Dual-Targeting Magnetic PLGA Nanoparticles for Codelivery of

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Dual-Targeting Magnetic PLGA Nanoparticles for Codelivery of Paclitaxel and Curcumin for Brain Tumor Therapy Yanna Cui,†,‡,∥ Meng Zhang,†,∥ Feng Zeng,†,§,⊥ Hongyue Jin,† Qin Xu,⊥ and Yongzhuo Huang*,† †

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China Key Laboratory of Primate Neurobiology, Institute of Neuroscience, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China § Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China ⊥ Institute of Tropical Medicine, Guangzhou University of Chinese Medicine, 12 Jichang Road, Guangzhou 501405, China

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‡

S Supporting Information *

ABSTRACT: Chemotherapy is one of the most important strategies for glioma treatment. However, the “impermeability” of the blood−brain barrier (BBB) impedes most chemotherapeutics from entering the brain, thereby rendering very few drugs suitable for glioma therapy, letting alone application of a combination of chemotherapeutics. Thereby, there is a pressing need to overcome the obstacles. A dual-targeting strategy was developed by a combination of magnetic guidance and transferrin receptor-binding peptide T7-mediated active targeting delivery. The T7-modified magnetic PLGA nanoparticle (NP) system was prepared with co-encapsulation of the hydrophobic magnetic nanoparticles and a combination of drugs (i.e., paclitaxel and curcumin) based on a “one-pot” process. The combined drugs yielded synergistic effects on inhibition of tumor growth via the mechanisms of apoptosis induction and cell cycle arrest, displaying significantly increased efficacy relative to the single use of each drug. Dual-targeting effects yielded a >10-fold increase in cellular uptake studies and a >5-fold enhancement in brain delivery compared to the nontargeting NPs. For the in vivo studies with an orthotopic glioma model, efficient brain accumulation was observed by using fluorescence imaging, synchrotron radiation X-ray imaging, and MRI. Furthermore, the antiglioma treatment efficacy of the delivery system was evaluated. With application of a magnetic field, this system exhibited enhanced treatment efficiency and reduced adverse effects. All mice bearing orthotopic glioma survived, compared to a 62.5% survival rate for the combination group receiving free drugs. This dual-targeting, co-delivery strategy provides a potential method for improving brain drug delivery and antiglioma treatment efficacy. KEYWORDS: brain targeting delivery, magnetic targeting, PLGA nanoparticles, paclitaxel, curcumin, T7 peptide

1. INTRODUCTION Malignant glioma is one of the most common and aggressive brain tumors in humans. Glioma patients have a median survival time of approximately 1 year, and only 5% survive more than 5 years.1 Due to the formidable blood−brain barrier (BBB), options for glioma chemotherapy (chemo) are very limited, and there are merely a few BBB-permeable chemo drugs (e.g., temozolomide, carmustine) clinically available.2 The strictly limited selection of drugs imposes a great challenge and, therefore, the development of BBB-penetrating drug delivery systems has been a major effort in antiglioma therapy. Indeed, achieving brain tumor targeting drug delivery with reduced unwanted drug exposure to healthy organs has become the holy grail earnestly pursued in the medical community. The brain is the most energy-consuming organ, with active material exchanges through the BBB. Nutrient transporters on the BBB are crucial in maintaining the normal functions of the © 2016 American Chemical Society

brain, by fetching essential, life-sustaining components such as amino acids/peptides, sugars, and proteins.3 Owing to the high expression of such nutrient transporters on the BBB and their potent transcellular transport ability, targeting the nutrient transporters has been an important design strategy for brain delivery.4 Indeed, these transporters can potentially serve as portals for brain uptake of drugs and have been widely explored, including the large-neutral amino acid transporter-1 (LAT-1), the glucose transporter-1 (GLUT-1), low-density lipoprotein (LDL), and transferrin receptors.5 For example, the substrates of the transporters (e.g., transferrin) have been used for surface modification of nanoparticles (NPs) serving as a Received: August 13, 2016 Accepted: November 3, 2016 Published: November 3, 2016 32159

DOI: 10.1021/acsami.6b10175 ACS Appl. Mater. Interfaces 2016, 8, 32159−32169

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ACS Applied Materials & Interfaces “Trojan horse” strategy, by which the camouflaged cargos can sneak into the tumors.6 Apart from the BBB penetration issue, the effective targeting of the BBB site is another primary challenge. Fluid dynamics in blood vessels is a major factor that influences the biofate of nanoparticles; they exhibit tumbling and rolling dynamics in the bloodstream, with different particle−cell interaction modes from that observed in cellular uptake studies in a static cellular culture model.7 Therefore, the interaction of nanoparticles with endothelial walls is a very important design consideration because the favorable particle−cell and receptor−ligand interactions facilitate extravasation through the fenestrated tumor vessels.7 Often, the sheer force drags the nanoparticles off their adhesion on the neovasculature endothelium and carries them back to the bloodstream, rendering insufficient targeting efficiency.8 Adult cerebral blood flow is about 750 mL per minute, accounting for 15% of the cardiac output.9 The swift cerebral blood flow could create a barrier against particle− cell binding interaction. To increase the binding affinity to the targeting cells and thus achieve improved active targeting delivery to the brain, scientists have made great efforts in developing various targeting ligands. However, the receptor/ligand binding mode-based “active targeting delivery” is arguably not active, and actually the nanoparticles are passively “trapped” by the receptors, instead of guiding themselves to a target.10 In this regard, improving the active functions could be a boost for delivery efficiency. To address the complicated situation, multiple targeting strategies have been explored. For example, magnetic-guided delivery is a powerful physical method with good controllability for active targeting.11 Guided by the applied magnetic field, the magnetic nanoparticles migrate toward the magnet,12 thus achieving the active guided delivery. In this presented work, we combined ligand-mediated and magnetic-guided targeting and developed a dual-functional tumor-specific delivery system. Transferrin receptors overexpressed on BBB and glioma are mainly responsible for transport of the Fe−transferrin complex.13 For the ligand-mediated targeting, the human transferrin receptor-binding peptide T7 (sequence HAIYPRH) was selected. Of importance, no competition was found between T7 and transferrin for receptor binding, indicating that their binding sites are distinct from one another.14 Therefore, the unique advantage of T7 is that it would not be affected by the endogenous transferrin. We previously reported the application of T7-mediated glioma targeting delivery of nanoprobes for tumor imaging, suggesting its potential in brain delivery. On this basis, we developed a T7modified, magnetic PLGA nanoparticulate system (MNP/T7PLGA NPs) by a single-emulsion solvent evaporation method, by which the hydrophobic magnetic nanoparticles (MNPs) were entrapped into the PLGA NPs. Compared to the conventional magnetic nanoparticles, a great benefit of such a magnetic PLGA system is its capability to co-encapsulate various drugs, providing the potential of combination therapy against glioma. This system was used for co-delivery of paclitaxel (PTX) and curcumin (CUR) via a T7-mediated, magnetic-guided dual-targeting mechanism (Scheme 1). The treatment efficiency was investigated by both in vitro and in vivo experiments.

Scheme 1. Schematic Illustration of BBB-Penetrating and Tumor-Targeting Delivery via the T7-Mediated and Magnetic-Guided, Dual-Targeting MNP/T7-PLGA NPs

2. RESULTS 2.1. Synthesis and Physicochemical Properties of MNP/T7-PLGA NPs. The NH2-PEG3500-T7 was synthesized via thiol-maleimide click reaction according to Scheme 2a, and the drug-loaded magnetic PLGA-PEG-T7 nanoparticles, termed MNP/T7-PLGA NPs, were prepared by the singleemulsion solvent evaporation method (Scheme 2b). The hydrophobic oleic acid-modified iron oxide nanoparticles (OA-MNPs) were co-encapsulated into the PLGA NPs along with the drugs. The structure of PLGA-PEG-T7 copolymer was confirmed by 1HNMR analysis. The characteristic signals are designated in Figure 1A. The characteristic peak at 1.54 ppm is attributed to the methyl hydrogen of D,L-lactide. The strong peak at 3.62 ppm corresponds to the methylene hydrogen of PEG. The peak at 4.81 ppm is attributed to the methylene hydrogen of glucolide. The peak at 5.20 ppm belongs to the methyl hydrogen of D,L-lactide. The characteristic signal at δ 6.55−6.75 of the maleimide peak disappeared, indicating that the MAL group of PLGA-PEG-MAL reacted with the thiol group (−SH) of T7. The morphology of MNP/T7-PLGA NPs was observed via transmission electron microscopy (TEM) (Figure 1B), showing a size around 100 nm. Blank dots dispersed in the PLGA NPs were the MNPs. The high content of MNP was associated with the large size and aggregation of MNP into the PLGA NPs, which resulted in the asymmetric distribution. If a low amount of MNP was encapsulated, the shape became regular, and the MNP exhibited an even distribution inside the PLGA NPs (Figure S1, Supporting Information). Despite the larger size of the PLGA NPs with higher content of MNP, it could benefit enhanced responsivity and targeting efficiency. The particle size and ζ-potential were measured by dynamic light scattering (DLS). The results showed that the sizes of the T7-PLGA NPs without MNPs, the MNP/T7-PLGA NPs with low-content iron, and the MNP/T7-PLGA NPs with highcontent iron, were 99, 113, and 130 nm, respectively (Figure 1C); the increased content of MNPs was related to the growing particle size. The ζ-potential of the MNP/T7-PLGA NPs with loaded drugs (PTX + CUR) was −15.9 mV. 32160

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ACS Applied Materials & Interfaces Scheme 2. Synthesis of PLGA-PEG-T7 Polymer (a) and Drug-Loaded MNP/T7-PLGA NPs (b)

Figure 1. Characterization of the MNP/T7-PLGA NPs: (A) 1H NMR spectrum of PLGA-PEG-T7 copolymer in chloroform-D; (B) transmission electron microscopy of MNP/T7-PLGA NPs; (C) size distribution; (D) stability study of MNP/T7-PLGA NPs with and without drugs at 10% FBS.

MNPs have been widely applied as T2 contrast agents in MRI. The relaxation ratios of the MNP/T7-PLGA NPs with various iron concentrations were measured by using magnetic resonance analyzer to investigate the signal enhancement for T2 weighted imaging. A good linear correlation between T2 relaxation rate (R2) and iron concentration was found. The R2 relaxivity coefficient value (r2) of the MNP/T7-PLGA NPs was 281.05 mM−1 s−1 (Figure 2B), which was higher than those of other reported magnetic nanocarriers.16,17 The R2 values of the MNP/T7-PLGA NPs were also higher than those of commercially available dextran-coated MNPs.18 The MNP/ T7-PLGA NPs with lower iron concentrations showed the brighter T2-weighted MR images that were measured by using an MRI equipment (3.0 T), displaying a negative relationship between the iron concentration and the MRI signal intensity (Figure 2C). This suggested that the MNP/T7-PLGA NPs effectively enhanced the transverse proton relaxation process and could be utilized as MRI negative contrast agents.

The stability of the NPs in the PBS supplemented with 10% FBS was investigated by monitoring changes in particle size. The results showed that the MNP/T7-PLGA NPs remained stable for 70 h at 37 °C (Figure 1D). It was interesting that incorporation of drugs led to a slight decrease in particle size. The hydrophobic interaction between drugs and PLGA may change the assembling process of the nanoparticles and their size; the small drugs could fill the gaps among the iron NPs and between the iron NPs and PLGA. The magnetic property of the MNP/T7-PLGA NPs was evaluated by using a vibrating sample magnetometer. Both the OA-MNPs and MNP/T7-PLGA NPs showed a superparamagnetic property at 300 K (Figure 2A). The saturation magnetizations (Ms) of the OA-MNPs and MNP/T7 PLGA NPs were 82 and 13 emu/g Fe, respectively, displaying higher Ms than the reported result (2 emu/g Fe).15 This indicated the potential of the MNP/T7-PLGA NPs for magnetic targeting application. 32161

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Figure 2. Magnetic properties of the MNP/T7-PLGA NPs: (A) magnetic hysteresis loop of OA-MNPs and the MNP/T7-PLGA NPs tested by vibration sample magnetometer, with saturation magnetization of 82 and 13 emu/g Fe; (B) transverse relaxation rate (1/T2 = R2, s−1) of the MNP/ T7-PLGA NPs with different Fe concentrations; (C) T2-weighted MR images of the MNP/T7-PLGA NPs. Dark contrast increased along with elevating Fe concentrations.

2.2. Drug Release Profiles of MNP/T7-PLGA NPs. The water-insoluble drugs PTX and CUR were loaded into the hydrophobic cores of T7-PLGA NPs via the hydrophobic interactions. The encapsulation efficiencies of PTX and CUR were 68 and 18%, respectively. Figure 3 shows the sustained

cellular uptake efficiency of the MNP/T7-PLGA NPs was >10 times higher than that of the NPs without T7 modification (mean fluorescence intensity, 2845 vs 262). If the cells were pretreated with free T7, the cellular uptake of MNP/T7-PLGA NPs was greatly inhibited (Figure 4C). A cell culture model of BBB was prepared in which the bEnd.3 cell monolayer was cultured in an upper chamber of the Transwell supports and the U87 cells in a lower chamber. The integrity of BBB monolayers was monitored by measuring the transepithelial electrical resistance (TEER). The transport efficiency across the BBB model was evaluated. At 4 h, the transport ratio showed 0.91% for free coumarin-6 (CM), 1.66% for MNP-PLGA NPs, 1.73% for MNP-PLGA NPs + MAG, 2.2% for MNP/T7-PLGA NPs, and 6.42% for MNP/T7-PLGA NPs + MAG (Figure 5A). This demonstrated that the transport efficiency through BBB was profoundly enhanced owing to the dual-targeting mechanisms (i.e., transferrin receptor-mediated targeting and magnetic guidance). Furthermore, to study the in vivo brain targeting efficiency, mice bearing orthotopic glioma (U87-Luc) were treated with the Cy5-labeled NPs via tail vein injection and then subjected to in vivo imaging. The group of MNP/T7-PLGA NPs with applied magnetic field (+ MAG) showed the highest brain accumulation among all groups (Figure 5B). The dissected brains displayed a clear tendency of the NP accumulation as follows: MNP/T7-PLGA NPs + MAG > MNP/T7-PLGA NPs > MNP/PLGA NPs + MAG > MNP/PLGA NPs. These results were consistent with the in vitro results in Figure 5A. Figure 5C shows the intratumoral infiltration of the MNP/T7-PLGA NPs in the glioma slices from the mice treated with the Cy5-labeled NPs under applied magnetic field. This demonstrated that the MNP/T7-PLGA NPs under the dual-targeting mediation successfully overcame the in vivo delivery barriers, especially the BBB, and accumulated in the glioma. An intense flux of electromagnetic radiation (i.e., synchrotron radiation) is produced by a circular accelerator, in which a circulating electron beam is deflected by the bending magnets in a storage ring. Synchrontron radiation has become one of the most promising modalities for imaging.19 For instance, a synchrotron radiation X-ray fluorescence technique can provide highly sensitive three-dimensional images with superior submicrometer spatial resolution for tracking metal elements in biological tissues. The synchrotron radiation X-ray technique

Figure 3. Release profiles of PTX and CUR from the MNP/T7-PLGA NPs at pH 7.2 (PBS) containing 0.2% Tween 80.

release patterns, in which the release rates of PTX and CUR from the MNP/T7-PLGA NPs were 76 and 60%, respectively, during the initial 36 h. The release rates of PTX and CUR were different. The release rate was influenced by the processes of swelling and erosion of the PLGA NPs. Generally, the drugs embedded close to the surface of the NPs would be released more quickly than those deep inside the NPs. Moreover, the dissolution rate of the drugs is also a key factor. 2.3. In Vitro and in Vivo Transport across the BBB by Dual-Targeting Mechanisms. The intracellular delivery was investigated in human malignant glioma U87 cells and mouse brain endothelial cell line bEnd.3. Both U87 and bEnd.3 cells showed substantial uptake of the MNP/T7-PLGA NPs. Of interest, it appeared that the magnetic field-driven cellular uptake was not a major factor for the MNP/T7-PLGA NPs. As shown in Figure 4A and 4B, for the MNP/PLGA NPs, the application of a magnetic field yielded limited enhancement effect on the uptake efficiency in both cell lines (i.e., 1.6- and 1.2-fold). This revealed that T7-mediated intracellular delivery was the primary mechanism for intracellular delivery of the NPs in the cultured cell models. Further evidence is that the U87 32162

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Figure 4. Cellular uptake experiments of different formulations on U87 and bEnd.3 cells: (A) flow cytometry results; (B) representative fluorescent images of cellular uptake (nuclei were stained blue with DAPI); (C) T7 modification significantly enhanced the cellular uptake of the NPs. Nanoparticles were labeled with coumarin-6 (green fluorescence).

Figure 5. Transporting efficiency across BBB in vitro and in vivo: (A) transport ratios across the in vitro BBB of different NPs at 4 h; (B) in vivo distribution of the nanoparticle systems by IVIS animal imaging system after injection via tail vein at 4 h; (C) image of brain tissue section by confocal laser scanning microscope; (D) iron distribution in brain examined by synchrotron radiation X-ray; (E) MR imaging of MNP/T7-PLGA NPs. The results demonstrated brain targeting mediated by dual targeting.

was applied in our studies to analyze MNP distribution in the brain. The iron element (the red signal) was observed by the synchrotron radiation facility (Figure 5D), displaying substantial brain accumulation of the MNP/T7-PLGA NPs. Detailed information about iron distribution inside the brain

can be clearly viewed in the 3-D stimulation modeling video (Supporting Information, clip 1). The brain-targeting effect was further confirmed by MRI and synchrotron radiation X-ray techniques. MNPs have been widely used as MRI contrast agents for tumor detection and 32163

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Figure 6. Viability of U87 cells after treatment with the drug-loaded NPs and free drugs: (A) U87 cells treated with dual-drug-loaded nanoparticles, MNP/T7-PLGA (PTX + CUR), MNP/T7-PLGA (PTX + CUR) + MAG, and MNP-PLGA (PTX + CUR), respectively; (B) U87 cells treated with free PTX + free CUR, MNP/T7-PLGA (PTX), and MNP/T7-PLGA (CUR), respectively.

characterized by high MMP show red fluorescence owing to the formation of JC-1 aggregates, whereas the apoptotic cells with low MMP show green fluorescence (JC-1 monomers). As shown in Figure 7, the MMP in the U87 cells was significantly changed after treatment of the MNP/T7-PLGA NPs, as evidenced by the increased green fluorescence, indicating induction of apoptosis.

imaging. The MRI signal was monitored at 4 h after tail vein injection of the MNP/T7-PLGA NPs (Figure 5E), displaying a clear MRI signal in the glioma site. These results corroborated the potential of the MNP/T7-mediated dual-targeting strategy for efficient transport across the BBB and accumulation in the brain tumor. The MNP/T7-PLGA NPs could serve as an effective T2-weighted MRI contrast agent for application in tumor diagnosis and brain-targeting evaluation. It should be mentioned that the targeting effect of T7mediated brain delivery was already supported by the biodistribution study (Figure 5), in which the group MNP/ PLGA + MAG without targeting ligand showed significantly lower drug accumulation in the brain, compared to T7-MNP/ PLGA + MAG. Therefore, the group MNP/PLGA + MAG without targeting ligand T7 was not included in the following treatment studies. 2.4. Tumor Cell Proliferation and Cellular Apoptosis Study. The synergistic effect of PTX and CUR was examined. Their optimized dose ratio was measured to be 1:1 (Figure S2, Supporting Information). This showed that the co-delivery of drugs (PTX + CUR) by the MNP/T7-PLGA NPs significantly increased the cytotoxicity, with an IC50 of 3.06 μg/mL, compared to 6.23 and 9.43 μg/mL for MNP/T7-PLGA (PTX + CUR) with magnetic field and MNP/PLGA (PTX + CUR), respectively (Figure 6 and Table 1). Of note, the in vitro Table 1. IC50 (Half-Maximal Inhibitory Concentration) Values in U87 Cells

IC50 (μg/mL)

MNP/ T7PLGA (CUR + PTX)

MNP/T7PLGA (CUR + PTX) + MAG

MNP/ PLGA (CUR + PTX)

free CUR + PTX

MNP/ T7PLGA (PTX)

MNP/ T7PLGA (CUR)

3.04

6.23

9.43

5.80

6.48

8.35

antitumor activity did not benefit from the application of the magnetic field, which may account for the ineffectiveness of the magnetic field on driving intracellular uptake in the static culture system. For further investigation of the cytotoxic effect, mitochondrial membrane potential (MMP) was measured by flow cytometry. The mitochondrion is the cell powerhouse. Chemo drugs can impair mitochondrial functions and induce apoptosis. MMP is an important indicator of mitochondrial function and can serve as an early marker of the onset of apoptosis.20 The JC-1 staining method was used for indicating the mitochondrial activity in this experiment. In normal conditions, the cells

Figure 7. Mitochondrial membrane potential (MMP) analysis of U87 cells after treatment of different drug-loaded NPs for 48 h by flow cytometry.

The cell cycle progression was measured using flow cytometry. The G2/M checkpoint is a major target for DOXbased chemotherapy.21,22 G2/M arrest was prominent in the cells exposed to free DOX or the DOX-loaded NPs. The percentage of G2/M phase in the U87 cells treated with the combo drug-loaded MNP/T7 PLGA NPs was highest, displaying a population of 63.6%, whereas that of the PTX32164

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Figure 8. Cell cycle analysis on U87 cells after treatment with (A) MNP/T7-PLGA (CUR), (B) MNP/T7-PLGA (PTX + CUR), (C) MNP/T7PLGA (PTX), (D) free CUR, (E) free PTX + free CUR, (F) free PTX, and (G) blank (as control) for 48 h, respectively. (H) Quantative results of cell cycle phases analyzed by flow cytometry. The percentage of G2/M phase was increased by combination treatment of the combo drugs.

by the dual-targeting group (MNP/T7-PLGA NPs + MAG), whereas other groups displayed relatively fast growth rates. To further assess the antiglioma efficiency, the overall survival time and body weight change of the glioma-bearing mice were measured (Figure 11). The control group (treated with PBS) showed a rapid loss in body weight and early death; no mice survived longer than 13 days. Of significance, no animal died in the group of MNP/T7-PLGA NPs + MAG during the experimental course (35 days), whereas the survival rates were 83% for the MNP/T7-PLGA NPs (PTX + CUR) without a magnetic field and 67% for the free combo drugs. Importantly, the body weight showed a very slight decrease after combination treatment based on dual-targeting delivery (the group of MNP/T7-PLGA NPs + MAG), whereas there was a >15% body weight loss found in the group treated with free combo drugs. This suggested a reduced adverse effect of the combination therapy using MNP/T7-PLGA NPs + MAG.

loaded MNP/T7 PLGA NPs was 39.7% (Figure 8). This indicated that combination therapy could improve the chemotherapeutic efficacy. Moreover, crystal violet was used for monitoring the druginduced change of U87 cloning morphology. A colony-forming ability assay is commonly used for determining the effects of chemo drugs on cancer cell growth.23 The MNP/T7-PLGA NPs with or without applied magnetic field showed 22 or 29% a colony-forming rate (Figure 9), respectively, significantly lower than those of other groups. This demonstrated that the combination therapy via the MNP/T7-PLGA NPs under the dual-targeting mediation was highly effective in inhibiting the proliferation of the U87 glioma cells. 2.5. In Vivo Antiglioma Efficacy. The in vivo antiglioma efficacy was investigated using the transplanted orthotopic U87Luc glioma model. Bioluminescence intensity indicated the size of the glioma. The groups receiving chemotherapy showed substantial treatment efficacy (Figure 10), with inhibition efficiency in the following order: MNP/T7-PLGA NPs + MAG > MNP/T7-PLGA NPs > MNP/T7-PLGA NPs (PTX) > MNP/T7-PLGA NPs (CUR) > free CUR + PTX. This demonstrated the great potential of the combination therapy under the dual-targeting mechanism. Comparison of the results from days 12 and 21 clearly showed that the glioma growth rate was significantly suppressed

3. DISCUSSION Due to the presence of the BBB, the brain accumulation of drugs is minimal. Much worse, the highly in vivo dynamics often diminishes the effective retainment of nanoparticles in the central nervous system. Single-targeting systems often show inadequate efficiency and specificity to brain tumor.24 Multifunctional nanoparticles with dual-targeting mechanisms have 32165

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target the same receptor, or target the two cell types in brain tumor.25 Recently, the combination of magnetic guidance and ligand-mediated targeting has also been emerging.26−28 Typically, magnetic delivery systems for in vivo applications are constructed using magnetic ferrofluids with coating of biocompatible materials, such as polymers (e.g., dextran, PEG, and polyacrylamide).29 However, it should be noted excessive release of free iron from MNPs facilitates the generation of free radicals (e.g., ROS) and causes oxidative stress and toxicities.30,31 A benefit of our system is that the encapsulation mode of MNPs@PLGA NP could effectively retard iron release and acute ROS generation. Importantly, the modification of a peptide ligand on the PLGA NPs is much more convenient due to its compatibility with organic solvents. T7 modification and encapsulation of MNPs and drugs can be done with a “one-pot” process by mixture of T7-PEG-PLGA with other polymer constituents, MNPs and drugs, offering great advantages in terms of its simple procedures. By contrast, modification of antibodies or other proteins that are organic solvent-phobic needs extra steps. For example, the first step is to prepare the drug-loaded PLGA NPs, and the second one is to activate the functional groups on the NPs and then conduct surface modification. An interesting phenomenon we observed was the inconsistency between the in vitro and in vivo investigations, in which the dual-targeting delivery was significant in animal studies but yielded only a marginally synergistic effect in cellular studies. The insufficiency of magnet-assisted transcellular penetration and cellular uptake in a static culture system could be accounted for with magnetically induced nanoparticle aggregates.32 The formation of large aggregates prevents intracellular delivery. However, highly dilute dispersions and serum environment could inhibit magnetically induced aggregation.33 Our results demonstrated the great synergistic effect of ligand-mediated and magnetic-guiding delivery, suggesting the in vivo targeting potential. The enhanced efficacy in vivo was probably associated with the prevention of aggregate formation under conditions of high serum concentration and dynamic bloodstream. The synergistic mechanisms of PTX and CUR have been reported. For example, CUR can improve the paclitaxelinduced apoptosis of cancer cells via the NF-κB-p53-caspase-3 pathway.34 CUR can improve PTX resistance mediated by P-gp transporter.35 The synergistic effect of CUR and PTX on human brain tumor stem cells was related to induction of apoptosis and suppression of cell migration, production of angiogenic factors, and network formation.36 Our results demonstrated that the synergistic effect was also associated with induction of apoptosis and mitochondrion damage. In terms of the complexity of brain delivery, multifunctional nanoparticle platforms could be an ideal solution to achieve optimal efficiency by incorporating various targeting mechanisms. Yet, still little is known about how to strategically combine the ligands and make them work synergistically, which largely relies on empirical evidence or experimental observation; nor is much known about the combination of physical and biochemical targeting mechanisms. Therefore, further detailed investigation would be helpful to provide insight on the rational design of brain-targeting systems. Moreover, it is worthwhile to explore dual ligands combined with magnetic guidance for further enhancing the efficiency of brain delivery.

Figure 9. Inhibition efficiency of colony formation of U87 glioma cells after treatment with (a) blank (as control), (b) MNP/T7-PLGA (CUR + PTX), (c) MNP/T7-PLGA (CUR + PTX) + MAG, (d) MNP/T7-PLGA (CUR), (e) MNP/T7-PLGA (PTX), (f) free CUR + PTX, or (g) MNP/PLGA (CUR + PTX) for 48 h, respectively.

Figure 10. Glioma growth inhibition in the Balb/c nude mice: (A) schedule of treatment and imaging; (B) increased fold of bioluminescence intensity at 12 and 21 days after treatment, respectively; (C) in vivo images of brain glioma before treatment (left column) and after treatment for 21 days (right column) (a1 and a2, MNP/T7PLGA NPs (CUR); b1 and b2, MNP/T7-PLGA NPs (CUR + PTX); c1 and c2, MNP/T7-PLGA NPs (CUR + PTX) + MAG; d1 and d2, MNP/T7-PLGA NPs (PTX); e1 and e2, free CUR + PTX).

become an attractive method to boost brain delivery efficiency. The dual-targeting mechanisms can be achieved by incorporation of different ligands to target the BBB and brain tumor, 32166

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Figure 11. (A) Change of animal body weight; (B) overall survival of glioma-bearing mice (n = 8). the active NHS ester. Finally, PLGA-PEG-T7 was obtained via the reaction of PLGA-NHS and NH2-PEG-T7 (molar ratio 1.5:1) in DMF for 24 h under nitrogen protection. The structure of PLGA-PEG-T7 copolymers dissolved in chloroform-D was confirmed by a NMR spectrometer (Bruker Avance III, 400 MHz). The hydrophobic MNPs were prepared according to the procedures previously reported.26 Preparation of magnetic PLGA-PEG-T7 nanoparticles (MNP/T7-PLGA NPs) with or without drugs was conducted through the single emulsion−solvent evaporation method. In brief, PLGA-PEG-T7 (20 mg), drugs (8 mg), and MNPs (120 μL, 20 mg/mL) in 2 mL of chloroform were dropwise added to 4 mL of an aqueous solution containing the surfactant (1% sodium cholate), followed by sonication for 2 min at 180 W. The thus-formed microemulsion was added into 10 mL of 0.5% sodium cholate dropwise under magnetic stirring. The organic solvent was evaporated, and the MNP/T7-PLGA NPs were obtained. Blank NPs were prepared as above without the addition of drugs. Fluorescein (coumarin, DIR, or Cy5)-labeled NPs were prepared with the same procedures by adding fluorescein as replacement for the drugs. 5.5. Characterization of Dual-Targeting Nanodrug Delivery System. The particle size and ζ-potential of NPs were measured by a dynamic light scattering instrument (Nano-ZS, Malvern, UK). The morphology of NPs was observed using transmission electron microscopy (TEM, Tecnai G2 Spirit, 120 kV). The magnetic property of NPs was measured using a vibrating sample magnetometer (VSM, HH series, Nanjing Nanda Co., China). The transverse relaxation rate was measured by using a magnetic resonance analyzer (Bruker mini Spec mq-60, German). The strength of the applied magnetic field used for in vitro and in vivo experiments was 100 mT, measured by a Gauss meter (WT10A, Magnetoelectricity Technology Ltd. Co., China). 5.6. Encapsulation and in Vitro Release Rate of Paclitaxel and Curcumin. The encapsulation efficiency (EE) of PTX and CUR was calculated by using the following equation:

4. CONCLUSIONS Overcoming the BBB via brain-targeting delivery strategies will dramatically expand the horizon of brain tumor therapy by providing many optional chemotherapeutics that otherwise are BBB-impermeable. The dual-targeting delivery system developed in this work was characterized by the receptor-mediated and magnet-based brain-targeting mechanism. The MNP/T7PLGA NPs can efficiently penetrate the BBB and enhance brain delivery efficiency, as demonstrated in both in vitro and in vivo studies. The system exhibited improved glioma therapy efficacy yet with reduced adverse toxicities. 5. MATERIALS AND METHODS 5.1. Materials. Carboxyl-terminated PLGA (50:50, MW 10K, inherent viscosity 0.15−3.0 dL/g) was purchased from Daigang Biotechnology Co. (Jinan, China). Amino-poly(ethylene glycol)maleimidyl (NH2-PEG-MAL, MW 3500) was obtained from Seebio Biotech, Inc. (Shanghai, China). T7 peptide (sequence HAIYPRH) was synthesized by Leon Chemical Co. Ltd. (Shanghai, China). Dichloromethane, trichloromethane, diethyl ether, ethanol, N,Ndiisopropylcarbodiimide (DIC), N-hydroxysuccinimide (NHS), acetonitrile (ACN), dimethylformamide (DMF), trifluoroacetic acid (TFA), sodium cholate, dimethyl sulfoxide (DMSO), potassium ferrocyanide, coumarin-6 (CM), and curcumin (CUR) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Paclitaxel (PTX) was obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). MTT reagent was purchased from Sigma (St. Louis, MO, USA) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DIR) from Biotium (Invitrogen, USA). Annexin VFITC apoptosis detection kit and Triton X-100 were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). 5.2. Cell Lines. U87 glioma cells genetically marked with a firefly luciferase reporter gene (U87-Luc) were kindly provided by Prof. J. Ding (Shanghai Institute of Materia Medica, Chinese Academy of Science (CAS)). Mouse brain endothelial cells (bEnd.3) were provided by Prof. J. X. Wang (School of pharmacy, Fudan University). The cells were cultured in incubators and maintained at 37 °C with 5% CO2 under fully humidified conditions. The cell culture medium was DMEM with high glucose, supplemented with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin sulfate. 5.3. Animals. Male BALB/C nude mice (4−5 weeks) were supplied by Shanghai Laboratory Animal Center, CAS, and acclimated at the SPF care facility under a 12 h light/dark cycle. All of the animal experiments were carried out according to the protocol approved by the Institutional Animal Care and Use Committee (SIMM, CAS). 5.4. Synthesis and Characterization of PLGA-PEG3500-T7 Copolymer. Synthesis of PLGA-PEG-T7 copolymer was performed as follows. First, NH2-PEG-T7 was obtained via the Michael-type addition reaction of NH2-PEG-MAL and the thiolated T7 peptide in DMF at room temperature for 4 h with a molar ratio of 2:1 under nitrogen protection. Second, carboxyl-terminated PLGA (PLGACOOH, 500 mg) was activated by using DIC and NHS to create

EE (%) =

amount of encapsulated drug in the nanoparticles × 100% amount of added drug

The in vitro PTX and CUR release from the NPs was conducted in PBS (pH 7.2, containing 0.2% Tween 80) with gentle shaking at 150 rpm at 37 °C using a dialysis membrane (MWCO 3500 Da). At the designated time points (2, 4, 6, 8, 12, 20, 24, 30, 36, 48, 60, 72, and 84 h), a portion of releasing medium (1 mL) was taken for HPLC analysis. Meanwhile, 1 mL of fresh releasing medium was replenished. The quantification of PTX and CUR was determined using reversed-phase high-performance liquid chromatography (HPLC) and fluorescence spectrophotometry, respectively. A certain quantity of samples was added to chloroform to extract the drugs. The organic solution was collected and evaporated. Acetonitrile was added to dissolve the residuals. The samples were subjected to measurement by HPLC and fluorescence spectrophotometer, respectively. For PTX analysis, the mobile phase was a gradient mixture of acetonitrile and distilled water. The volume ratio of acetonitrile was changed from 40 to 80% between 0 and 25 min at a flow rate of 1.0 mL/min and with a detection wavelength of 227 nm. The PTX 32167

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48 h. The final concentrations of CUR and PTX were 30 and 10 nM, respectively. The cells were stained with the JC-1 mitochondrial membrane potential assay kit (Beyotime Co. Ltd.) according to the manufacturer’s protocol and analyzed by flow cytometry. 5.11. Colony Formation Assay. The U87 cells were seeded onto a 6-well plate at a density of 300−500 cells/well and incubated at 37 °C for 24 h. The cells were incubated with the different NPs. After 48 h of incubation, the medium was replaced with fresh complete medium and the cell colonies were allowed to grow for 8 days. Then, the colonies were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 30 min. The stained cell colonies were thoroughly washed with PBS three times and observed under a microscope. The colonies were analyzed using ImageJ software. 5.12. In Vivo Antiglioma Treatment. The orthotopic gliomabearing mouse model was established. A total of 3 × 105 U87-Luc cells was slowly implanted into the right striatum at a location 1.5 mm lateral to the bregma and at 3.5 mm depth at the injection speed of 1 μL/min using a stereotactic fixation device. After surgery, the mice were maintained under the standard housing conditions for 15 days, and the xenografted glioma was monitored using IVIS Spectrum after intraperitoneal injection of luciferin (15 mg/mL). The mice were randomly divided into six groups. Each group had six mice. The glioma-bearing mice were intravenously administered PBS (blank, control group), MNP/T7-PLGA (CUR + PTX) + MAG, MNP/T7PLGA (CUR + PTX), MNP/T7-PLGA (CUR), MNP/T7-PLGA (PTX), or free CUR + free PTX, respectively, at a dose of 4 mg drugs/ kg body weight with a dosage frequency of every 3 days. At days 12 and 21 after treatment, antiglioma effects were measured by IVIS Spectrum. Survival rate was calculated over a treatment course of 35 days. 5.13. Statistical Analysis. All data were the mean ± standard deviation (SD). For in vitro experiments the number of repetitions was 6. For in vivo treatment, each group included eight mice. The unpaired Student’s t test was used to calculate the statistical significance of differences between the antiglioma groups and the control groups. *, P < 0.05, and **, P < 0.01.

concentration was calculated by using the predetermined calibration curve. For CUR analysis, the fluorescence spectrophotometer (F-4600, Hitachi, Japan) was used to detect the absorbance at 552 nm with the excitation wavelength of 420 nm. The voltage during measurement of all samples was 800 V. CUR concentration in samples was calculated according to the predetermined standard equation. 5.7. Cellular Uptake Efficiency. The bEnd.3 or U87 cells were seeded into a 24-well plate with 104 cells/well. After incubation for 12 h, the medium was replaced with fresh complete DMEM containing different coumarin-labeled nanoparticles with an equivalent concentration of coumarin at 2 nM. The cells were incubated for 2 h with or without an applied magnetic field and then washed with PBS, fixed with 4% paraformaldehyde for 20 min, and stained with 0.5 μg/mL DAPI for 10 min. The stained cells were imaged via confocal laser scanning microscopy (Fluoview FV1Oi Olympus, Japan). For cell uptake efficiency measured by flow cytometry, the cells (bEnd.3 cells or U87 cells) were seeded into a 6-well plate at a density of 105 cells/well and incubated for 12 h before use. The cells were incubated with different coumarin-labeled NPs with an equivalent concentration of coumarin at 2 nM for 2 h with or without a magnetic field. The strength of the magnetic field was 100 mT. The cells were washed with PBS, harvested by trypsinization, and resuspended in PBS for flow cytometry assay (FACSCalibur, BD, USA). 5.8. In Vitro and in Vivo Transport across the BBB by DualTargeting Mechanisms. For in vitro transport efficiency across the BBB, first, establish the BBB model in vitro. Briefly, bEnd.3 cells were seeded into the 24-well Transwell upper chambers at a density of 5 × 104 cells/well. The integrity of the bEnd.3 monolayer was tested by measuring the TEER (>200 Ω·cm2) using an impedance instrument. The U87 cells were planted in the lower chambers. The coumarinlabeled NPs (equal to 2 μM coumarin per well) were added into the upper chambers of the Transwell supports, followed by incubation at 37 °C for 4 h. The U87 cells at the lower chambers were collected, and the cell lysis was subjected to fluorometry (Hitachi F4000 fluorometer, Japan) to assess the transport efficiency across the BBB. For evaluation of the in vivo brain-targeting effect, we employed three methods as follows: (1) Fluorescence Image Technique. Mice bearing orthotopic glioma were randomly divided into various groups. Four hours after tail vein injection of the different DIR labeled NPs, animals were subjected to in vivo imaging to examine the distribution of NPs under the different targeting mechanisms through IVIS spectrum system (Caliper PerkinElmer, Hopkinton, MA, USA). The final DIR concentration was 2.5 μM. (2) Synchrotron Radiation X-ray Imaging Technique. After injection of the MNP/T7-PLGA NPs at a dose of 4 mg Fe/kg body weight via the tail vein and application of a magnetic field for 4 h, mice were killed, and the brains were dissected and fixed in 4% paraformaldehyde for 48 h. Synchrotron radiation X-ray fluorescence imaging of iron element in the brain tissue was conducted by Synchrotron-based X-ray fluorescence imaging technique at Shanghai Synchrotron Radiation Facility (SSRF), CAS. Parameters were set as follows: energy, 12.5 keV; pixel, 3.25 μm; flat gap number, 100. (3) T2-Weighted Magnetic Resonance Imaging (MRI) Technique. After injection of the MNP/T7-PLGA NPs at a dose of 4 mg Fe/kg body weight via the tail vein and application of a magnetic field for 4 h, mice were scanned for T2-weighted MR imaging (Siemens 3.0 T, Germany) with the following parameters: TR = 4000 ms, TE = 105 ms, slice thickness = 0.8 mm. The mice with injection of PBS were used as control group. 5.9. Antiproliferative Activity against U87 Glioma Cells. U87 cells were seeded in a 96-well plate at a density of 6 × 103 cells/well and incubated for 24 h. The U87 cells were then treated with 100 μL of medium containing NPs or free drugs for 48 h. Then, the cell viability was measured by a standard MTT method. 5.10. Mitochondrial Membrane Potential Assay. The effect of drugs (PTX, CUR) on the mitochondrial membrane potential was assessed in U87 glioma cells. The cells were seeded onto a 6-well plate and incubated for 24 h. The cells were then treated with the NPs for

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b10175. Additional figures (PDF) Stimulation modeling video (MPG)

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AUTHOR INFORMATION

Corresponding Author

*(Y.H.) Phone: +86-21-20231000, ext. 1401. Fax: +86-2120231981. E-mail: [email protected]. ORCID

Yongzhuo Huang: 0000-0001-7067-8915 Author Contributions ∥

Y.C. and M.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by 973 Program, China (2013CB932503, 2014CB931900), NFSC (81373357, 81402885, 81422048, 81673382), and China’s Postdoctoral Science Foundation (133646 and 2014M551475). The synchrotron X-ray fluorescence imaging was performed at the BL15U beamline of Shanghai Synchrotron Radiation Facility (SSRF, China). We thank Dr. Guangzhao Zhou (SSRF) for assistance with imaging process. 32168

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(21) Ling, Y. H.; el-Naggar, A. K.; Priebe, W.; Perez-Soler, R. Cell Cycle-Dependent Cytotoxicity, G2/M Phase Arrest, and Disruption of P34cdc2/Cyclin B1 Activity Induced by Doxorubicin in Synchronized P388 Cells. Mol. Pharmacol. 1996, 49, 832−841. (22) Bar-On, O.; Shapira, M.; Hershko, D. D. Differential Effects of Doxorubicin Treatment on Cell Cycle Arrest and Skp2 Expression in Breast Cancer Cells. Anti-Cancer Drugs 2007, 18, 1113−1121. (23) Rafehi, H.; Orlowski, C.; Georgiadis, G. T.; Ververis, K.; ElOsta, A.; Karagiannis, T. C. Clonogenic Assay: Adherent Cells. J. Visualized Exp. 2011, DOI: 10.3791/2573. (24) Zhang, J.; Chen, N.; Wang, H.; Gu, W.; Liu, K.; Ai, P.; Yan, C.; Ye, L. Dual-Targeting Superparamagnetic Iron Oxide Nanoprobes with High and Low Target Density for Brain Glioma Imaging. J. Colloid Interface Sci. 2016, 469, 86−92. (25) Gao, H. Perspectives on Dual Targeting Delivery Systems for Brain Tumors. J. Neuroimmune Pharmacol. 2016, 1−11. (26) Cui, Y.; Xu, Q.; Chow, P. K.; Wang, D.; Wang, C. H. Transferrin-Conjugated Magnetic Silica PLGA Nanoparticles Loaded with Doxorubicin and Paclitaxel for Brain Glioma Treatment. Biomaterials 2013, 34, 8511−8520. (27) Fang, J. H.; Chiu, T. L.; Huang, W. C.; Lai, Y. H.; Hu, S. H.; Chen, Y. Y.; Chen, S. Y. Dual-Targeting Lactoferrin-Conjugated Polymerized Magnetic Polydiacetylene-Assembled Nanocarriers with Self-Responsive Fluorescence/Magnetic Resonance Imaging for In Vivo Brain Tumor Therapy. Adv. Healthcare Mater. 2016, 5, 688−695. (28) Fang, J. H.; Lai, Y. H.; Chiu, T. L.; Chen, Y. Y.; Hu, S. H.; Chen, S. Y. Magnetic Core-Shell Nanocapsules with Dual-Targeting Capabilities and Co-Delivery of Multiple Drugs to Treat Brain Gliomas. Adv. Healthcare Mater. 2014, 3, 1250−1260. (29) Mody, V. V.; Cox, A.; Shah, S.; Singh, A.; Bevins, W.; Parihar, H. Magnetic Nanoparticle Drug Delivery Systems for Targeting Tumor. Appl. Nanosci. 2014, 4, 385−392. (30) Patil, U. S.; Adireddy, S.; Jaiswal, A.; Mandava, S.; Lee, B. R.; Chrisey, D. B. In Vitro/In Vivo Toxicity Evaluation and Quantification of Iron Oxide Nanoparticles. Int. J. Mol. Sci. 2015, 16, 24417−24450. (31) Singh, N.; Jenkins, G. J.; Asadi, R.; Doak, S. H. Potential Toxicity of Superparamagnetic Iron Oxide Nanoparticles (SPION). Nano Rev. 2010, 1, 10.3402/nano.v1i0.5358 (32) Min, K. A.; Shin, M. C.; Yu, F.; Yang, M.; David, A. E.; Yang, V. C.; Rosania, G. R. Pulsed Magnetic Field Improves the Transport of Iron Oxide Nanoparticles Through Cell Barriers. ACS Nano 2013, 7, 2161−2171. (33) Min, K. A.; Yu, F.; Yang, V. C.; Zhang, X.; Rosania, G. R. Transcellular Transport of Heparin-coated Magnetic Iron Oxide Nanoparticles (Hep-MION) Under the Influence of an Applied Magnetic Field. Pharmaceutics 2010, 2, 119−135. (34) Dang, Y. P.; Yuan, X. Y.; Tian, R.; Li, D. G.; Liu, W. Curcumin Improves the Paclitaxel-Induced Apoptosis of HPV-Positive Human Cervical Cancer Cells via the NF-KappaB-P53-Caspase-3 Pathway. Exp. Ther. Med. 2015, 9, 1470−1476. (35) Ganta, S.; Amiji, M. Coadministration of Paclitaxel and Curcumin in Nanoemulsion Formulations to Overcome Multidrug Resistance in Tumor Cells. Mol. Pharmaceutics 2009, 6, 928−939. (36) Hossain, M.; Banik, N. L.; Ray, S. K. Synergistic anti-Cancer Mechanisms of Curcumin and Paclitaxel for Growth Inhibition of Human Brain Tumor Stem Cells and LN18 and U138MG Cells. Neurochem. Int. 2012, 61, 1102−1113.

REFERENCES

(1) Gallego, O. Nonsurgical Treatment of Recurrent Glioblastoma. Curr. Oncol. 2015, 22, e273−e281. (2) Wang, Z.; Sun, H.; Yakisich, J. S. Overcoming the Blood-Brain Barrier for Chemotherapy: Limitations, Challenges and Rising Problems. Anti-Cancer Agents Med. Chem. 2014, 14, 1085−1093. (3) Spector, R. Nutrient Transport Systems in Brain: 40 Years of Progress. J. Neurochem. 2009, 111, 315−320. (4) Allen, D. D.; Geldenhuys, W. J. Molecular Modeling of BloodBrain Barrier Nutrient Transporters: in Silico Basis for Evaluation of Potential Drug Delivery to the Central Nervous System. Life Sci. 2006, 78, 1029−1033. (5) Mittapalli, R. K.; Manda, V. K.; Adkins, C. E.; Geldenhuys, W. J.; Lockman, P. R. Exploiting Nutrient Transporters at the Blood-Brain Barrier to Improve Brain Distribution of Small Molecules. Ther. Delivery 2010, 1, 775−784. (6) Daniels, T. R.; Bernabeu, E.; Rodriguez, J. A.; Patel, S.; Kozman, M.; Chiappetta, D. A.; Holler, E.; Ljubimova, J. Y.; Helguera, G.; Penichet, M. L. The Transferrin Receptor and the Targeted Delivery of Therapeutic Agents against Cancer. Biochim. Biophys. Acta, Gen. Subj. 2012, 1820, 291−317. (7) Blanco, E.; Shen, H.; Ferrari, M. Principles of Nanoparticle Design for Overcoming Biological Barriers to Drug Delivery. Nat. Biotechnol. 2015, 33, 941−951. (8) Doshi, N.; Prabhakarpandian, B.; Rea-Ramsey, A.; Pant, K.; Sundaram, S.; Mitragotri, S. Flow and Adhesion of Drug Carriers in Blood Vessels Depend on Their Shape: A Study Using Model Synthetic Microvascular Networks. J. Controlled Release 2010, 146, 196−200. (9) Arslan, O. Neuroanatomical Basis of Clinical Neurology, 2nd ed.; CRC Press: 2015. (10) Bae, Y. H.; Park, K. Targeted Drug Delivery to Tumors: Myths, Reality and Possibility. J. Controlled Release 2011, 153, 198−205. (11) Wang, J.; Huang, Y.; David, A. E.; Chertok, B.; Zhang, L.; Yu, F.; Yang, V. C. Magnetic Nanoparticles for MRI of Brain Tumors. Curr. Pharm. Biotechnol. 2012, 13, 2403−2416. (12) Pedram, M. Z.; Shamloo, A.; Alasty, A.; Ghafar-Zadeh, E. Toward Epileptic Brain Region Detection Based on Magnetic Nanoparticle Patterning. Sensors 2015, 15, 24409−24427. (13) Moos, T.; Morgan, E. H. Transferrin and Transferrin Receptor Function in Brain Barrier Systems. Cell. Mol. Neurobiol. 2000, 20, 77− 95. (14) Lee, J. H.; Engler, J. A.; Collawn, J. F.; Moore, B. A. Receptor Mediated Uptake of Peptides that Bind the Human Transferrin Receptor. Eur. J. Biochem. 2001, 268, 2004−2012. (15) Liu, Q.; Zhang, J.; Xia, W.; Gu, H. Magnetic Field Enhanced Cell Uptake Efficiency of Magnetic Silica Mesoporous Nanoparticles. Nanoscale 2012, 4, 3415−3421. (16) Cheng, Z.; Dai, Y.; Kang, X.; Li, C.; Huang, S.; Lian, H.; Hou, Z.; Ma, P.; Lin, J. Gelatin-Encapsulated Iron Oxide Nanoparticles for Platinum (IV) Prodrug Delivery, Enzyme-Stimulated Release and MRI. Biomaterials 2014, 35, 6359−6368. (17) Chan, N.; Laprise-Pelletier, M.; Chevallier, P.; Bianchi, A.; Fortin, M. A.; Oh, J. K. Multidentate Block-Copolymer-Stabilized Ultrasmall Superparamagnetic Iron Oxide Nanoparticles with Enhanced Colloidal Stability for Magnetic Resonance Imaging. Biomacromolecules 2014, 15, 2146−2156. (18) Corot, C.; Robert, P.; Idee, J. M.; Port, M. Recent Advances in Iron Oxide Nanocrystal Technology for Medical Imaging. Adv. Drug Delivery Rev. 2006, 58, 1471−1504. (19) Li, J.; Zhang, C.; Yang, K.; Liu, P.; Xu, L. X. SPIO-RGD Nanoparticles as A Molecular Targeting Probe for Imaging Tumor Angiogenesis Using Synchrotron Radiation. J. Synchrotron Radiat. 2011, 18, 612−616. (20) Liang, J. M.; Zeng, F.; Zhang, M.; Pan, Z. Z.; Chen, Y. Z.; Zeng, Y. N.; Xu, Y.; Xu, Q.; Huang, Y. Z. Green Synthesis of Hyaluronic Acid-Based Silver Nanoparticles and Their Enhanced Delivery to CD44(+) Cancer Cells. RSC Adv. 2015, 5, 43733−43740. 32169

DOI: 10.1021/acsami.6b10175 ACS Appl. Mater. Interfaces 2016, 8, 32159−32169