatment groups, but we did not observe nanoparticles in the kidney. In addition, numerous cytoplasmic vesicles and lysosomes contained spiky Fe3O4@Au NPs, while the nuclei, mitochondria, or the Golgi complex structures have not find spiky Fe3O4@Au NPs in the ultra-histological structure of liver and spleen. the spiky Fe3O4@Au NPs were discharged into the circulatory system and were subsequently devoured via the mononuclear phagocytic system of liver and spleen after intravenous injection. The spiky Fe3O4@Au NPs, as invaded blood-borne matter, came 13



into intimate contact with immune cells in the liver and spleen, such as hepatic macrophages (Kupffer cells)

40-43.

It is well known that Kupffer cells are specialized

macrophages located in the liver that phagocytose and scavenge foreign invading substances.44-46 Thus, they resulted in a gradual increase in spiky Fe3O4@Au NPs in the liver and spleen. This result unambiguously showed that spiky Fe3O4@Au NPs cumulated predominately in liver and spleen and preserved long-term post-exposure without biodegradation. Many previous studies speculated that Au NP excretion by the kidney system gives rise to destroy to the glomeruli, thus increasing its permeability 47. In the current study, the conspicuous histopathological abnormities in the kidney after histological assessment was not detected in Figure 4G–4I. As observed in Figure 2, Au was not detected in the urine of mice treated with either short- or long-branched spiky Fe3O4@Au NPs on the first day of intravenous administration. The above results revealed that neither type of spiky Fe3O4@Au NP was excreted through the renal system. As reported by many researchers, hydrated diameters of particles smaller than 5.5 nm can be eliminated by the kidney in mice 5. In the current experiment, no spiky Fe3O4@Au NPs can be found in the kidney, demonstrating that the spiky Fe3O4@Au NPs cannot infiltrate the glomeruli basilar membrane (Figure 4H and 4I). It is highly probable that large spiky Fe3O4@Au NPs were not excreted through the urine, but were purged from the circulation via the mononuclear phagocyte system and thus tended to cumulate in the liver and spleen

48,49.

Therefore, the above results revealed that the

morphology of spiky Fe3O4@Au NPs can increase their interaction with cells, thus 14


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inducing their accumulation in the spleen and liver.

Figure 4. TEM images of the liver (A-C), spleen (D-F), and kidney (G-I) of mice sacrificed on the seventh day after intravenous administration of short- and longbranched spiky Fe3O4@Au NPs.

Generally, the potential in vivo toxicity of the spiky Fe3O4@Au NPs is a considerable concern because of their non-biodegradability 50. Thus, we measured the variation of body weight as a direct index of in vivo toxicity, as illustrate in Figure S3 51.

The body weight of mice treated with spiky Fe3O4@Au NPs were not obviously

changed compared with that of saline-treated mice during seven days. In addition, no death occurred in the experimental animals. It demonstrated that the particles did not exhibit overall side-effect on mice. The relative change of the major organs weight was also considered as a toxicity indicator 52. The relative weights of the collected organs on the seventh day after intravenous administration of spiky Fe3O4@Au NPs are shown 15



in Figure 5. The observations suggested that the spiky Fe3O4@Au NPs have no effect on the fluctuation of the organs weight of mice. Overall, mice tolerated the spiky Fe3O4@Au NP injections well and there are no behavioral disorders during whole experiment. Moreover, there was no clinical symptom after injection of the spiky Fe3O4@Au NPs in mice, such as illness, stress discomfort, or death in the study. Compared to the saline-treated mice, evidence of inflammation, including infectionfighting cells, were not detected in necropsy in two spiky Fe3O4@Au NP-treated mouse group.

Figure 5. Relative organ weights of mice on the seventh day after treatment of spiky Fe3O4@Au NPs

The liver plays a key role in metabolizing harmful xenobiotics, such as drugs and toxic chemicals

53.

These excessive agentia could lead to hepatic damage, which is

usually detected via abnormal liver function, hydropic degradation, apoptosis, or 16


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necrosis of hepatic cell 54. Thus, in order to investigate liver toxicity induced by spiky Fe3O4@Au NPs, the biochemical activities of substances within the serum, including alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), were analyzed on the first and seventh day after intravenous administration (Figure 6A–6D). Among them, ALT and AST activity were important indicators of liver function. The results can be used to distinguish various kinds of hepatic dysfunction and measure in the range of foregone hepatic injury. ALT levels in control, short, and long groups showed no significant differences on the first and seventh day after intravenous administration of spiky Fe3O4@Au NPs. The levels of AST and LDH in both short and long groups were low compare with the control group, which indicated that both types of spiky Fe3O4@Au NPs inhibited the activity of these two enzymes to some extent. Kidney function was evaluated by analyzing serum blood urea nitrogen (BUN) and creatinine (CRE) on the first and seventh day after intravenous administration (Figure 6E–6F). There were no obvious changes in the markers of kidney function (BUN and CRE) in both short and long groups, indicating that the spiky Fe3O4@Au NPs did not significantly affect the renal function of the mice. All serum biochemical activity levels were within the normal range in mice and could not sensitively reflect liver and kidney damage, suggesting the good in vivo biocompatibility of spiky Fe3O4@Au NPs. Although more research is necessary to understand the long-term toxicity intensively, our small-scale pilot toxicity study provided a basis for further exploration of these nanomaterials.

17



Figure 6. Blood biochemistry data including markers of liver function: (A) ALT, (B) AST, (C) ALP, and (D) LDH and kidney function: (E) BUN and (F) CRE.

Based on our results, we postulated that spiky Fe3O4@Au NPs possessed good biocompatibility and low toxicity, which is beneficial to apply in biomedical fields, e.g. genetic transmission, drug targeted delivery system, biosensing, bioimaging, as well as photothermal treatment. We envisaged that the use of spiky Fe3O4@Au NPs as targeting nanovehicles of therapeutic drugs to the liver and spleen is possible. Despite showing great potential, the use of these nanomaterials requires more investigation before they can be used in a clinical setting. 18


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4. Conclusions In this work, the effects of short- and long-branched spiky Fe3O4@Au NPs with different branch lengths and numbers were studied concerning biodistribution, clearance, as well as biocompatibility via intravenous injection in mice. Organ distributions showed that both types of spiky Fe3O4@Au NPs cumulated primary in liver and spleen after treatment of spiky NPs. Apparent impact of particle geometry on the in vivo behavior were exhibited. Short-branched spiky Fe3O4@Au NPs were highly accumulated in the spleen compared with long-branched NPs. This was attributed to the high-aspect-ratio of long-branched spiky Fe3O4@Au NPs, resulting in slower internalization than that of short-branched spiky Fe3O4@Au NPs. Moreover, both types of spiky Fe3O4@Au NPs were found in a lot of cytoplasmic vesicles, lysosomes of liver Kupffer cells and spleen macrophages in TEM images of ultra-histological structures. Serum biochemistry results demonstrated that the spiky Fe3O4@Au NPs did not cause distinct toxicity in vivo and could not sensitively reflect liver and kidney dysfunction. These findings provided substantial evidence that nanoparticle shape is an important contributor to tissue distribution and cellular interactions in vivo.

Supporting Information The supporting information is available free of charge on the ACS Publications website. Physicochemical characteristics of spiky Fe3O4@Au NPs, preparation and characterization of spherical GI-Au NPs, biodistribution of spherical GI-Au NPs at 1, 19



2, and 8 days after brain injection, mice body-weight-change curves over a span of 7 days after intravenous injection with saline solution, short-, or long-branched spiky Fe3O4@Au NPs (PDF).

Author Information Corresponding Author * Email: [email protected] (J. Lee)

Author contributions †These

authors contributed equally to this work.

Notes The authors declare no competing financial interest.

Acknowledgment This work was supported by the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant HI16C1553, HI17C1260), the National Natural Science Foundation of China (Grant 51502296), Anhui Provincial Natural Science Foundation (1808085MH268), and the National Research Foundation of Korea grant funded by the Korea government (MSIP) (Grant 2016R1A2B4012072, 2017R1A41015627). We are also grateful for the support of Hefei leading talent in 2017 for Fengming Zou. 20


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419x212mm (96 x 96 DPI)


In Vivo Study of Spiky Fe3O4@Au Nanoparticles with Different Branch

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