Dual-targeting and microenvironment-responsive micelles as a gene delivery system to improve the sensitivity of glioma to radiotherapy

Dbait is a small double-stranded DNA molecule that has been utilized as a radiosensitizer to enhance the sensitivity of glioma to radiotherapy (RT). However, there is no effective drug delivery system to effectively overcome the blood—brain barrier (BBB). The aim of this study was to develop a gene delivery system by using the BBB and glioma dual-targeting and microenvironment-responsive micelles (ch-Kn(s-s)R8-An) to deliver Dbait into glioma for RT. Angiopep-2 can target the low-density lipoprotein receptor-related protein-1 (LRP1) that is overexpressed on brain capillary endothelial cells (BCECs) and glioma cells. In particular, due to upregulated matrix metalloproteinase 2 (MMP-2) in the tumor microenvironment, we utilized MMP-2-responsive peptides as the enzymatically degradable linkers to conjugate angiopep-2. The results showed that ch-Kn(s-s)R8-An micelles maintained a reasonable size (80–160 nm) with a moderate distribution and a decreased mean diameter from the cross-linking as well as exhibited low critical micelle concentration (CMC) with positive surface charge, ranging from 15 to 40 mV. The ch-K5(s-s)R8-An/pEGFP showed high gene transfection efficiency in vitro, improved uptake in glioma cells and good biocompatibility in vitro and in vivo. In addition, the combination of ch-K5(s-s)R8-An/Dbait with RT significantly inhibited the growth of U251 cells in vitro. Thus, ch-K5(s-s)R8-An/Dbait may prove to be a promising gene delivery system to target glioma and enhance the efficacy of RT on U251 cells.

(80-160 nm) with a moderate distribution and a decreased mean diameter from the cross-linking as well as exhibited low critical micelle concentration (CMC) with positive surface charge, ranging from 15 to 40 mV. The ch-K5(s-s)R8-An/pEGFP showed high gene transfection efficiency in vitro, improved uptake in glioma cells and good biocompatibility in vitro and in vivo. In addition, the combination of ch-K5(s-s)R8-An/Dbait with RT significantly inhibited the growth of U251 cells in vitro. Thus, ch-K5(s-s) R8-An/Dbait may prove to be a promising gene delivery system to target glioma and enhance the efficacy of RT on U251 cells.

Introduction
Glioblastoma multiforme (GBM) and malignant glioma are the most common primary malignant brain tumors 1,2 . GBM is commonly treated with surgery followed by radiotherapy (RT) and chemotherapy. However, GBM cells are likely to infiltrate extensively into the surrounding normal brain tissue, making it difficult to completely eradicate the tumor by surgery 3 . Glioma is often resistant to radiotherapy (RT) due to its enhanced DNA repair activity 4 , while increased dose of RT may increase injury to cognition and other normal functions of the brain 5 . In addition, the blood-brain barrier (BBB) and blood-brain tumor barriers (BBTB) are also factors that may affect the efficacy of most therapeutic agents 6,7 . Therefore, developing alternative approaches to overcome these problems and improve the therapeutic efficacy for glioma may be necessary.
Recent studies have shown that Dbait as a radiosensitizer combination with RT is effective for treating glioma, skin melanoma, colorectal cancer metastasis, and head and neck squamous cell carcinoma among others [8][9][10] . Dbait is a new class of DNA doublestrand breaks (DSBs) repair inhibitors consisting of a short doublestranded DNA molecule 10 . It can mimic DSBs to provide a "false" DNA damage signal and ultimately inhibit recruitment of many proteins involved in DSB repair pathways at the damage site 11,12 . However, Dbait cannot easily enter cells or the BBB due to its relatively large size (20.153 kDa) and high negative charge content 13 . Thus, target delivery of Dbait into glioma cells is challenging.
To enhance the therapeutic efficacy, dual targeting delivery systems are designed to target both the BBB and glioma cells. Many types of receptors are overexpressed in brain capillary endothelial cells (BCECs) that form the BBB and on glioma cells, including insulin receptors, transferrin receptors and low-density lipoprotein receptor-related protein-1 (LRP1) 14,15 . Angiopep-2, a member of the Kunitz domain-derived peptide family that can target the LRP1, exhibits a high transcytosis capacity and parenchymal accumulation as compared with transferrin, lactoferrin and avidin 16 . Angiopep-2 has been modified for use in delivery systems to facilitate penetration of the BBB and target glioma cells [17][18][19] . In addition, tumor microenvironment-responsive release of nanoparticle (NP) constitutes an important strategy that can specifically deliver drugs or genes to tumors without causing drugassociated adverse effects on normal tissues 18,20 . In particular, specific tumor microenvironment characteristics such as upregulated matrix metalloproteinase 2 (MMP-2) activity and high glutathione concentrations in the cytosol of tumor cells are reported to promote the development of tumor microenvironment-responsive drug delivery systems 21,22 .
Recent studies have demonstrated that polypeptides and cellpenetrating peptides (CPPs) can serve as gene and drug delivery systems due to their inherent bioactivity, biodegradability and biocompatibility [23][24][25] . Polyarginine molecules, which consist of 5-11 sequentially arranged arginines, have been shown to effectively penetrate the cell membrane, and R8 peptide can be internalized into cells most efficiently 25 . However, studies [26][27][28] showed that although CPPs could produce efficient cellular uptake in vitro, they could not accumulate in the target site in vivo effectively due to the lack of target cell specificity; as a result, the drug distributed in both tumor and healthy cells, producing undesirable effects during cancer treatment. To overcome this bottleneck, we chose the MMP-2 responsive peptide Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys (GPLGVRGK) as an enzymatically degradable linker to conjugate angiopep-2 thereby reducing the cell-penetrating properties of CPPs in the circulation, and then expressed it intrinsically in the tumor. In the circulation, angiopep-2 could facilitate penetration of the BBB and accumulate at the tumor site, at which point the shielding effect would be eliminated upon the cleavage of the linker by MMP-2. Hence, the exposed CPPs subsequently internalized the delivery system into tumor cells.
In this study, we utilized the unique features of the tumor microenvironment to design novel daul-targeting and microenvironment-responsive micelles as gene delivery system (Scheme 1). In particular, we chose MMP-2-responsive peptides as the enzymatically degradable linkers to conjugate angiopep-2. The micelles are expected to effectively activate the target glioma and then further penetrate into the core of the tumor by exposing R8 following cleavage of the linker by MMP-2. To evaluate the tumor targeting and penetration abilities of the micelles, we first synthesized and characterized ch-Kn(s-s)R8-An, and examined the in vitro transfection efficiency, cytotoxicity, cellular uptake and BBB penetration. In addition, the effects and mechanisms of ch-K5(s-s)R8-An/Dbait in combination with RT on cell apoptosis were investigated to evaluate the antitumor activity of the micelles in vitro. Finally, acute toxicity and in vivo distribution of ch-K5(ss)R8-An/pDNA in U251 orthotropic GBM-bearing were evaluated.

Materials
Poly ( Male BALB/c nude mice (18-22 g) and male BALB/c mice (18-22 g) were purchased from the Department of Experimental Animals of the Second Military Medical University (Shanghai, China). All animal experiments were performed in accordance with the ethics and regulations of animal experimentation ethics and regulations of the Shanghai Jiao Tong University.

Synthesis and characterization of polymers
Schematic of synthesizing ch-KnR8 (n ¼ 3, 5 and 7) graft copolymer is shown in Scheme 1 A using the F-moc-solid phase peptide synthesis method 29 . Lysine-arginine peptide (KnR8) was synthesized using the F-moc-solid phase peptide synthesis method. Second, we coupled cholesterol with the N-terminus of the lysine to obtain ch-KnR8. In addition, we chose the MMP-2 responsive peptide (GPLGVRGK) as the enzymatically degradable linker for conjugation to angiopep-2 using the F-moc-solid phase peptide synthesis method to obtain the angiopep-2-modified graft copolymer (ch-KnR8-An, n ¼ 3, 5 and 7). The products were then purified by reverse-phase high-performance liquid chromatography. The precise molecular weight of ch-KnR8 and ch-KnR8-An (n ¼ 3, 5 and 7) were measured using matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) (Bruker Daltonik GmbH, Bremen, HB, Germany).

Preparation and characterization of blank micelles
The polymeric micelles with shell-specific disulfide cross-links were prepared by a membrane dialysis method and a subsequent shell cross-linking reaction (Scheme 1B) 30,31 . The ch-KnR8 (n ¼ 3, 5 and 7) polymer (10 mg) was dissolved in DMSO. The solution was dialyzed against 1 L of deionized water for 48 h using a dialysis membrane bag with a molecular weight cut-off (MWCO) of 3000 Da (Pierce Co, Rockford, IL, USA). The blank non-cross-linked micelles (ch-KnR8, ch-KnR8-An, n ¼ 3, 5 and 7) were then collected. Next, DTSSP used as a disulfidecontaining cross-linking agent was added to the solution at the feed molar ratio of [DTSSP]:[Lys] ¼ 1:1. The reaction was maintained for 4 h at pH 8.0 and the solution was then dialyzed for 4 h to remove residual DTSSP. For the brain-targeting micelle preparation, 50% (mol of total copolymers) ch-KnR8-An was added to the copolymer-DMSO solution. Subsequently, blank cross-linked micelles (ch-Kn(s-s)R8, ch-Kn(s-s)R8-An, n ¼ 3, 5 and 7) were obtained after lyophilization. The specific absorption peaks of the disulfide bond range between 240 and 300 nm of different micelles was detected by ultraviolet and visible spectrophotometry (UV 2450/2550, Shimadzu, Japan). The particle size and zeta potential of blank micelles were measured in a PBS solution (pH 7.4) by dynamic light scattering (Zetasizer Nano ZS90, Malvern Instruments, Malvern, UK). The morphology of the ch-Kn(s-s)R8-An was examined using a transmission electron microscope (TEM) (Hitachi, Tokyo, Japan) at an acceleration voltage of 75 kV.

Critical micelle concentration (CMC)
The CMC of micelles was determined using a pyrene fluorescence method as described previously 32 . Briefly, pyrene solution (6 Â 10 À 5 mol/L in acetone) was transferred into a series of volumetric flasks and the acetone was evaporated under a gentle nitrogen gas stream for 4 h at room temperature. The ch-KnR8-An and ch-Kn(s-s)R8-An solutions in a series of concentrations ranging from 1 Â 10 À 5 to 2 mg/mL in deionized water were added into each flask with a final pyrene concentration of 6 Â 10 À 6 mol/L. The fluorescence spectrum was obtained using a F-7000 fluorescence spectrometer (Hitachi, Japan). Intensity ratios of the first (374 nm) to the third (384 nm) vibronic peaks (I 1 /I 3 ) were plotted as a logarithm function of the micelles concentration. The CMC value was taken from the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at low concentrations.

Preparation and characterization of micelles/DNA
pEGFP was used as a model plasmid. The ch-Kn(s-s)R8-An (n ¼ 3, 5 and 7) micelles were mixed with pEGFP plasmid (60 μL per 2 μg DNA) at different N/P ratios from 1:1 to 15:1 in 1 mL PBS (pH 7.4). The particle size and zeta potential of ch-Kn(s-s)R8-An/pEGFP at different N/P ratios were measured in a PBS solution (pH 7.4) by dynamic light scattering.

Agarose gel electrophoresis
The ability of the micelles to condense pDNA was determined by agarose gel electrophoresis. The ch-Kn(s-s)R8-An/pEGFP (n ¼ 3, 5 and 7) was prepared at different N/P ratios (0-5). After 30 min of incubation the samples were analyzed on a 1.0% agarose gel by staining with Gelred™ fluorescent dye and Tris-acetate EDTA buffer for 40 min at 4 1C. Then, the samples were electrophoresed at 100 V for 40 min. pDNA was visualized using a UV transilluminator. To compare the stability of the ch-Kn(s-s)R8-An/pEGFP under reductive environments, ch-Kn(s-s)R8-An/pEGFP at different N/P ratios in the presence of 100-fold DTT (mole ratio to ch-KnR8-An monomer) was analyzed using agarose gel electrophoresis. pEGFP bands were visualized under UV light. ch-Kn(s-s)R8-An/pEGFP stability was evaluated using a heparin competition assay. The ch-Kn(s-s)R8-An/pEGFP was prepared at an N/P ratio of 10. Then, increasing amounts of heparin were added to the micelles/pEGFP. After treatment for 1 h, the samples were assessed using agarose gel electrophoresis under the same conditions.

Gene transfection assay
HEK293 and U251 cells were seeded in 24-well plates at a density of 2 Â 10 5 and 5 Â 10 4 cells/well, respectively. After 24 h, when the cells achieved 70%-80% confluence, the culture medium was removed. Cells were exposed to fresh serum-free culture medium containing ch-Kn(s-s)R8-An/pEGFP with different N/P ratios and incubated for another 6 h, after which the medium was replaced with DMEM containing 10% FBS and cells were incubated an additional 42 h. The expression of pEGFP in cells was observed by fluorescence microscopy (Leica, CMS, GmbH, Germany) and quantified by flow cytometry (Becton Dickinson, San Jose, CA, USA). Naked pEGFP and bPEI-25K/pEGFP were used as controls. The experiment was repeated three times.

Cytotoxicity assay
The cytotoxicity of the ch-Kn(s-s)R8-An/pEGFP (n ¼ 3, 5 and 7) toward BCECs and U251 cells was evaluated by CCK-8 assay. BCECs and U251 cells in 100 μL DMEM containing 10% FBS were seeded in 96-well plates at a density of 8000 cells/well. After 24 h, the cells were transfected as described above and the medium was replaced with fresh culture medium containing various concentrations of ch-Kn(s-s)R8-An/pEGFP at an N/P ratio of 10. The final concentrations of the polymer ranged from 5-200 μg/mL. After 24 h, the medium was then replaced by 90 μL of fresh complete medium and 10 μL of CCK-8 solution. The cells were incubated for an additional hour. The absorbance of each well was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The absorbance of untreated cells was set at 100%, and cell viability was expressed as the percentage relative to the absorbance of the untreated cells. bPEI-25K/pEGFP were used as controls. The experiment was repeated four times.

2.7.
In vitro cellular uptake assay 2.7.1. Cellular uptake determined by flow cytometry HEK293 cells and BCECs were seeded in 12-well plates at a density of 3 Â 10 5 cells per well. Prior to preparation of the micelles/pEGFP at an N/P ratio of 10, pEGFP was labeled with YOYO-1 for 30 min. After replacing the culture medium, the ch-K5(s-s)R8-An/pEGFP-YOYO-1 solution was added to HEK293 cells and BCECs, with a final pEGFP-YOYO-1 concentration of 75 nmol/L. After 3 h of incubation, the cells were trypsinized, centrifuged, washed, and resuspended in PBS. The fluorescence intensity of cells was analyzed on a flow cytometer. bPEI-25k/ pEGFP-YOYO-1, ch-K5(s-s)R8/pEGFP-YOYO-1 and pEGFP-YOYO-1 were used as controls. The experiment was replicated three times.

BBB penetration in vitro
The transportation study was performed using an in vitro BBB model that was established using the method described previously. BCECs in 500 μL DMEM containing 10% FBS were seeded at a density of 1 Â 10 6 cells/mL on the upper side of 24-transwell plate filters. Then, 1 mL DMEM-free FBS was added in the lower side of the 24-transwell plate filters. The culture medium was replaced every 2 days. After 4 days, U251 cells were seeded in the lower side of the 24-transwell plate filters at a density of 2 Â 10 5 cells/well and grown overnight. To evaluate their ability as carriers across the BBB, bPEI-25k/pEGFP-YOYO-1, ch-K5(ss)R8/pEGFP-YOYO-1, and ch-K5(s-s)R8-An/pEGFP-YOYO-1 were added into the corresponding insert. In addition, to evaluate the effect of MMP-2 on U251 cellular uptake, MMP-2 was added into the lower side (the size and TEM images of ch-K5(s-s)R8-An micelles after MMP-2 treated was measured by the methods mentioned earlier) and ch-K5(s-s)R8-An/pEGFP-YOYO-1 were added into the upper side. After 12 h of incubation, the fluorescence intensity of cells was analyzed using flow cytometry. The experiment was replicated three times.

Confocal laser scanning microscopy (CLSM)
To investigate the cellular uptake profiles of micelles, 1 Â 10 5 cells/well U251 cells were seeded onto sterilized microscope cover slips placed in 24-well plate and incubated for 24 h. Seeded cells were then treated with naked pEGFP, bPEI-25K/pEGFP-YOYO-1, ch-K5(s-s)R8/pEGFP-YOYO-1 or ch-K5 (s-s)R8-An/pEGFP-YOYO-1 and incubated for 1, 2 or 3 h (untreated cells were used as control). Afterwards, the cells were washed with PBS and incubated with Lysotracker red for 30 min. Then the cells were washed with PBS, fixed in 4% paraformaldehyde, treated with DAPI for staining of the nucleus, sealed with mounting medium, and imaged using a confocal laser scanning microscope (TCS SP8, Leica, Wetzlar, Germany).

Colony formation assay
The X-ray radiation treatment was performed at increasing doses from 0 to 4 Gy, as reported previously. Briefly, U251 cells in DMEM containing 10% FBS and an appropriate amount of MMP-2 were seeded into 6-well plates at 400 cells mL/2 mL. After 24 h, cells were treated with ch-K5(s-s)R8-An, Dbait and ch-K5(s-s)R8-An/Dbait. After 6 h, cells were irradiated with 0, 2 or 4 Gy, respectively (dose rate ¼ 0.3 Gy/min). About 14 days later, cell clones were fixed with ice-cold acetic acid and incubated for 15 min, then stained with crystal violet. All colonies with over 50 cells were counted and the data were analyzed using Graph Pad Prism software. Untreated cells were considered as controls. The plating efficiency (PE) was calculated by dividing the number of colonies by the number of cells plated.

Cell apoptosis assay
In order to determine the rate of apoptosis, U251 cells in DMEM containing 10% FBS and an appropriate amount of MMP-2 were seeded in 12-well plates at a density of 1.5 Â 10 5 cells/well. After 24 h, cells were treated with ch-K5(s-s)R8-An, Dbait and ch-K5(ss)R8-An/Dbait. After 6 h, cells were irradiated with 2 Gy (dose rate ¼ 0.3 Gy/min). After 48 h, the cells were washed twice with ice-cold PBS, trypsinized, centrifuged and then re-suspended in cold binding buffer. The cells were stained with Annexin V-APC and propidium iodide (PI), followed by incubation in the dark for 10 min. A total of at least 10,000 cells from each sample were analyzed using an analytical flow cytometer (FACSCalibur; BD Biosciences, UK). Untreated cells were used as controls. The tests were carried out in triplicate.

Western blot
U251 cells were seeded and treated as described above in the cell apoptosis assay. After 48 h, cells were harvested and resuspended in RIPA lysis buffer with protease inhibitor and phosphatase inhibitor, followed by incubation on ice for 30 min. The lysates were centrifuged at 12,000 rpm (Thermo ST40, USA) for 10 min at 4 1C, the supernatants were collected and examined for protein concentrations using the BCA protein assay kit (Thermo, USA). Proteins were separated by SDS-PAGE, electro-transferred to polyvinylidene fluoride membrane (Millipore, USA), and blocked using 5% non-fat dried milk at RT for 1 h. Membranes were incubated overnight at 4 1C with the following primary antibodies: γ-H2AX (Abcam, UK), DNA-PKcs (Bioworld, China), phosphorylated p53 (Ser15) (Abcam, UK) and GAPDH (Bioworld, China). After washing, membranes were then incubated with HRP-conjugated secondary antibodies (Cell Signaling, USA) for 1 h at room temperature. Visualization was performed as previously described.

In vivo biodistribution
Micelles/pEGFP-YOYO-1 were used to investigate the distribution of NPs in U251 orthotropic GBM-bearing nude mice, as described previously. Male nude mice were divided randomly into three groups. Each mouse was anesthetized with 1% pelltobarbitalum natricum and placed in a stereotaxic apparatus. U251 cells (1 Â 10 5 cells/5 μL) were then injected into the right brain of each mouse (1 mm lateral to bregma and 3.0 mm deep from the dura). Two weeks later, tumorbearing mice were injected intravenously through the tail vein with 200 μL of PBS, YOYO-1, ch-K5(s-s)R8/pEGFP-YOYO-1, or ch-K5 (s-s)R8-An/pEGFP-YOYO-1 at a dose of 50 mg pEGFP per mouse. After 24 h, mice were sacrificed and the principal organs (heart, liver, spleen, lung, kidneys, and brain) were carefully removed. Biodistribution was evaluated using in vivo fluorescence imaging (Xenogen IVIS-200, Caliper Life Sciences, Hopkinton, MA, USA).

In vivo safety assay
BALB/c mice were divided randomly into 2 groups and injected with PBS and blank ch-K5(s-s)R8-An micelles through the tail vein at a dose of 30 mg/kg every 2 days totaling 4 injections. The mice were sacrificed 24 h after the last injection to excise the heart, liver, spleen, lung, kidney, and brain. The excised organs were fixed in 4% paraformaldehyde, and then subjected to hematoxylin and eosin (H&E) staining.

Statistical analysis
All values are presented as the means7standard deviation (SD). One-way analysis of variance (ANOVA) was used for the statistical analysis. The differences were considered significant for * P o 0.05.

Synthesis and characterization of polymers
As a self-assembling block copolymer for the core-shell polymer micelles, the ch-KnR8 (n ¼ 3, 5 and 7) and ch-KnR8-An (n ¼ 3, 5 and 7) copolymers were synthesized via the F-moc-solid phase peptide synthesis method (Scheme 1A) and a subsequent purification process. The copolymers were synthesized at 95% purity and the precise molecular weight of ch-K5R8 and ch-K5R8-An are shown in ESI Supporting Information Table S1. The MALDI-TOF-MS data demonstrate that ch-KnR8-An (n ¼ 3, 5 and 7) was successfully synthesized.

Preparation and characterization of blank micelles
Ch-KnR8 self-assembled and formed a core-shell-corona polymer micelle with three well-defined distinct domains: the inner hydrophobic cholesterol core, the cationic Kn middle shell, and the cationic R8 outer corona. For cross-linked micelle formation, DTSSP with activated bifunctional NHS groups could react with amino groups of the pLys segment to form intramicellar disulfide linkages 31 . Cross-linked micelles ch-Kn(s-s)R8-An (n ¼ 3, 5 and 7) were confirmed to contain disulfide bonds as demonstrated with an absorption peak of 270 nm (Supporting Information Fig. S1). The micelle size and zeta potential are shown in Table 1. The micelles maintained a reasonable size (80-160 nm) with a moderate distribution and a decreased mean diameter from the crosslinking, which may facilitate the accumulation of micelles in tumor tissues. The zeta potential of the blank micelles was different, ranging from 15 to 40 mV. Notably, after the cross-linking, the values of the positive zeta potential of ch-KnR8 and ch-KnR8-An were decreased. Among the micelles, ch-K5(s-s)R8-An is presented as an example to show an appropriate size and zeta potential ( Fig. 1A and B), with a particle size and zeta potential of 119.574.31 nm and 25.072.70 mV, respectively. Fig. 1A presents the TEM images of the ch-K5(s-s)R8-An micelle. It shows that the micelle assumed a spherical morphology and almost homogeneous distribution.

The CMC of micelles
To confirm the formation of micelles self-assembled from the ch-KnR8-An block copolymer, the CMC values of ch-KnR8-An and ch-Kn(s-s)R8-An (n ¼ 3, 5 and 7) micelles were measured by fluorescence spectroscopy. As the micelle concentration increased, the intensity ratio exhibited a substantial decrease at a certain concentration, suggesting that pyrene molecules were incorporated into the hydrophobic core region during micelle formation. Therefore, CMC values were determined from the crossover point at the low concentration. As shown in Fig. 1C and D, the CMC values of ch-KnR8-An and ch-Kn(s-s)R8-An (n ¼ 3, 5 and 7) micelles were approximately 0.00794, 0.00891, 0.0138, 0.00750, 0.00116 and 0.00284 mg/mL, respectively. The cross-linked micelles had lower CMC values compared with the non-crosslinked ones and K5(s-s)R8-An had the lowest CMC value, indicating that they had the best stability in solution.

Stability of ch-Kn(s-s)R8-An
The kinetic stability of ch-Kn(s-s)R8-An micelles were further assessed in PBS (pH 7.4) with or without 10% FBS. As shown in Supporting information Fig. S2, ch-Kn(s-s)R8-An micelles maintained their size in a range of 90-160 nm and zeta potential in a range of 10-40 mV in PBS (pH 7.4) with or without 10% FBS for up to 24 h, and there is no significant differences between two kinds of medium, indicating that the micelles are stable in serum.

Preparation and characterization of micelles/DNA
In the present study, the particle size and zeta potential of ch-Kn(ss)R8-An/pEGFP (n ¼ 3, 5 and 7) with different N/P ratios are shown in Fig. 1E. All ch-Kn(s-s)R8-An/pEGFP carried a positive surface charge with zeta potential ranging from 10 to 30 mV when the N/P ratio was higher than 5. Fig. 1F shows that the particle size of the ch-Kn(s-s)R8-An/pEGFP was less than 200 nm at an N/P ratio higher than 2.5. When the N/P ratio was higher than 5, most ch-Kn(s-s)R8-An/pEGFP had a higher zeta potential and a smaller particle size.

Agarose gel electrophoresis
Fig . 1G shows the result of agarose gel electrophoresis for pDNA binding affinity for ch-Kn(s-s)R8-An/pEGFP (n ¼ 3, 5 and 7). pEGFP was retarded completely at an N/P ratio of 5, 3, and 2 for ch-Kn(s-s)R8-An/pEGFP (n ¼ 3, 5 and 7) respectively, whereas pEGFP was not completely retarded at an N/P ratio of 5 for ch-K3R8-An/pEGFP and was retarded at an N/P ratio of 5 and 3 for ch-KnR8-An/pEGFP (n ¼ 5, 7), indicating that the binding affinity of ch-Kn(s-s)R8-An was improved by disulfide bond  cross-linking. To verify this finding, DTT was used as a reducing agent to break the disulfide bond 33 . As shown in Fig. 1G, ch-Kn(ss)R8-An/pEGFP (n ¼ 3, 5 and 7) had weaker pEGFP binding affinity in the presence of DTT due to depolymerization of ch-Kn (s-s)R8-An. The stability of the ch-Kn(s-s)R8-An/pEGFP (n ¼ 3, 5 and 7) was measured by heparin competition assays (Fig. 1H). Increasing amounts of heparin were added to the ch-Kn(s-s)R8-An/pEGFP (n ¼ 3, 5 and 7) solution and dissociation of the ch-Kn (s-s)R8-An/pEGFP (n ¼ 3, 5 and 7) was measured by gel electrophoresis. Ch-K3(s-s)R8-An released pEGFP in the presence of 5 μg heparin whereas ch-K5(s-s)R8-An and ch-K7(s-s)R8-An released pEGFP upon the addition of 10 μg heparin. In addition, with the amount of heparin increasing, pEGFP was replaced to a greater degree. These results suggest that ch-K5(s-s)R8-An/pEGFP and ch-K7(s-s)R8-An/pEGFP were more stable than ch-K3(s-s) R8-An/pEGFP.

Gene transfection assay
Gene transfection efficiency of the micelles in HEK293 and U251 cells was evaluated using pEGFP as the model plasmid. Naked pEGFP and bPEI-25K were used as controls. The result showed that the gene transfection efficiency was dependent on the N/P ratio as observed for HEK293 cells ( Fig. 2A). As shown in Fig. 2A, naked pEGFP exhibited the lowest gene transfection efficiency, and ch-Kn(s-s)R8-An/pEGFP (n ¼ 5 and 7) showed significantly higher EGFP fluorescence intensities than ch-K3(s-s) R8-An/pEGFP at N/P ratios of 5, 7.5, 10, and 15. The fluorescence intensity of ch-Kn(s-s)R8-An/pEGFP (n ¼ 5 and 7) at an N/P ratio of 10 was comparable with that of bPEI-25K/pEGFP. In addition, the EGFP fluorescence intensity following transfection by ch-Kn (s-s)R8-An/pEGFP (n ¼ 5 and 7) and bPEI-25K/pEGFP at an N/P ratio of 10 in U251 cells was also higher than that of ch-K3(s-s) R8-An/pEGFP and naked pDNA (Fig. 2B). Flow cytometry assay determination of the mean fluorescence intensity in U251 cells also confirmed this tendency (Fig. 2C), most probably because of the enhanced gene compression of the ch-Kn(s-s)R8-An/pEGFP (n ¼ 5 and 7) 34 . Notably, ch-Kn(s-s)R8-An/pEGFP (n ¼ 5 and 7) showed optimal gene transfection efficiency at an N/P ratio of 10.
3.6. In vitro cellular uptake assay 3.6.1. Cellular uptake determined by flow cytometry The in vitro internalization efficiency was evaluated using a cellular uptake study. As shown in Fig. 3A, cellular uptake efficiency was significantly higher for ch-K5(s-s)R8 than that for pEGFP, bPEI-25K, and ch-K5(s-s)R8-An in HEK293 cells. Specifically, mean fluorescence intensity of ch-K5(s-s)R8/pEGFP was approximately 2.50-fold and 2.15-fold higher than that of ch-K5(s-s)R8-An/pEGFP and bPEI-25k/pEGFP, respectively (both P o 0.05). At the same time, cellular uptake efficiency of ch-K5(s-s)R8/pEGFP and ch-K5(s-s)R8-An/ pEGFP was significantly higher than that for bPEI-25K/pEGFP and naked pEGFP in BCECs (Fig. 3B). Here, mean fluorescence intensity of ch-K5(s-s)R8/pEGFP and ch-K5(s-s)R8-An/pEGFP was approximately 3.17-fold and 3.36-fold higher than that of bPEI-25k/pEGFP (P o 0.05). These flow cytometric analysis results indicate that the cholesterol hydrophobic moiety enhanced the membrane affinity for the micelles/pEGFP. In addition, compared with the non-targeting ch-K5(s-s)R8/pEGFP, the targeting ch-K5(s-s)R8-An/pEGFP entered BCECs much more efficiently as represented by significantly intensified intracellular fluorescence in BCECs which was the opposite for HEK293 cells. These findings may be due to surface modifications of the micelles. Specifically, non-targeting micelles with a polyarginine shell were positively charged and could better penetrate the cell membrane, albeit not selectively. However, Angiopep-2 recognized and bound to LPR1 that was overexpressed on the BCECs, thus retaining more pEGFP in the BCECs.

CLSM
To investigate the cellular uptake profiles of micelles, CLSM was performed to analyze the cellular uptake of micelles/pEGFP-YOYO-1 in U251 cells at different time point. As shown in Fig. 4, the fluorescence intensity of pEGFP-YOYO-1 transfected by ch-K5(s-s) R8/pEGFP-YOYO-1 and ch-K5(s-s)R8-An/pEGFP-YOYO-1 was higher than that of pEGFP-YOYO-1 and bPEI-25K/pEGFP-YOYO-1 in U251 cells. Therefore, these CLSM images confirmed that pEGFP was simultaneously delivered into cells by the micelles, which is consistent with the result of the cellular uptake assay. In addition, after 1 h of incubation with micelles/pEGFP-YOYO-1, the red fluorescence (lysosomes) and pEGFP-YOYO-1 were overlapped. The yellow fluorescence, resulting from the overlay of red and green fluorescence in the merged image, indicated that pEGFP was effectively delivered into U251 cells. After 3 h of incubation, the green fluorescence was observed in the nuclear and perinuclear regions of the cytoplasm, indicating that pEGFP-YOYO-1 escaped from the endosome and entered the nucleus successfully. There was no significant difference between the control and the ch-K5(s-s)R8-An group, suggesting that ch-K5(s-s)R8-An exhibited no radiosensitization, and that the radiosensitization of ch-K5 (s-s)R8-An/Dbait was derived from the Dbait. At a single irradiation dose of 2 Gy, the viability of U251 cells in the control, ch-K5(s-s)R8-An, Dbait and the ch-K5(s-s)R8-An/Dbait group was 94.07%, 89.29%, 81.35% and 72.90%, respectively. U251 cell viability was decreased significantly in ch-K5(s-s)R8-An/ Dbait group at a low irradiation dose. This may be attributable to the sustained release of Dbait from ch-K5(s-s)R8-An/Dbait, and the enhanced cellular uptake of Dbait delivered by micelles.

Colony formation assay
Knowing that clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to form colony 37 , we performed a clonogenic assay to determine the radiosensitization effect of ch-K5(s-s)R8-An/Dbait by cell growth inhibition. As shown in Fig. 5B, the plating efficiency in ch-K5(s-s)R8-An, Dbait and ch-K5(s-s)R8-An/Dbait groups without RT was similar to the control group, suggesting that they had no inhibitory effect on colony formation in U251 cells. In contrast, the plating efficiency of ch-K5(s-s)R8-An/DbaitþRT group was decreased significantly at 2 Gy and 4 Gy (41.0372.88% and 26.3273.34%) as compared with the other groups (P o 0.05), suggesting that ch-K5(s-s)R8-An/Dbaitþlow-dose RT could effectively enhance the radiosensitization. In summary, the rapid internalization and sustained release of Dbait from micelles may substantially improve the anticancer effect of RT at the same dose.
Knowing that high-dose RT would impair the cognitive function and other normal brain functions 5,38 , we chose the low-dose (2 Gy) RT for the subsequent experiments.

Cell apoptosis assay
Apoptosis is an important indicator of DNA damage caused by ionizing radiation 13 . To investigate the effect of ch-K5(s-s)R8-An/ Dbait þ RT on the apoptosis of U251 cells, Annexin-V binding and PI staining assays were performed using a flow cytometer. U251 cells showed an early apoptosis level of 1.16% and a late apoptosis level of 1.10% in the control group (Fig. 5C). This type of apoptosis is normal for untreated cells since these cells would likely have undergone apoptosis under these growth conditions 37

Western blot analysis
In higher eukaryotic cells, the appearance of γ-H 2 AX in the cell nucleus is often used as an indicator of the presence of DNA DSBs 39,40 . γ-H 2 AX foci are formed at DSB damage sites and resolved when the damage is repaired 41 . Therefore, we used Western blot analysis to see whether ch-K5(s-s)R8-An/Dbait þ RT could increase γ-H 2 AX in U251 cells. As shown in Fig. 6A, U251 cells treated with ch-K5(s-s)R8-An/Dbait þ RT expressed significantly higher levels of γ-H 2 AX compared with non-treated cells. The level of γ-H 2 AX induced by ch-K5(s-s)R8-An/Dbait combined with 2 Gy RT was seven-fold higher than that induced by 2 Gy RT alone, and two times higher than that induced by Dbaitþ2 Gy RT ( Fig. 6A and B). This suggests that the DNA repair ability of U251 cells was decreased after treatment with ch-K5(s-s)R8-An/Dbaitþ2 Gy RT. We also examined the effect of ch-K5(s-s)R8-An/Dbaitþ2 Gy RT on other DNA repair proteins including DNA-PKcs and phospho-p53, and found that ch-K5(s-s)R8-An/Dbaitþ2 Gy RT also induced relatively high levels of DNA-PKcs and phospho-p53 in U251 cells (Fig. 6A, C and D). These results indicate that ch-K5 (s-s)R8-An/Dbait þ RT could inhibit DNA repair, which in turn improved the RT efficacy.

In vivo biodistribution
The ch-K5(s-s)R8/pEGFP-YOYO-1 and ch-K5(s-s)R8-An/ pEGFP-YOYO-1 were injected intravenously into U251 orthotropic GBM-bearing nude mice to trace in vivo distribution and tumor-targeting characteristics. Fig. 7A shows the fluorescence intensity in the whole body at 1, 12, and 24 h after injection. The ch-K5(s-s)R8-An/pEGFP-YOYO-1 group (d) exhibited much stronger fluorescent signals and a directional aggregation ability at the brain tumor site as compared with the YOYO-1 (b) and ch-K5(s-s)R8/pEGFP-YOYO-1 (c) treated group, which displayed less fluorescence in the other organs tested. In the control group (b and c), YOYO-1 accumulated in other parts of the body. The excised organs were sectioned 24 h after injection to further study the in vivo micelle location. As shown in Fig. 7B, ch-K5(s-s)R8- Figure 4 Cellular uptake analysis of micelles/pEGFP-YOYO-1 using CLSM, naked YOYO-1-labeled pEGFP (green) was used as blank control. U251 cells, with an incubation time of 1, 2 and 3 h, and Lysotracker Red was used to label the lysosomes. Scale bar, 20 μm.
An/pEGFP-YOYO-1 (d) showed a high level of accumulation at the boundary and interior of the tumor; whereas YOYO-1 (b) and ch-K5(s-s)R8/pEGFP-YOYO-1 (c) accumulated mainly in the liver with less fluorescence in the brain. Thus, brain-targeting ch-K5(s-s)R8-An/pEGFP-YOYO-1 accumulation at the tumor site was markedly increased and the accumulation in the lungs, spleen, and other organs was substantially decreased. The results indicate that angiopep-2 modification could effectively enhance micelle accumulation in the tumor due to its positive brain-targeting characteristics.

In vivo toxicity assay
After staining with H&E (Fig. 7C), the images showed that the results of the heart, liver, spleen, lung, kidney and brain tissues in the group receiving blank ch-K5(s-s)R8-An micelles were similar to those of the PBS control group with no apparent inflammatory response or damage being observed in any of the blank-micelletreated group. This indicates that blank ch-K5(s-s)R8-An micelles had no obvious systemic toxicity.

Discussion
To overcome the insensitivity of glioma cells to RT, Dbait was used as a radiosensitizer to increase the sensitivity of U251 cells to RT. In the present study, we have developed a novel BBB and glioma dual-targeting and tumor microenvironment-responsive gene delivery system by using an MMP-2 responsive peptide as the enzymatically degradable linker to conjugate angiopep-2 and incorporated disulfide cross-linking in the middle shells to enhance cellular internalization and DNA release into the cytoplasm and nucleus.
Ch-Kn(s-s)R8-An forms core-shell-corona polymer micelles with three well-defined distinct domains: the inner hydrophobic cholesterol core, the cationic KnR8 middle shell, and the angiopep-2 targeting ligand in the outer corona. Successful disulfide cross-linking of ch-Kn(s-s)R8-An was detected through the characteristic absorption peaks at 270 nm in UV spectra (Supporting Information Fig. S1). Cross-linking decreased the mean diameter of the micelles, indicating a condensed structure caused by chemical network formation in the micelle middle shells (Table 1). Notably, after cross-linking, the positive zeta potential of ch-KnR8 and ch-KnR8-An was decreased. This decreased zeta potential reflects the conversion of primary amine groups of the middle pLys shells to amide linkages 30 . This result also demonstrated that the successful disulfide cross-linking of ch-Kn(s-s)R8-An. As suggested previously, cross-linking could overcome the drawbacks of non-crosslinked micelles by decreasing the CMC of micelles, thus endowing them with more useful properties and performance for biomedical applications 42 . In the study, DTSSP was used as a disulfide-containing cross-linking agent to obtain the cross-linked micelles. The CMC results showed that the crosslinked micelles were compact and intact in an aqueous solution ( Fig. 1C and D). The CMC values for non-crosslinked micelles increased gradually along with the lysine concentration increasing, probably due to the increased electrostatic repulsion between lysine moieties. However, the decrease in CMC values of crosslinked micelles with the amount of lysine increasing might be caused by the higher degree of cross-linking. Furthermore, the particle size and zeta potential did not change significantly with the lapse of time in PBS (pH 7.4) with or without 10% FBS (Supporting Information Fig. S2). These results indicated that the cross-links in the middle shells may not only enhance the stability of micelles in the blood but reduce drug leakage before they reach the target tissue 30 .
In this study, we have explored the possibility of gene delivery across the BBB by brain-targeted drug delivery system based on ch-Kn(s-s)R8-An micelles to treat glioma. The rich external amine groups on the surface of micelles offer plenty reaction sites for functional modifications as well as positive charge for therapeutic gene encapsulation. The results of agarose gel electrophoresis suggest that disulfide cross-linked ch-K5(s-s)R8-An/pEGFP and ch-K7(s-s)R8-An/pEGFP were more stable, probably due to rich positive charges (Fig. 1G). In addition, ch-Kn(s-s)R8-An/pEGFP (n ¼ 3, 5 and 7) showed a weaker pEGFP binding affinity in a reducing environment (Fig. 1H). As reported, the reductive glutathione concentration in the cytosol of tumor cells was 100-1000 times higher than that in normal cells 43 . Therefore, the presence of disulfide bonds may act as an effective means to maintain stability in the extracellular context and promote the release of the gene into tumor cells. In addition, the in vitro gene expression of different micelles/pEGFP in HEK293 and U251 cells showed that ch-Kn(s-s)R8-An/pEGFP (n ¼ 5 and 7) displayed an optimal gene transfection efficiency at an N/P ratio of 10, most probably because of enhanced gene compression ( Fig. 2A-C).
According to previous reports 44 , classical cationic polymers such as PEI generally exhibit a certain degree of heterogeneity because of the nature of the polymer chemistry. However, in the present study, ch-Kn(s-s)R8-An/pEGFP had more favorable biocompatibility with both cell lines than PEI (Fig. 2D and E). In addition, histological examination also indicated that the blank ch-K5(s-s)R8-An micelle had no significant systemic toxicity (Fig. 7C) which is consistent with the in vitro result. This benefit may derive from the micelles in the material, including cholesterol, lysine and arginine, all of which are known to have good biocompatibility as has been confirmed in clinical settings.
Angiopep-2 has been reported to play a key role in targeting the BBB and glioma 18,45 . In this study, the angiopep-2 modified micelle entered the cells much more efficiently in BCECs but not in HEK293 cells, compared with the angiopep-2 non-modified micelle (Fig. 3). In addition, BBB penetration of Angiopep-2 modified micelle treated with MMP-2 was significantly enhanced. Linker MMP-2 responsive peptides are broken in the presence of MMP-2 to expose the R8. This simultaneously reduces the particle size and drives the micelles into glioma cells with up-regulated MMP-2. Non-modified micelles with an R8 shell exhibited the ability to penetrate the cell membrane. However, without tumor targeting and selectivity, the R8 shell had lower cellular uptake into tumor cells in vivo 46 . For example, CPPs initially binds to the local vasculature and redistributes to the liver after intravenous administration, where more than 90% of the injected dose accumulates after 30 min 47 . In this study, non-modified micelles accumulated mainly in the liver with less fluorescence in the brain. It is probably that long-circulating decoration helps to divert a fraction of nanoparticles to peripheral tissues, but a large fraction of nanoparticles are still captured by the macrophage-rich organs 48,49 . In addition, these results indicate that modification of angiopep-2 played a role in targeting the BBB, and that cleavage of angiopep-2 subsequently enhanced internalization in tumor cells. Also, the in vivo imaging results provided convincing evidence that this tumor-triggered mechanism makes the delivery system increase the pDNA content at the glioma site and decrease the pDNA content in the macrophage-rich organs such as liver ( Fig. 7A and B).
In our study, ch-K5(s-s)R8-An was used as a delivery nanocarrier to transport the radiosensitizer Dbait to U251 cells. A previous study reported by Yao et al. 41 showed that upon irradiation, the Dbait molecule could further improve the RT efficacy for PCa cell lines. For the first time, we demonstrated that ch-K5(s-s)R8-An/DbaitþRT was able to increase the sensitivity of U251 cells to RT, inhibited colony formation in U251 cells, and promoted cell apoptosis (Fig. 5). Moreover, Western blot analysis showed that ch-K5(s-s)R8-An/Dbait þ RT inhibited the DNA damage repair signaling pathway by mimicking DNA doublestrand breaks (Fig. 6), which is consistent with previous studies 41 . Thereby, ch-K5(s-s)R8-An/Dbait combined with RT exerts a strong antitumor phenotype by impairment of proliferation and induction of apoptosis in tumor cells.

Conclusions
In summary, we have developed a novel dual-targeting and microenvironment-responsive micelle (ch-Kn(s-s)R8-An/pDNA) for gene delivery during glioma treatment. The microenvironmentresponsive micelles showed higher gene transfection efficiency, improved brain-penetrating and glioma-targeting efficiency, better biocompatibility and improved therapeutic efficacy during glioma radiotherapy in vitro. In vivo study also demonstrated good micelleglioma targeting with no obvious toxicity toward normal tissues. All these results demonstrate the tremendous potential of this tumor- triggered micelle system for efficient delivery of gene therapeutic agents during oncotherapy. Further work will focus on investigation and development of ch-K5(s-s)R8-An/Dbait as a radiosentizer in glioma treatment in vivo.