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Superparamagnetic iron oxide nanoparticles modified with Tween 80 pass through the intact blood-brain barrier in rats under magnetic field Yinping Huang, Baolin Zhang, Songbo Xie, Boning Yang, Qin Xu, and Jie Tan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02838 • Publication Date (Web): 19 Apr 2016 Downloaded from http://pubs.acs.org on April 23, 2016

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ACS Applied Materials & Interfaces

Superparamagnetic iron oxide nanoparticles modified with Tween 80 pass through the intact blood-brain barrier in rats under magnetic field Yinping Huang, † Baolin Zhang,*,† Songbo Xie, † Boning Yang,*,‡ Qin Xu, § and Jie Tan*,§ †

State Key Laboratory Breeding Base of Nonferrous Metals and Specific Materials Processing, College of

Materials Science and Engineering, Guilin University of Technology, Jian Gan Road 12, Guilin 541004, China. ‡

Guangxi Collaborative Innovation Center for Biomedicine and Department of Human Anatomy, Guangxi Medical

University, 22 Shuang Yong Road, Nanning 530021, China. §

Guangxi Key Laboratory of Brain and Cognitive Neuroscience, Guilin Medical University, 109 North 2nd Huan

Cheng Road, Guilin 541004, China.

ABSTRACT: The methods for the delivery of theranostic agents across the blood-brain barrier (BBB) are highly required. Superparamagnetic iron oxide nanoparticles (SPIONs) coated with PEG (poly (ethylene glycol)), PEI (poly (ethylene imine)) and Tween 80 (polysorbate 80) (TweenSPIONs) were prepared. We demonstrate the effective passage of tail-vein injected TweenSPIONs across normal BBB in rats under an external magnetic field (EMF). The quantitative analyses show significant accumulation of SPIONs in the cortex near the magnet, with progressively lower accumulation in brain tissues far from the magnet. A transmission electron microscopy picture of an ultra-thin section of the rat brain displays Tween-SPIONs crossing the BBB. The comparative study confirms that both the Tween-80 modification and EMF play crucial roles in the effective passage of SPIONs across the intact BBB. Only the magnetic force cannot drag the SPIONs coated with PEI/PEG polymers through the BBB. The results indicate the Tween-SPIONs cross the BBB via an active penetration facilitated by EMF. This work is encouraging for further study on the delivery of drug or diagnostic agents

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into the parenchyma of the brain for dealing with neurological disorders by using TweenSPIONs carriers under EMF. KEYWORDS: Iron oxide nanoparticles, blood-brain-barrier (BBB), Tween 80, magnetic field, Rat 1. INTRODUCTION The BBB (blood-brain-barrier) inhibits effective entering of most drugs into the brain parenchyma. The methods for the delivery of theranostics agents across the BBB are highly required.1-3 The applications of SPIONs (superparamagnetic iron oxide nanoparticles) as potential drug delivery vehicles have been explored actively.4-7 A large number of researches focused on the surface modification of SPIONs with ligands or antibodies to exploit receptormediated endo- or transcytosis.7,8 However, little or no detectable penetration of SPIONs across the BBB is evidenced in in vivo models.9,10 Most studies have been carried out by using tumor-bearing rodents, but the BBB of these rats has been changed and the status of the BBB integrity is unclear.11 The technique of osmotic disruption is utilized to open BBB transiently, but this approach can allow toxins to enter into the brain.12 Studies were performed on using an external magnetic field (EMF) to enhance the penetration of SPIONs across in vitro BBB models.13-14 Nevertheless, the use of EMF for SPIONs to enter the animal brain is certainly required. Kong et al carried out study to apply EMF to drag magnetic nanospheres to pass through the BBB, and the magnetic nanospheres accumulated in the brain parenchyma near blood vessels.15 Nd-Fe-B magnets were either implanted intracranially or placed on the outside skull of mice.15 Although this is the current state of the art, it has some limitations, they used polystyrene composite nanospheres containing many magnetite nanoparticles and fluorophores with a diameter of ~100 nm and a 0.63 T magnetic field. The dragging of the large sized nanospheres by a magnetic force limited the trafficking of the magnetic nanospheres into the deep brains and also this passive process could impair the brain cells. In this work, we explore a strategy based on Tween 80 modified monodispersed SPIONs and EMF for SPIONs to actively cross the intact BBB of rats. We use monodisperse SPIONs with an average size of 11.5 ± 2.2 nm and 0.3 T magnetic field. Monodisperse SPIONs can be


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synthesized via the thermal decomposition method.16-21 We prepared the SPIONs modified with PEG (poly (ethylene glycol)) and PEI (poly (ethylene imine)) using the thermal decomposition method. Intravenously administrated SPIONs modified with Tween 80 to actively cross the BBB of rats under EMF was observed and significant amount of SPIONs was found in the cortex. The comparative study was carried out to infer the mechanism of the passage of the Tween-SPIONs across the BBB. 2. EXPERIMENTAL SECTION 2.1. Preparation of PEI/PEG-SPIONs and Tween-SPIONs. The raw materials used and the synthesis are reported in our previous published work22. In brief, PEI/PEG-SPIONs were synthesized by the decomposition of 0.7 g Fe(acac)3 (Tokyo Chemical Industry) in 15.0 g PEG (Aladdin, China) mixed with 0.3 g PEI (Aladdin, China) at 260 °C for 1 h in argon atmosphere. The reactants cooled to 60 °C were washed three times successively with toluene and acetone, and PEI/PEG-SPIONs were collected by a magnet placed under the container. Tween-SPIONs was prepared by mixing 0.2 g Tween 80 with 0.4 g PEI/PEG-SPIONs dispersed in 40 mL deionized water at 50 °C for 60 min and dialyzing the mixture against deionized water for 120 h (MWCO 100,000 dialysis bag, Spectrumlabs). 2.2. Characterization. The morphology of the nanoparticles was observed by TEM (Transmission electron microscopy, JEM-2100F, Japan) at 200 kV. The zeta potential and hydrodynamic diameters of the nanoparticles were measured using a ZetaSizer Nano ZS90 (Malvern Instruments). The saturation magnetizations of PEI/PEG-SPIONs and Tween-80SPIONs were determined by the SQUID (Quantum Design MPMS XL-7 superconducting quantum interference device, QuantumDesign, America). 2.3. Cytotoxicity assay in vitro. MTT assay was used to evaluate cytotoxicities of nanoparticles against C6 rat glioma cells. The cells were seeded in 96-well plates at 1×104 cells per well in 200 µL of complete DMEM and incubated at 37 °C in 5 % CO2 atmosphere for 24 h. After removing the culture medium Tween-SPIONs in complete DMEM with different concentrations (0-100 µg/mL) were added. Cell viabilities after incubation with Tween-SPIONs for 24-72 h were measured by MTT assay.



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2.4. Animal experiment. Animal procedures were in agreement with the guidelines of the Institutional Animal Care and Use Committee. Adult female Sprague-Dawley rats (250-300 g) were anesthetized with an intraperitoneal injection of 10% chloral hydrate (1.2 mL/kg). SPIONs dispersed in phosphate buffer saline (PBS) (2 mg/mL) were tail-vein injected into the rats. For all experiments, 5 groups (n = 3 per group) of rats were divided: Group 1: control group, rats were tail-vein injected with 0.9% saline (10 mL/kg rat weight). Group 2: rats were tail-vein injected with PEI/PEG-SPIONs (10 mg Fe/kg rat weight). Group 3: rats were tail-vein injected with Tween-SPIONs (10 mg Fe/kg rat weight). Group 4: rats were tail-vein injected with PEI/PEG-SPIONs (10 mg Fe/kg rat weight), and subjected to a magnetic force. A Nb-Fe-B disk-shaped magnet (25.40 mm diameter×6.35 mm thick, 0.3 T) was placed on the rat skull (Figure 1). Group 5: rats were tail-vein injected with Tween-SPIONs (10 mg Fe/kg rat weight), and subjected to the magnetic force as described above. The magnet was placed above the rat head for 2 h after the injection, rats were again deeply anesthetized and transcardially perfused with 500 mL saline solution and then with a 500 mL buffered solution of 4% paraformaldehyde (PFA). Isolated brains were fixed in 4% PFA.

Figure 1. The photo shows the position of the magnet fixed on the rat head. 2.5. Determination of iron contents in rat brains. The iron contents of brain tissues were assessed by ICP-OES (Inductively Coupled Plasma-Optical Emission Spectroscopy, Optima 8000). For the ICP-OES measurements of iron contents, the brain tissues (olfactory bulb,


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temporal lobe, frontal cortex, thalamus, brain stem) were dried at 105 °C to constant weight, 1 mL of concentrated nitric and 0.2 mL of concentrated hydrochloric acid were added to the weighted sample (100 mg) in a tube, and heated at 80 °C for 2 h, then 0.1 mL Triton X-100 was added to the tube for 1 h. After digestion the solution was diluted to 10 mL for ICP-OES tests. 2.6. SPIONs distribution in rat brains observed by TEM. The specimens were observed on TEM (H-7650 and JEM-2100F). JEM-2100F installed with EDS (energy dispersive X-ray spectroscopy) was used to identify iron oxide nanoparticles in tissues. For biological TEM analysis, the removed tissues from rat brains were fixed in 2.5% glutaraldehyde, then were cut into ~1 mm3 pieces, and further fixed with 1% osmium tetroxide in 0.1 M cacodylate buffered solution for 2 h. The specimens were then dehydrated in 25% to 100% ethanol. The tissue specimens were embedded in Epon-Araldite, and then heat-treated at 37 °C for 12 h, then at 48 °C for 12 h, and finally at 60 °C for 48 h. The samples were cut into ultra-thin section with an ultramicrotome. The ultra-thin sections were put on copper grids (200 mesh) and dropped with uranyl acetate and lead citrate, then subjected to TEM observation. 3. RESULTS Figure 2 shows the TEM image and size distribution of Tween-SPIONs, the average size of Tween-SPIONs is 11.5 ± 2.2 nm. The magnetite nanoparticles are monodisperse with narrow size distribution. Physico-chemical features of the nanoparticles are listed in Table 1. The zeta potential and hydrodynamic diameter of Tween-SPIONs are 19.0 mV and 28.7 nm, respectively. The saturation magnetization of the Tween-SPIONs is 47.3 emu/g. PEI/PEGSPIONs or Tween-SPIONs dispersed well in water for 1 month without obvious change of their hydrodynamic diameters. The PEI/PEG polymers provide the SPIONs dispersibility in aqueous solution, and allow further conjugation of Tween 80 to the SPIONs surface (Scheme 1) 22.



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Figure 2. The TEM image and size distribution of Tween-SPIONs. Table 1. Physico-chemical properties of PEI/PEG-SPIONs and Tween-SPIONs Size (nm) Zeta potential (mV) Hydrodynamic diameter in PBS (nm) Saturation magnetization Msat (emu/g)

PEI/PEG-SPIONs 10.3 35.0 22.0 50.4

Tween-SPIONs 11.5 19.0 28.7 47.3

Scheme 1. The synthesis and surface coating of PEI/PEG-SPIONs and Tween-SPIONs.


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The cytotoxicity of Tween-SPIONs on C6 rat glioma cells is shown in Figure 3. The cells incubated with Tween-SPIONs show a relative high viability after 24-72 h. This results indicate Tween-SPIONs are low cytotoxic.

Figure 3. Cell viabilities of C6 rat glioma cells after incubation with 3.1-100 µg/mL TweenSPIONs for 24-72 h.

Figure 4. The distributions of iron in different brain areas of rats 2 h after tail vein injection. The inset indicates the magnet position.

Table 2. The iron contents (µg Fe/g tissue, mean ± s.e.m, n=3 animals per group) in the olfactory bulbs, temporal lobes, frontal cortexes, thalami and brain stems extracted from five groups of rats that were injected with saline, PEI/PEG-SPIONs and Tween-SPIONs in the presence or in the absence of EMF.



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Group Saline

Olfactory bulb 109.3±90.6

Temporal lobe 73.37±44.0

Frontal cortex 60.8±15.4

Thalamus 73.7±28.0

Brain stem 91.7±60.1

PEI/PEG-SPIONs No magnet

122.6±68.6

110.8±66.0

83.9±38

109.9±60.1

90.7±71.0

PEI/PEG-SPIONs + magnet

125.7±59.9

114.1±86.4

95.2±41.4

115.5± 52.7

114.8±50.9

Tween-SPIONs No magnet

261.4±145.6

244.0±128.8

218.9±143.3

117.3±15.2

125.7±31.0

Tween-SPIONs + magnet

280.4±183.5

270.2±107.8

597.6±121.6

144.7±53.1

130.7±41.9

Figure 4 shows the impacts of the surface modification and EMF on the distribution of iron in rat brains evaluated by the intravenous administration of PEI/PEG-SPIONs and Tween-SPIONs, in the presence and in the absence of EMF, as described in the animal experiment section. The iron accumulations in the brain tissues (frontal cortex, olfactory bulb, brain stem, temporal lobe and thalamus) at 2 h after injection were quantified by ICP-OES (Table 2). For the rats intravenously injected with Tween-SPIONs in the presence of EMF, the iron content in the frontal cortex, which is near the magnet, is 597.6 ± 121.6 µg (Fe/g tissue), which is more than the iron content in the frontal cortex of rats injected with TweenSPIONs in the absence of EMF (218.9±143.3 µg (Fe/g tissue)) (1.73-fold increase), and much more than the iron content in the frontal cortex of the control rats treated with saline (60.8 ± 15.4 µg (Fe/g tissue) (8.83-fold increase). The iron contents in the brains of rats treated with PEI/PEG-SPIONs were found to be nearly the same as those of the rats injected with saline, and no obvious change was observed in the presence of EMF. In contrast, the iron contents in the brain areas of rats injected with Tween-SPIONs even in the absence of EMF are increased compared with the control rats injected with saline (1.39-fold increase for olfactory bulbs, 2.33-fold increase for temporal lobes, 2.60-fold increase for frontal cortexes, 0.59-fold increase for thalami and 0.37-fold increase for brain stems). EMF applied greatly increases the iron contents in the rat brain areas as compared with those of the control rats (1.56-fold increase for olfactory bulbs, 2.68 fold increase for temporal lobes, 8.83-fold increase for frontal cortexes, 0.97 fold increase for thalami and 0.43 fold increase for brain stems), especially in the frontal cortex region which is near the magnet, but the iron contents in the rat brain areas are progressively decreased with the distance from the magnet applied (Figure 4).


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This result indicates that both Tween 80 and EMF play crucial roles in the effective passage of SPIONs across the BBB.

End

clusters Asp

d

c

Figure 5. Sub-cellular distribution of the Tween-SPIONs in the frontal cortex in the presence of EMF. (a) SPIONs enter the brain by crossing BBB. Asp: astrocyte processes, End: endothelial cell. (b) Higher magnification of the figure, the inset shows the size of SPIONs. (c) Nanoparticle clusters were found near the axons of neurons. (d) EDS analysis of electrondense black clusters shows the presence of Fe.

It is necessary to ascertain whether Tween-SPIONs actually cross the intact BBB and distribute in cerebral parenchyma, so TEM was used to observe the ultra-thin sections of rat brains. Figure 5a shows the distribution of the SPIONs in the cerebral parenchyma of the frontal cortex taken from the rat injected with Tween-SPIONs in the presence of EMF. Figure 5a shows the nanoparticle clusters in the foot processes of astrocytes. Figure 5c shows the clusters around the axons of neurons which were relatively far from cerebral vessels. By comparison, SPIONs were hardly found by TEM in the brain tissues of the rats injected with Tween-SPIONs in the absence of EMF or injected with the PEI/PEG-SPIONs both in the



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presence and in the absence of EMF. The TEM observations along with the results of ICPOES analysis indicate the effective penetration of Tween-SPIONs into the brain under EMF. Rats were transcardially perfused first with 500 mL of 0.9% saline then with 500 mL buffered solution of 4.0% PFA, most SPIONs in the blood were removed by the saline and paraformaldehyde perfusion, but yet a serendipitous TEM picture of a vessel in the frontal cortex has been found that shows Tween-SPIONs being internalized, this group of SPIONs was not removed by the perfusion due to its attaching to the vessel wall. It should be noted that very few SPIONs were found by TEM in the vessels as in this case because most SPIONs in blood were removed by the perfusion. It can be seen that these SPIONs, although encapsulated in a substance from the blood, are well dispersed. It is known that nanoparticle surfaces are covered with proteins when nanoparticles enter into blood23. These proteins around nanoparticles are called the protein corona. The property of the nanoparticles may affect the composition of the protein corona, which determines the interaction of the nanoparticles with the living systems23. Figure 5a shows that a group of SPIONs encapsulated are isolated from blood, and the SPIONs in the encapsulated group are penetrating the BBB. The inset in Figure 5b shows the size of SPIONs, and their diameters measured are 11.2, 13.5 and 15.2 nm, respectively, in consistence with the average diameter of Tween-SPIONs (11.5 ± 2.2 nm) (Figure 2). EDS analysis was employed out in JEM 2100F to identify the magnetic nanoparticles. A representative X-ray spectrum collected while the electron beam was directed on the electrondense black clusters (Figure 5d) shows the Fe Kα peak, confirming that these electron-dense black clusters are indeed magnetic nanoparticles instead of dyes or other impurities. The peaks of copper Kα, osmium Kα, uranium Kα and lead Kα are from copper grids and chemicals for fixing and staining the tissues. 4. DISCUSSION The results demonstrate tail-vein injected Tween-SPIONs effectively crossing the intact BBB with the aid of EMF. The high iron content in the frontal cortex (Figure 4, Table 2) corresponds to nanoparticle clusters of Tween-SPIONs in brain parenchyma (Figure 5a.


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Figure 5c). The TEM picture shows that most of the Tween-SPIONs are still individual nanoparticles in the vessel (Figure 5b). This implies that the clusters are formed after the nanoparticles enter the brain. The BBB is composed of cerebral vascular endothelial cells resting on a basal lamina that is further supported by the foot processes of local astrocytes3. The presence of Tween-SPIONs in rat brains evidences that these nanoparticles can pass the BBB structure and enter into the cerebral parenchyma. The comparative study confirms that both the Tween 80 and EMF play crucial roles in the effective passage of the SPIONs across the BBB, the Tween-SPIONs in the absence of EMF and the PEI/PEG-SPIONs in the presence of EMF, both are difficult to cross the BBB, even though PEI/PEG-SPIONs have higher positive zeta potential (Table 1) and are supposed to be more attractive to negatively charged endothelial cell surfaces13 than Tween-SPIONs, this suggests a receptor-mediated endocytosis is the most possible pathway. It can be proposed for this active penetration of the BBB that the Tween-SPIONs enter into the capillary endothelial cells via the receptor mediated endocytosis with the help of EMF, the magnetic attractive force on SPIONs increases the contact between SPIONs and endothelial cells, and promotes the transportation of SPIONs during the endocytosis process. It should be noticed that only the magnetic force cannot drag the SPIONs through the BBB as can be seen in the case for PEI/PEG-SPIONs, the data in Table 2 confirm that the PEI/PEG-SPIONs cannot be dragged by the magnet through the BBB via a passive penetration. It is shown in Figure 5a and Figure 5b the endothelial membrane is intact. It can be observed that the SPIONs are covered by biomolecules, possibly proteins from the plasma (Figure 5a, Figure 5b). Such a particleprotein complex could stay in the vessel as a unified structure, which is sufficiently stable. This is in agreement with the finding that particle-protein complexes are capable of isolating themselves from the medium.23 Encapsulated in this structure Tween-SPIONs might utilize the formation of protein coronas to cross the BBB. It has been proposed that low density lipoprotein receptor (LDLR) mediates the transport of nanoparticles to cross the BBB.24 LRP 1 and LRP 2 (LDLR-related proteins) expressed on the BBB are receptors for low density lipoprotein (LDL).24 Tween 80 enables the coated nanoparticles to adsorb apolipoprotein E or



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B in the blood.25 Nanoparticles coated with apolipoproteins are endocytosed by the brain blood capillary endothelial cells through a LDLR-meditated endocytosis and then transport through the BBB into brain.26, 27 The results in this work show that EMF also plays a crucial role in the passage of Tween-SPIONs across the BBB. The process as illustrated in Scheme 2 can be suggested that the positively charged Tween-SPIONs adsorb proteins, especially the apolipoproteins from the blood, with the crucial assistance of EMF to increase the contact with the endothelial cells and promote the transportation of SPIONs during the endocytosis process, actively cross the BBB via the receptor-mediated endocytosis. The magnetic force also promotes the transportation of SPIONs into the deep brain after the SPIONs have crossed the BBB, Nevertheless, further work should be carried out to clarify the exact process. The effective passage of SPIONs across intact BBB demonstrated in this work is particularly encouraging for further study on the drug delivery into the brain for dealing with neurological disorders and the study of magnetothermal deep brain stimulation in the intact brain for revealing structural and functional brain circuits.28, 29

Scheme 2. Schematic illustration for Tween-SPIONs crossing the BBB in the presence of a magnet. 5. CONCLUSION We demonstrate the effective passage of tail-vein injected Tween-SPIONs across the intact BBB of rats under the magnetic field. Significant accumulations of SPIONs were found in the brain cortex of rats. The comparative study confirms that both the Tween 80 and the


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magnetic field play crucial roles in the passage of SPIONs across the BBB. The potential applications of this method are drug delivery into the brain for dealing with neurological disorders. AUTHOR INFORMATION *Corresponding Authors Tel: +86 773 5896771; Fax: +86 773 5896436. E-mail: [email protected] (B. L. Zhang); [email protected] (B. N. Yang); [email protected] (J. Tan) Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 51162003, 51562007). REFERENCES (1) Li, J.; Cai, P.; Shalviri, A.; Henderson, J. T.; He, C.; Foltz, W. D.; Prasad, P.; Brodersen, P. M.; Chen, Y.; DaCosta, R.; Rauth, A. M. A Multifunctional Polymeric Nanotheranostic System Delivers Doxorubicin and Imaging Agents Across the Blood–Brain Barrier Targeting Brain Metastases of Breast Cancer. ACS Nano 2014, 8, 9925-9940. (2) Tabatabaei, S. N.; Girouard, H.; Carret, A. S.; Martel, S. Remote Control of the Permeability of the Blood–Brain Barrier by Magnetic Heating of Nanoparticles: A Proof of Concept for Brain Drug Delivery. J. Controlled Release 2015, 206, 49-57. (3) Lajoie, J. M.; Shusta, E. V. Targeting Receptor-Mediated Transport for Delivery of Biologics Across the Blood-Brain Barrier. Annu. Rev. Pharmacol. 2015, 55, 613-631. (4) Farr, T. D.; Lai, C. H.; Grünstein, D.; Orts-Gil, G.; Wang, C. C.; Boehm-Sturm, P.; Seeberger, P. H.; Harms, C. Imaging Early Endothelial Inflammation Following Stroke by Core Shell Silica Superparamagnetic Glyconanoparticles that Target Selectin. Nano

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