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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10175 • Publication Date (Web): 03 Nov 2016 Downloaded from http://pubs.acs.org on November 4, 2016

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ACS Applied Materials & Interfaces

Dual-Targeting Magnetic PLGA Nanoparticles for Codelivery of Paclitaxel and Curcumin for Brain Tumor Therapy

Yanna Cui a,b,1, Meng Zhang a,1, Feng Zeng a,c,d, Hongyue Jin a, Qin Xu d, Yongzhuo Huang a* a

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Rd, Shanghai 201203, China b Key Laboratory of Primate Neurobiology, Institute of Neuroscience, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Rd, Shanghai 200031, China c Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, 826 Zhangheng Rd, Shanghai 201203, China d Guangzhou University of Chinese Medicine, Institute of Tropical Medicine, 12 Jichang Rd, Guangzhou 501405, China 1. These authors contributed equally to this work.

*Corresponding author: HUANG, Yongzhuo, Ph.D., Prof. Shanghai Institute of Materia Medica Chinese Academy of Sciences Tel +86-21-20231000 ext 1401; Fax: +86-21-20231981 Email: [email protected]

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ABSTRACT: Chemotherapy is one of the most important strategies for glioma treatment. However, the “impermeability” of the blood-brain barrier (BBB) impedes most chemotherapeutics to enter the brain, thereby rendering very few drugs suitable for

glioma

therapy,

letting

along

application

of

combination

of

chemotherapeutics. Thereby, it is a pressing need to overcome the obstacles. A dual-targeting strategy was developed by combination of magnetic-guidance and the transferrin receptor-binding peptide T7-mediated active targeting delivery. The T7-modified magnetic PLGA nanoparticle system was prepared with co-encapsulation of the hydrophobic magnetic nanoparticles and a combo 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 than single use of each drug. Dual-targeting effects yielded over 10-fold increase in cellular uptake studies, and over five-fold enhancement in brain delivery compared to the non-targeting 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 anti-glioma treatment efficacy of the delivery system was evaluated. With application of magnetic field, this system exhibited enhanced treatment efficiency and reduced adverse effects. All mice bearing orthotopic glioma survived, compared to 62.5% survival rate for the combination group receiving free drugs. This dual-targeting, codelivery strategy provides a potential method for improving brain drug delivery and anti-glioma treatment efficacy.

KEYWORDS: Brain targeting delivery, magnetic targeting, PLGA nanoparticles, paclitaxel, curcumin, T7 peptide


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1. Introduction Malignant glioma is one of the most common and aggressive brain tumors in human. Glioma patients have median survival time of approximate 1 year and only 5% survive more than 5 years.1 Due to the formidable blood-brain barrier (BBB), options for glioma chemotherapy 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, to develop BBB-penetrating drug delivery systems has been a major effort in anti-glioma therapy. Indeed, to achieve brain-tumor targeting drug delivery with reduced unwanted drug exposure to health organs has become a holy grail earnestly pursued in medical community. Brain is the most energy-consuming organ, with active material exchanges through BBB. Nutrient transporters on the BBB are crucial in maintaining the normal functions of the brain, by fetching the essential, life-sustaining components such as amino acids/peptides, sugars, and proteins.3 Owing to the high expression of such nutrient transporters on BBB and their potent transcellular transport ability, targeting to 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 serving as a “Trojan Horse” strategy, by which the camouflaged cargos can sneak into the tumors.6 Apart from the BBB penetration issue, to effective target to 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 blood stream, with different particle-cell interaction modes from that observed in the cellular uptake studies in a static cellular culture model.7 Therefore, it is a very important design consideration about the interaction of



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nanoparticles with endothelial walls, 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 blood stream, 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 the 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 effort 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, to improve 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 towards 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 Fe-transferrin complex.13 For the ligand-mediated targeting, the human transferrin receptor-binding peptide T7 (sequence: HAIYPRH) was selected. Of important, no competition was found between T7 and transferrin for receptor binding, indicating that their bind sites are distinct from each other.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 the basis of this, we developed

a

T7-modified,

magnetic

PLGA

nanoparticulate


system

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(MNP/T7-PLGA 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 magnetic PLGA system is its capability of co-encapsulation of various drugs, providing potential of combination therapy against glioma. This system was used for codelivery 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 single-emulsion 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.



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Scheme 2 Synthesis of PLGA-PEG-T7 polymer (a) and drug-loaded MNP/T7-PLGA NPs (b)

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 4.81 ppm is attributed to the methylene hydrogen of glucolide. The peak at 5.20 ppm belongs to the methane hydrogen of D, L-lactide. The characteristic signal at δ = 6.55−6.75 ppm of 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 large size and the aggregation of MNP into the PLGA NPs, which resulted in the asymmetric distribution. If 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 of the larger size of the PLGA NPs with higher content of MNP, it could be benefits for enhanced responsivity and targeting efficiency. The particle size and ζ-potential were measured by dynamic light scattering (DLS). The results showed that the size of the T7-PLGA NPs without MNPs, the MNP/T7-PLGA NPs with low-content iron, and the MNP/T7-PLGA NPs with high-content iron, were 99 nm, 113 nm, 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.


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Stability of the NPs in the PBS supplemented with 10% FBS were investigated by monitoring changes in particle size. The results showed that the MNP/T7-PLGA NPs remained stable in 70 h at 37°C (Figure 1D). It was interesting that incorporation of drugs led to 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 in the gaps among the iron NPs and between the iron NPs and PLGA.

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.

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 superparamagnetic property at 300 K (Figure 2A). The saturation



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magnetization (Ms) of the OA-MNPs and MNP/T7 PLGA NPs was 82 emu/g Fe and 13 emu/g Fe, respectively, displaying higher Ms than the reported result (2 emu/g Fe).15 It indicated the potential of the MNP/T7-PLGA NPs for magnetic targeting application. MNPs have been widely applied as the 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. It was found that a good linear correlation between T2 relaxation rate (R2) and iron concentration. 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 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). It suggested that the MNP/T7-PLGA NPs effectively enhanced the transverse proton relaxation process and could be utilized as MRI negative contrast agents.

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 the


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saturation magnetization of was 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 efficiency of PTX and CUR measured by HPLC was 68% and 18%, respectively. Figure 3 shows the sustained release patterns, in which the release rate of PTX and CUR from the MNP/T7-PLGA NPs was 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 faster than those deep inside the NPs. Moreover, the dissolution rate of the drugs is also a key factor.

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

2.3 In vitro and in vivo transport across 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



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that the magnetic field-driven cellular uptake was not a major factor for the MNP/T7-PLGA NPs. As shown in Figure 4(A-B), for the MNP/PLGA NPs, the application of magnetic field yielded limited enhancement effect on the uptake efficiency in both cell lines (i.e., 1.6 and 1.2 folds). It revealed that T7-mediated intracellular delivery was the primary mechanism for intracellular delivery of the NPs in the cultured cell models. Another evidence is that the U87 cellular uptake efficiency of the MNP/T7-PLGA NPs was more than 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).

Figure 4 Cellular uptake experiments of different formulations on U87 and bEnd.3 cells. Nanoparticles were labeled with coumarin-6 (green fluorescence). (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.


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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 trans-epithelial 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, 6.42% for MNP/T7-PLGA NPs + MAG (Figure 5A). It 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, in order to study the in vivo brain targeting efficiency, the 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 the 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. It 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,

synchrotron radiation X-ray fluorescence technique can provide high sensitive three-dimensional images with the superior submicrometer spatial resolution for tracking metal elements in biological tissues. Synchrotron radiation X-ray



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technique was applied in our studies to analyze the MNP distribution in the brain. The iron element (the red signal) was observed by synchrotron radiation facility (Figure 5D), displaying substantial brain accumulation of the MNP/T7-PLGA NPs. The 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 imaging. The MRI signal was monitored at 4 h after tail vein injection of the MNP/T7-PLGA NPs (Figure 5E), displaying clear MRI signal in the glioma site. These results corroborated the potential of the MNP/T7-mediated dual-targeting strategy for efficient transport across BBB and accumulation in 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 T7-mediated brain delivery was already supported by the bio-distribution study (Fig 5), in which the group of (MNP/PLGA+MAG) without targeting ligand showed significantly lower drug accumulation in the brain, compared to that of T7-MNP/PLGA+MAG. Therefore, the group of (MNP/PLGA+MAG) without targeting ligand T7 was not included in the following treatment studies.


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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 the brain targeting mediated by dual targeting.

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). It showed that the codelivery 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 anti-tumor activity did not benefit from the application of magnetic field, which may account for the ineffectiveness of magnetic field on driving intracellular uptake in the static culture system.



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

Table 1 IC50 (half maximal inhibitory concentration) values in U87 cells

IC50

MNP/T7-

MNP/T7-PLGA

MNP/PLGA

Free

MNP/T7-

MNP/T7-

PLGA

(CUR+PTX) +

(CUR+PTX)

CUR +

PLGA (PTX)

PLGA (CUR)

(CUR+PTX)

MAG

3.04

6.23

6.48

8.35

PTX 9.43

5.80

(μg/ml)

For further investigation of cytotoxic effect, mitochondrial membrane potential (MMP) was measured by flow cytometry. Mitochondrion is the cell powerhouse. Chemo drugs can impair mitochondrial functions and induce apoptosis. MMP is an important indicator of mitochondria function and can serve as an early marker of the onset of apoptosis.20 JC-1 staining method was used for indicating the mitochondrial activity in this experiment. In the normal conditions, the cells 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.


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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. G2/M checkpoint is a major target for DOX-based 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 drugs-loaded MNP/T7 PLGA NPs was highest, displaying a population of 63.6%, while that of the PTX-loaded MNP/T7 PLGA NPs was 39.7% (Figure 8). This indicated that the combination therapy could improve the chemotherapeutic efficacy.



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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/T7-PLGA (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.

Moreover, the crystal violet was used for monitoring the drug-induced change of U87 cloning morphology. 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% of the colony-forming rate (Figure 9), respectively, significantly lower than other groups. It demonstrated that the combination therapy via the MNP/T7-PLGA NPs


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under the dual-targeting mediation was highly effective in inhibiting the proliferation of the U87 glioma cells.

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.

2.5 In vivo anti-glioma efficacy The in vivo anti-glioma efficacy was investigated using the transplanted orthotopic U87-Luc glioma model. Bio-luminescence intensity indicated the size of the glioma.

The groups received chemotherapy showed substantial

treatment efficacy (Figure 10), with inhibition efficiency as 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. It demonstrated the great potential



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of the combination therapy under the dual-targeting mechanism. By comparison of the results from day 12 and 21, it clearly showed that the glioma growth rate was significantly suppressed by the dual targeting group (MNP/T7-PLGA NPs + MAG), while other groups displayed relative fast growth rate.

Figure 10 Glioma growth inhibition in the Balb/c nude mice. (A) Schedule of treatment and imaging. (B) Increased fold of bio-luminescence 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 & a2: MNP/T7-PLGA NPs (CUR); b1 & b2: MNP/T7-PLGA NPs (CUR + PTX); c1 & c2: MNP/T7-PLGA NPs (CUR + PTX) + MAG; d1 & d2: MNP/T7-PLGA NPs (PTX); e1 & e2: free CUR + PTX.

To further assess the anti-glioma efficiency, the overall survival time and body


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weight change of the glioma-bearing mice were measured (Figure 11A & B). 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), while the survival rates were 83% for the MNP/T7-PLGA NPs (PTX+CUR) without magnetic field, 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 >15% body weight loss found in the group treated with free combo drugs. It suggested that the reduced adverse effect of the combination therapy using MNP/T7-PLGA NPs + MAG.

Figure 11 (A) Change of animal body weight; and (B) overall survival of glioma-bearing mice (n = 8).

3. Discussion Due to the presence of 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 become an attractive method to boost the brain delivery efficiency. The dual-targeting mechanisms can be achieved by incorporation of different ligands to target BBB and brain



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tumor, target to the same receptor, or target to the two cell types in brain tumor.25 Recently, 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 caused oxidative stress and toxicities.30-31 A benefit of our system is that the encapsulation mode of MNPs@PLGA NP could effective retard the iron release and acute ROS generation. Importantly, the modification of a peptide ligand on the PLGA NPs is much convenient due to its compatibility to 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 inconsistence between the in vitro and in vivo investigations, in which the dual-targeting delivery was significant in animal studies, but only yielded 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 magnetically-induced nanoparticle aggregates.32 The formation of large aggregates prevents intracellular delivery. However, high 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 from


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aggregate formation under conditions of the high serum concentration and dynamic blood stream. The synergistic mechanisms of PTX and CUR have been reported. For example, CUR can improve the paclitaxel-induced apoptosis of cancer cells via the NF-ĸb-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, it is still little known about how to strategically combine the ligands and make them work synergistically, which largely relies on empirical evidence or experimental observation; nor was for the combination of physical and biochemical targeting mechanisms, too. Therefore, further detailed investigation would be helpful for providing insight on 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.

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/T7-PLGA NPs can efficiently penetrate the BBB and enhance brain delivery efficiency, as demonstrated both in vitro and in vivo studies. The system exhibited improved glioma therapy efficacy yet with reduced adverse toxicities.



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5. Materials and methods 5.1 Materials Carboxyl-terminated PLGA (50:50, MW 10 k, inherent viscosity 0.15−3.0 dL/g) was purchased from

Daigang

Biotechnology Company (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,

N-diisopropylcarbodiimide (DIC), N-Hydroxysuccinimide (NHS), acetonitrile (ACN), dimethylformamide (DMF), trifluoroacetic acid (TFA), sodium cholate, dimethylsulfoxide (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 V-FITC 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, 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 medium with high glucose, supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin sulfate. 5.3 Animals


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Male BALB/C nude mice (4−5 weeks) were supplied by Shanghai Laboratory Animal Center, CAS and acclimated at SPF care facility under a 12-h light/dark cycle. All 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. Firstly, 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 the nitrogen protection. Secondly, carboxyl-terminated PLGA (PLGA-COOH, 500 mg) was activated by using DIC and NHS to create 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 nano-drug delivery system



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The particle size and zeta 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 Company, China). The transverse relaxation rate was measured by using a magnetic resonance analyzer (Bruker mini Spec mq-60, German). The strength of applied magnetic field used for in vitro and in vivo experiments was 100 mT, measured by 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 paclitaxel (PTX) and curcumin (CUR) was calculated by the following equation: EE (%) =

× 100%

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 dialysis membrane (MWCO 3500 Da). At the designated time points (2, 4, 6, 8, 12, 20, 24, 30, 36, 48, 60, 72, 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 were determined using the reversed-phase high

performance

liquid

chromatography

(HPLC)

and

fluorescence

spectrophotometer, respectively. A certain quantity of samples were 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 gradient mixture of acetonitrile and distilled water. The volume ratio of acetonitrile was changed from 40% to 80% between 0 min and 25 min at a flow rate of 1.0 ml/min and with detection


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wavelength of 227 nm. The PTX 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 measuring all the 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

equivalent

concentration of coumarin at 2 nM. The cells were incubated for 2 h with or without 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 the density of 105 cells/well, and incubated for 12 h before use. The cells were incubated with different coumarin-labeled NPs with equivalent concentration of coumarin at 2 nM for 2 h with or without magnetic field. The strength of 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 BBB by dual-targeting mechanisms For in vitro transport efficiency across BBB, firstly, establish the BBB model in vitro. Briefly, bEnd.3 cells were seeded into the 24-well Transwell upper chambers at the density of 5×104 cells/well. The integrity of bEnd.3 monolayer was tested by measuring the transendothelia electrical resistance (TEER, >200 Ω·cm2) using impedance instrument. The U87 cells were planted in the lower



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chambers. The coumarin-labeled 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 fluorimetry (Hitachi F4000 fluorometer, Japan) to assess the transport efficiency across the BBB. For evaluation of 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 mechanism 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 tail vein and application of 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 tail vein and application of magnetic field for 4 h, the mice were scanned for T2-weighted MR imaging (Siemens 3.0 T, German) 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


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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 48 h. The final concentrations of CUR and PTX were 30 nM and 10 nM, respectively. The cells were stained with the JC-1 mitochondrial membrane potential assay kit (Beyotime Co. Ltd., Shanghai, China) according to the manufacture’s protocol, and analyzed by flow cytometry. 5.11 Colony formation assay The U87 cells were seeded onto a 6-well plate at the 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 for 3 times and observed under a microscope. The colonies were analyzed using ImageJ software. 5.12 In vivo anti-glioma treatment The orthotopic glioma-bearing mice model was established. The 3 × 105 U87-Luc cells were slowly implanted into the right striatum at the location of 1.5 mm lateral to the bregma and 3.5 mm of depth at the injection speed of 1 μl/min using a stereotactic fixation device. After surgery, the mice were maintained at 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 6 mice.



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The glioma-bearing mice were intraveneously administered with PBS (blank, control group), MNP/T7-PLGA (CUR + PTX) + MAG, MNP/T7-PLGA (CUR + PTX), MNP/T7-PLGA (CUR), MNP/T7-PLGA (PTX), or free CUR + free PTX, respectively, at the dose of 4 mg drugs/kg body weight with a dosage frequency of every three days. At the 12th and 21st day after treatment, anti-glioma 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 repetition was 6. For in vivo treatment, each group included 8 mice. The unpaired Student’s t test was used to calculate the statistical significance of differences between the anti-glioma groups and the control groups. *P < 0.05, **P < 0.01.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.xxxxxxx. Additional figures and stimulation modeling video are provided. Author Information Corresponding author *E-mail: [email protected] Notes The authors declare no competing financial interest. Acknowledgment: This work was supported by 973 Program, China (2013CB932503, 2014CB931900), NFSC (81373357, 81402885, 81422048, 81673382) and China's Post-doctoral Science Foundation (133646 & 2014M551475). The


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TOC


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Dual-Targeting Magnetic PLGA Nanoparticles for Codelivery of

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