PEGylated Polyamidoamine dendrimer conjugated with tumor homing peptide as a potential targeted delivery system for glioma

Colloids and Surfaces B: Biointerfaces 147 (2016) 242–249

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PEGylated Polyamidoamine dendrimer conjugated with tumor homing peptide as a potential targeted delivery system for glioma Yan Jiang 1 , Lingyan Lv 1 , Huihui Shi, Yabing Hua, Wei Lv, Xiuzhen Wang, Hongliang Xin ∗ , Qunwei Xu ∗ Department of Pharmaceutics, School of Pharmacy, Nanjing Medical University, Nanjing, 211166, China

a r t i c l e

i n f o

Article history: Received 14 June 2016 Received in revised form 30 July 2016 Accepted 2 August 2016 Available online 3 August 2016 Keywords: Glioblastoma Polyamidoamine dendrimer Interleukin 13 receptor ␣2 Pep-1 peptide Targeted nanocarrier

a b s t r a c t Glioblastoma multiforme (GBM) is the most common and aggressive primary central nervous system (CNS) tumor with a short survival time. The failure of chemotherapy is ascribed to the low transport of chemotherapeutics across the Blood Brain Tumor Barrier (BBTB) and poor penetration into tumor tissue. In order to overcome the two barriers, small nanoparticles with active targeted capability are urgently needed for GBM drug delivery. In this study, we proposed PEGylated Polyamidoamine (PAMAM) dendrimer nanoparticles conjugated with glioma homing peptides (Pep-1) as potential glioma targeting delivery system (Pep-PEG-PAMAM), where PEGylated PAMAM dendrimer nanoparticle was utilized as carrier due to its small size and perfect penetration into tumor and Pep-1 was used to overcome BBTB via interleukin 13 receptor ␣2 (IL-13R␣2) mediated endocytosis. The preliminary availability and safety of Pep-PEG-PAMAM as a nanocarrier for glioma was evaluated. In vitro results indicated that a significantly higher amount of Pep-PEG-PAMAM was endocytosed by U87 MG cells. In vivo fluorescence imaging of U87MG tumor-bearing mice confirmed that the fluorescence intensity at glioma site of targeted group was 2.02 folds higher than that of untargeted group (**p < 0.01), and glioma distribution experiment further revealed that Pep-PEG-PAMAM exhibited a significantly enhanced accumulation and improved penetration at tumor site. In conclusion, Pep-1 modified PAMAM was a promising nanocarrier for targeted delivery of brain glioma. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Glioblastoma multiforme (GBM) has been considered as an incurable disease which is the most aggressive brain and central nervous system (CNS) tumor with a median survival of 14.6 months [1]. Chemotherapy plays an important role in auxiliary treatment for glioma, however, several physiologic barriers including blood–brain barrier (BBB), blood–brain tumor barrier (BBTB) and poor penetration into glioma tissue lead to the failure [2]. The BBB serves as a major impediment to effective delivery of many therapies from the bloodstream into the brain parenchyma [3]. During the last decades, to deliver therapeutic agents across the BBB, some strategies have been adopted such as receptor-mediated endocytosis, adsorptive-mediated transcytosis, transporter uptake and membrane permeation of lipophilic molecules [4]. Meanwhile, to overcome BBTB, numerous strategies to improve the delivery

∗ Corresponding authors. E-mail addresses: [email protected] (H. Xin), [email protected] (Q. Xu). 1 These authors contributed equally to this manuscript. http://dx.doi.org/10.1016/j.colsurfb.2016.08.002 0927-7765/© 2016 Elsevier B.V. All rights reserved.

of agents across it and increase the uptake at tumor site have been developed, including osmotic blood–brain barrier disruption, bradykinin receptor-mediated BBTB opening, inhibition of multidrug efflux transporters, receptor-mediated transport systems and physiological circumvention of the BBTB [2]. As for drug delivery system, only nanocarriers smaller than 12 nm can pass fenestrated microvessels constituting BBTB [5]. Polyamidoamine (PAMAM) dendrimer is one of the most studied starburst macromolecules [6], which is based on an ethylene diamine core and an amidoamine repeat branching structure [7]. PAMAM has been most widely considered for biomedical use because of several characteristics including hyperbranch, monodispersion, threedimension and having defined molecular weight and host-guest entrapment properties [8]. Concretely speaking, PAMAM has a high amino group density, empty internal cavities and many functional end groups, which are responsible for high solubility and reactivity. Accordingly, the use of PAMAM as carriers for the delivery of drugs has attracted increasing attention [9–12]. More importantly, PEGylation of PAMAM which is synthesized in well-defined sizes (3–12 nm) can prolong the in vivo circulation

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time [13] and significantly decrease the toxicity [14,15] due to the low reticuloendothelial system (RES) recognition. As we know, the enhanced permeability and retention (EPR) effect is much weaker than that of peripheral tumors owing to the special glioma microenvironment [16]. Besides, nanoparticles larger than 200 nm are mainly recognized and non-specific uptake by reticuloendothelial system [17–19]. Therefore, the PEGylation of PAMAM nanoparticles would be more useful for glioma drug delivery because of the super fine particle size and good penetration ability into glioma tissue. Interleukin-13 plays a major role in regulating immune responses and immune microenvironment not only during normal physiological conditions but also in cancer [20,21]. The interleukin13 receptor ␣2 (IL-13R␣2), one of the subunits of the interleukin-13 receptor, is overexpressed in established glioma cell lines and primary glioblastoma cell culture [22–24], thus making it an important target for glioma therapy. Pep-1, a linear peptide with 9 amino acid residues (CGEMGWVRC), is a specific ligand of IL-13R␣2 [25], which can bind to IL-13R␣2 with high affinity and specificity, so as to target glioma via IL-13R␣2-mediated endocytosis [26,27]. In this study, dendrimer nanoparticles modified with Pep-1 (Pep-PEG-PAMAM) were developed, where Pep-1 was used as an anchor to enhance delivery across the BBTB and home to glioma and PEGylated PAMAM was used as drug carrier with small size and good penetration into tumor. In order to demonstrate the targeting efficacy of Pep-PEG-PAMAM, FITC and Cy5.5 were used as in vitro and in vivo tracer, respectively. In vitro, the cellular uptake and localization in U87MG cells were investigated, as well as the cytotoxicity. At last, the in vivo fluorescence imaging of GBM bearing nude mice and the glioma distribution were further observed to evaluate the targeting efficiency of Pep-PEG-PAMAM.

2. Materials and methods 2.1. Materials The PAMAM G5 was purchased from Weihai CY Dendrimer Technology Co., Ltd (Shandong, China). Pep-1 (CGEMGWVRC) peptide was synthesized by GL Biochem Co., Ltd (Shanghai, China). Mal-PEG-NHS was obtained from Jenkem Technology Co., Ltd (Beijing, China). FITC was from Aladdin (Shanghai, China). Cy5.5 was from Fanbo Biochemicals Co., Ltd (Beijing, China). DMEM medium, fetal bovine serum (FBS) and trypsin solution were bought from Gibco BRL (Gaithersberg, MD, USA). The other chemical reagents were of analytical grade and used as received.

2.2. Animals and cell line Balb/c nude mice (male, 4–5 weeks, 20 ± 2 g) were obtained from BK Lab Animal Ltd. (Shanghai, China) and housed at 25 ± 1 ◦ C with free access to food and water. All animal experiments were performed in accordance with protocols evaluated and approved by the ethics committee of Nanjing Medical University. U87MG cells (Human malignant glioblastoma cells) were obtained from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and BCEC cells (rat’s brain capillary endothelial cell line) were kindly gifted by Fudan University. The cell lines were cultured in DMEM medium, and supplemented with 10% (v/v) FBS, 100 U/mL penicillin and 100 U/mL streptomycin at 37 ◦ C in a humidified atmosphere of 5% CO2 .

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2.3. Preparation of Pep-1 conjugated PAMAM dendrimer nanoparticle (Pep-PEG-PAMAM) In a typical synthesis, PAMAM (1.1 ␮mol) was dissolved in NaHCO3 buffer (pH 9.0, 0.2 M, 1 mL) followed by adding FITC solution (4.88 ␮mol in 0.2 mL of DMSO) under stirring at room temperature. After 1 h, the reaction mixture was then dialyzed against water using a dialysis membrane with MWCO of 14000 for 2 days to remove unconjugated FITC, followed by lyophilization to obtain the product PAMAM-FITC. For the Cy5.5 labeling, 23.1 mg PAMAM-FITC was dissolved in NaHCO3 buffer (pH 8.0, 0.1 M, 1 mL) followed by adding Cy5.5 solution (0.1 ␮mol in 0.1 mL of DMSO) under stirring. The mixture was reacted for 2 h at the room temperature and then dialyzed against water using a dialysis membrane with MWCO of 14000 for 2 days to remove unconjugated Cy5.5, followed by lyophilization to obtain the product PAMAM-FITC/Cy5.5. For PEGylation of PAMAM, Mal-PEG-NHS (6.9 mg in 60 ␮L of DMSO) was dissolved in PBS buffer (pH 8.0, 0.01 M, 3 mL) in the presence of 5.5 mg PAMAM-FITC/Cy5.5 under stirring. After 1 h, the reaction mixture was then dialyzed against water using a dialysis membrane with MWCO of 14000 for 2 days, followed by lyophilization to obtain the product Mal-PEG-PAMAM-FITC/Cy5.5. Finally, for the Pep-1 modification, Mal-PEG-PAMAMFITC/Cy5.5 (5.4 mg) was mixed with Pep-1 peptide (1.8 mg) in PBS buffer (pH 7.4, 0.01 M, 4 mL) and reacted for 2 h at the room temperature. The mixture was then dialyzed against water using a dialysis membrane with MWCO of 14000 for 2 days, followed by lyophilization to obtain the product Pep-PAMAM-FITC/Cy5.5. 2.4. Characterization of Pep-PEG-PAMAM 2.4.1. 1 H NMR, particle size and zeta potential 1 H NMR spectra of the dendrimer was recorded on a Bruker AV400 nuclear magnetic resonance spectrometer. The particle size and zeta potential were performed using dynamic light scattering (DLS) (Zs90, Malvern, U.K.). 2.4.2. Ultraviolet (UV) and fluorescence spectrum scanning To determine whether FITC and Cy5.5 were conjugated to the PAMAM dendrimer nanoparticles successfully, PAMAM-FITC/Cy5.5 was scanned by UV–vis spectrophotometer and fluorescence spectrophotometer. 2.5. Cellular uptake of Pep-PEG-PAMAM U87MG cells were seeded in 6-well plates at a density of 3 × 105 cells/well, allowing for attachment for 24 h. Afterwards, the medium was replaced with different concentrations (10, 20, 30 ␮g/mL) of Mal-PEG-PAMAM-FITC and Pep-PEG-PAMAM-FITC, respectively. After incubation for 1 h at 37 ◦ C, the cells were washed twice with cold PBS (pH 7.4), fixed with 4% formaldehyde for 15 min, and finally subjected to fluorescent microscopy analysis (Imager A1, Zeiss, Germany). 2.6. In vitro cytotoxicity In vitro cytotoxicity of Mal-PEG-PAMAM and Pep-PEG-PAMAM was evaluated by MTT assay with BCEC and U87MG cells. Briefly, BCEC and U87MG cells were seeded in a 96-well plate at the density of 2 × 103 cells/well. After incubation for 24 h, the cells were then treated with a series of different concentration (from 0.1 to 10 ␮M) of Mal-PEG-PAMAM and Pep-PEG-PAMAM for 24 h, respectively. Then, 20 ␮L MTT solution (5 mg/mL in PBS) was added to each well and the cells were incubated for another 4 h at 37 ◦ C. Afterwards, medium was gently removed and 200 ␮L DMSO was added to each

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Fig. 1. 1 H NMR spectra of PAMAM, Mal-PEG-PAMAM and Pep-PEG-PAMAM in D2 O (A). The particle-size distribution of dendrimer nanoparticles (B). UV absorption spectrum (C) and fluorescence emission spectrum (E) of PAMAM-FITC. UV absorption spectrum (D) and fluorescence emission spectrum (F) of PAMAM-FITC/Cy5.5.

well to dissolve the formazan crystals for 15 min at room temperature away from light. Furthermore, the absorbance was detected using a microplate reader (Thermo Multiskan MK3, USA), with a test wavelength of 490 nm.

2.7. Confocal microscope U87MG cells were seeded on 14-mm2 glass cover slips that were placed in 6-well plate at the density of 1 × 105 cells/well, allowing for attachment for 24 h. And then cells were exposed to Pep-PEGPAMAM-FITC for 30 min or 60 min at 37 ◦ C followed by incubation with organelle-selective dyes. Cells were incubated with 10 mM

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Fig. 2. U87MG uptake of Mal-PEG-PAMAM-FITC (A, B, C) and Pep-PEG-PAMAM-FITC (D, E, F) at the concentration of 10 ␮g/mL (A, D), 20 ␮g/mL (B, E) and 30 ␮g/mL (C, F) was examined by fluorescent microscopy after 1 h incubation. Original magnification: ×20.

Hoechst 33342 for 10 min and 75 nM Lysotracker Green for 30 min, respectively. Afterwards, the solution was removed and the cell monolayers were washed three times with cold PBS, and then examined by confocal laser scanning microscopy (Leica TCS SP2, Germany).

sacrificed at 24 h post-injection and the glioma-bearing brain and major organs were harvested for fluorescent imaging, respectively.

2.9. Biodistribution of Pep-PEG-PAMAM in intracranial glioma-bearing mice

2.8. In vivo fluorescence imaging In vivo fluorescence imaging analysis was used to track the real-time distribution of the nanoparticles. U87 MG cells (5 × 105 cells/5 ␮L in pH 7.4 PBS) were implanted into the right striatum (1.8 mm lateral to the bregma and 3 mm of depth) of male Balb/c nude mice by using a stereotactic fixation device with mouse adaptor. The glioma-bearing mice were injected with Mal-PEGPAMAM-FITC/Cy5.5 or Pep-PEG-PAMAM-FITC/Cy5.5 (1.2 ␮mol/kg) via tail vein 21 days after implantation. And then the mice were anesthetized and imaged at predetermined time points (4 h and 24 h) via an in vivo imaging system (Caliper, USA). The mice were

The glioma-bearing mice model was established as described above. On the 21 st day after implantation, Mal-PEG-PAMAMFITC/Cy5.5 or Pep-PEG-PAMAM-FITC/Cy5.5 was injected into the tail vein of mice at a dose of 1.2 ␮mol/kg, respectively. After circulation for 24 h, the mice were anesthetized, and heart perfused with saline for 10 min and 4% paraformaldehyde for 20 min, respectively. Then the mice were sacrificed and the brains were harvested, fixed in 4% paraformaldehyde for 24 h, dehydrated with 15% sucrose solution for 24 h and 30% sucrose solution for 24 h sequentially. Thereafter, the brains were frozen in OCT embedding medium (Sakura, Torrance, CA, USA) at −80 ◦ C, sectioned into 5 ␮m. Finally,

Fig. 3. In vitro cytotoxicity of dendrimer nanoparticles against BCEC cells (A) and U87 cells (B) with varying concentrations from 0.1 to 10 ␮M (n = 4).

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Fig. 4. Cellular location of Pep-PEG-PAMAM-FITC, photos were taken after cells incubated with Pep-PEG-PAMAM-FITC for 0.5 h (A) or 2 h (B). Nanoparticles and acidic vesicles were distinguished using FITC (green) Hoechst 33342 (blue) and Lyso Tracker (red), respectively. Bar: 10 ␮m (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

the sections were stained with DAPI for 10 min at room temperature and examined by the fluorescence microscope. 3. Results and discussion 3.1. Characterization of Pep-PEG-PAMAM The structure of PAMAM, Mal-PEG-PAMAM and Pep-PEGPAMAM were confirmed by 1 H NMR spectroscopy. As shown in Fig. 1A, the solvent peak of D2 O was present at 4.79 ppm. The characteristic peak of methylene protons of PAMAM was between 2.4 ppm to 3.4 ppm, and the peak at 3.7 ppm was attributed to methylene protons in PEG. In the spectrum of Mal-PEGPAMAM, the maleimide group has a characteristic peak at 6.7 ppm, whereas disappeared after reaction with Pep-1 in the spectrum of Pep-PEG-PAMAM, suggesting that Pep-1 was conjugated with Male-PEG-PAMAM successfully [26,28,29]. As shown in Fig. 1B and Table 1, the mean particle size of Mal-PEG-PAMAM and Pep-PEG-PAMAM were 5.1 ± 0.1 nm and 8.0 ± 0.2 nm, respectively, and both of the nanoparticles exhibited a decent PDI, which indicated that the conjugation with Pep-1 slightly increased the particle size of nanoparticles. The Zeta potential of Mal-PEG-PAMAM and Pep-PEG-PAMAM were 3.15 ± 0.21 mV and 2.07 ± 0.14 mV, respectively, indicating that Pep-1 modification slightly decreased the Zeta potential which may due to the peptide contained negative charge amino acids. In addition, such nanoparticles may accumulate more readily in tumor due to the EPR

effect and are easier to avoid opsonized and Kupffer cells clearance [30,31] and distribute deeply and homogenously in tumor [32–34]. In order to confirm the conjugation of FITC and Cy5.5 to PAMAM, UV–vis and fluorescence spectrum scanning was performed, respectively. As shown in Fig. 1C, the peak of PAMAM-FITC in UV absorption spectrum (500 nm) was basically consistent with the free FITC (499 nm), meanwhile the peak of fluorescence emission spectrum (Fig. 1E), 535 nm, was close to the free FITC (530 nm). As shown in Fig. 1D, the peaks in PAMAM-FITC/Cy5.5 UV absorption spectrum (500, 632 and 678 nm) were basically consistent with the free FITC (499 nm), free Cy5.5 (628 and 676 nm). Moreover, the peaks of PAMAM-FITC/Cy5.5 in fluorescence emission spectrum (537 and 708 nm) were shown in Fig. 1F, were also close to free FITC (533 nm) and free Cy5.5 (704 nm).

3.2. Cellular uptake of nanoparticles by U87MG cells In this study, the cellular uptake of Mal-PEG-PAMAM-FITC and Pep-PEG-PAMAM-FITC against U87MG cells were investigated using fluorescent microscopy to determine whether or not Pep1 modification can increase the uptake. The results showed that the fluorescent intensity of Mal-PEG-PAMAM-FITC or Pep-PEGPAMAM-FITC displayed a concentration-dependent mode (Fig. 2), and at each concentration point (10, 20, 30 ␮g/mL), U87MG cells incubated with Pep-PEG-PAMAM-FITC emitted stronger fluorescence compared with Mal-PEG-PAMAM-FITC, obviously.

3.3. In vitro cell cytotoxicity Table 1 Number size and zeta potential of dendrimer nanoparticles.

Number size(nm) Polydispersity index Zeta potential(mV)a

Mal-PEG-PAMAM

Pep-PEG-PAMAM

5.1 ± 0.1 0.18 ± 0.02 3.15 ± 0.21

8.0 ± 0.2 0.20 ± 0.03 2.07 ± 0.14

a Measured in NaCl solution (1 mM). Data were presented as the means ± standard deviation; n = 3.

The cytotoxicity of nanoparticles both on BCECs and U87MG cells was evaluated using the MTT assay. As shown in Fig. 3, MalPEG-PAMAM and Pep-PEG-PAMAM did not exhibit cytotoxic effects to BCECs or U87MG cells. In particular, most cells were viable up to 10 ␮M of Mal-PEG-PAMAM and Pep-PEG-PAMAM, suggesting that the nanoparticles were not harmful to the cells at the study concentration.

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Fig. 5. In vivo fluorescence imaging of GBM-bearing nude mice administrated with Mal-PEG-PAMAM-FITC/Cy5.5 (left) and Pep-PEG-PAMAM-FITC/Cy5.5 (right) at 4 and 24 h (A). Image of the brains (B) and tissues (C) sacrificed 24 h after intravenous injection of Mal-PEG-PAMAM-FITC/Cy5.5 (up) and Pep-PEG-PAMAM-FITC/Cy5.5 (down). The corresponding semi-quantitative radiant efficiency of brains (D) and organs (E). **p < 0.01 compared with the untargeted group. Untargeted: Mal-PEG-PAMAM-FITC/Cy5.5. Targeted: Pep-PEG-PAMAM-FITC/Cy5.5.

3.4. Intracellular localization of the Pep-PEG-PAMAM-FITC The intracellular localization of Pep-PEG-PAMAM-FITC in U87MG cells was evaluated by confocal laser scanning microscopy. As shown in Fig. 4, after incubation for 30 min, almost all Pep-PEGPAMAM-FITC was found to localize in the lysosome of U87MG cells.

However, at 60 min, Pep-PEG-PAMAM-FITC showed a decrease colocalization with the red fluorescence of Lyso Tracker red which indicating that Pep-PEG-PAMAM-FITC was released from lysosome into the cytoplasm which may because the exposed cationic amine groups of the PAMAM induced endosomal escape via the proton sponge effect [35,36].

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Fig. 6. In vivo tumor distribution of Mal-PEG-PAMAM-FITC/Cy5.5 (A, B and C) and Pep-PEG-PAMAM-FITC/Cy5.5 (D, E and F) at 24 h after administration. Image C is the merger of A and B; image F is the merger of D and E. Blue: cell nuclei stained by DAPI. Green: FITC-labeled dendrimer nanoparticles. White dash lines: border of the glioma. Scale bar: 200×.

3.5. In vivo fluorescence imaging In vivo real-time imaging was performed to evaluate the targeting ability of Pep-PEG-PAMAM-FITC/Cy5.5 on intracranial U87MG tumor-bearing mice. It is obviously shown that Pep-PEGPAMAM-FITC/Cy5.5 accumulated much more at tumor site at each imaging time (4 h and 24 h) when compared with that of Mal-PEG-PAMAM-FITC/Cy5.5 (Fig. 5A), and with time increasing, the distribution of Mal-PEG-PAMAM-FITC/Cy5.5 and Pep-PEGPAMAM-FITC/Cy5.5 in the brain also increased. Ex vivo imaging of brains 24 h post-injection also confirmed that the fluorescence intensity at glioma site of Pep-PEG-PAMAM-FITC/Cy5.5 was 2.02 folds higher than that of Mal-PEG-PAMAM-FITC/Cy5.5 (Fig. 5B and D). What’s more, in vitro fluorescence imaging and corresponding semi-quantitative results of major organs revealed that both MalPEG-PAMAM-FITC/Cy5.5 and Pep-PEG-PAMAM-FITC/Cy5.5 were mainly distributed in kidney (Fig. 5C and E), instead of liver and spleen. That is to say, this dendrimer nanoparticle can avoid the uptake by RES effectively and be eliminated mainly through kidney which was conducive to the accumulation in tumor tissue and reduce the in vivo toxicity. 3.6. In vivo glioma distribution The biodistribution and brain targeting capability of PepPEG-PAMAM-FITC/Cy5.5 was studied qualitatively by fluorescence microscopy. Results showed that only a slight distribution of MalPEG-PAMAM-FITC/Cy5.5 in the glioma site due to EPR effect (Fig. 6) while there was a clearly higher fluorescence intensity of Pep-PEGPAMAM-FITC/Cy5.5 was observed in the glioma when compared with that of Mal-PEG-PAMAM-FITC/Cy5.5, suggesting that Pep-1 modification could enhance the brain targeting capability via IL13R␣2-mediated endocytosis. 4. Conclusion In conclusion, we established an effective nanocarrier for glioma drug delivery. We conjugated the Pep-1 peptide to the surface of

PEGylated PAMAM via a maleimide-thiol reaction and confirmed that Pep-PEG-PAMAM displayed an effective targeting ability. Cellular assay showed that Pep-PEG-PAMAM significantly enhanced cellular uptake than that of Mal-PEG-PAMAM. In vivo experiments indicated that Pep-PEG-PAMAM could penetrate into deeper section and achieve a broader glioma site distribution than that of Mal-PEG-PAMAM. In summary, the small size of PAMAM can reduce the RES recognition in liver and increase the penetration in tumor, and Pep-1 modification mproved the active targeting effect to glioma. Therefore, Pep-1 modified dendrimer nanoparticle has the potential to serve as a safe theranostic nanocarrier for glioma drug targeted delivery.

Acknowledgements This work was supported from the National Natural Science Foundation of China (81302710, 81273457), Natural Science Foundation of Jiangsu Province-Excellent Young Scientist Fund (BK20160096), the Ordinary University Natural Science Research Project of Jiangsu Province (13KJB350004) and the Excellent Young Teacher Project of Nanjing Medical University (2015RC16). We are acknowledged the Qing Lan Project of Jiangsu Province.

References [1] A.A. Stavrovskaya, S.S. Shushanov, E.Y. Rybalkina, Problems of glioblastoma multiforme drug resistance, Biochemistry (Moscow) 81 (2016) 91–100. [2] O. van Tellingend, B. Yetkin-Arikc, M.C. de Gooijer, P. Wesseling, T. Wurdinger, H.E. de Vries, Overcoming the blood–brain tumor barrier for effective glioblastoma treatment, Drug Resist. Updat. 19 (2015) 1–12. [3] W. Kamphorst, A.G. de Boer, P.J. Gaillard, Brain drug targeting: the future of brain drug development, J. Clin. Pathol. 55 (2002) (158). [4] Y. Liu, W. Lu, Recent advances in brain tumor-targeted nano-drug delivery systems, Expert Opin. Drug Deliv. 9 (2012) 671–686. [5] J.M. Squire, M. Chew, G. Nneji, C. Neal, J. Barry, C. Michel, Quasi-periodic substructure in the microvessel endothelial glycocalyx: a possible explanation for molecular filtering, J. Struct. Biol. 136 (2001) 239–255. [6] M. Nasr, M. Najlahc, A. D’Emanuele, A. Elhissi, PANAM dendrimers as aerosol drug nanocarriers for pulmonary delivery via nebulization, Int. J. Pharm. 461 (2014) 242–250.

Y. Jiang et al. / Colloids and Surfaces B: Biointerfaces 147 (2016) 242–249 [7] D.A. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos, S. Martin, J. Roeck, J. Ryder, P. Smith, A new class of polymers: starburst-Dendritic macromolecules, Polym. J. 17 (1985) 117–132. [8] M. Sowinska, Z.U. Lipkowska, Advances in the chemistry of dendrimers, N. J. Chem 38 (2014) 2168–2203. [9] S. Svenson, D.A. Tomalia, Dendrimers in biomedical applications?reflections on the field, Adv. Drug Deliv. Rev. 57 (2005) 2106–2129. [10] E.R. Gillies, M.J. Fréchet, Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications, Drug Discov. Today 10 (2005) 35–43. [11] Y. Wang, L. Li, J.J. Li, B.G. Yang, C.Y. Wang, W.C. Fang, F. Ji, Y. Wen, F.L. Yao, Stable and pH-responsive polyamidoamine based unimolecular micelles capped with a zwitterionic polymer shell for anticancer drug delivery, RSC Adv. 6 (2016) 17728–17739. [12] Z.J. Xu, Y. Wang, Z.Y. Ma, Z.J. Wang, Y. Wei, X.R. Jia, A poly(amidoamine) dendrimer-based nanocarrier conjugated with Angiopep-2 for dual-targeting function in treating glioma cells, Polym. Chem. 7 (2016) 715–721. [13] Y.F. Sun, Y.F. Jiao, Y. Wang, D.R. Lu, W.L. Yang, The strategy to improve gene transfection efficiency and biocompatibility of hyperbranched PAMAM with the cooperation of PEGylated hyperbranched PAMAM, Int. J. Pharm. 465 (2014) 112–119. [14] W. Wong, W. Xiong, J.L. Wan, X.H. Sun, H.B. Xu, X.L. Yang, The decrease of PAMAM dendrimer-induced cytotoxicity by PEGylation via attenuation of oxidative stress, Nanotechnology 20 (2009) 105103. [15] R. Jevprasesphant, J. Penny, R. Jalal, D. Attwood, N.B. McKeown, A. D’Emanuele, The influence of surface modification on the cytotoxicity of PAMAM dendrimers, Int. J. Pharm. 252 (2003) 263–266. [16] H. Nakamura, F. Jun, H. Maeda, Correction to: development of next-generation macromolecular drugs based on the EPR effect: challenges and pitfalls, Expert Opin. Drug Deliv. 12 (2015) 1–12. [17] S.M. Moghimi, A.C. Hunter, J.C. Murray, Long-circulating and target-specific nanoparticles: theory to practice, Pharmacol. Rev. 53 (2001) 283–318. [18] S.M. Moghimi, C.J.H. Porter, I.S. Muir, L. Illum, S.S. Davis, Non-phagocytic uptake of intravenously injected microspheres in rat spleen: influence of particle size and hydrophilic coating, Biochem. Biophys. Res. Commun. 177 (1991) 861–866. [19] L.T. Chen, L. Weiss, The role of the sinus wall in the passage of erythrocytes through the spleen, Blood 41 (1973) 529–537. [20] A. Minty, P. Chalon, J.M. Derocq, X. Dumont, J.C. Guilemot, M. Kaghad, C. Labit, P. Leplatois, P. Liauzun, B. Miloux, C. Minty, P. Casellas, G. Loison, J. Lupker, D. Shire, P. Ferrara, D. Caput, Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses, Nature 362 (1993) 248–250. [21] S. Fichtner-Feigl, W. Strober, K. Kawakami, R.K. Puri, A. Kitani, IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis, Nat. Med. 12 (2006) 99–106.

249

[22] W. Debinski, D.M. Gibo, S.W. Hulet, J.R. Connor, G.Y. Gillespie, Receptor for interleukin 13 is a marker and therapeutic target for human high-grade gliomas, Clin. Cancer Res. 5 (1999) 985–990. [23] C.E. Brown, R. Starr, B. Aguilar, A.F. Shami, C. Martinez, M. D’Apuzzo, M.E. Barish, S.J. Forman, M.C. Jensen, Stem-like tumor-initiating cells isolated from IL13R␣2 expressing gliomas are targeted and killed by IL13-zetakine-redirected T Cells, Clin. Cancer Res. 18 (2012) 2199–2209. [24] C.E. Brown, C.D. Warden, R. Starr, X. Deng, B. Badie, Y.C. Yuan, S.J. Forman, M.E. Barish, Glioma IL13R␣2 is associated with mesenchymal signature gene expression and poor patient prognosis, PLoS One 8 (2013) e77769. [25] H. Pandya, D.M. Gibo, S. Garg, S. Kridel, W. Debinski, An interleukin 13 receptor ␣ 2-specific peptide homes to human Glioblastoma multiforme xenografts, Neuro. Oncol. 14 (2012) 6–18. [26] B.Y. Wang, L.Y. Lv, Z.Y. Wang, Y. Zhao, L. Wu, X.L. Fang, Q.W. Xu, H.L. Xin, Nanoparticles functionalized with Pep-1 as potential glioma targeting delivery system via interleukin 13 receptor ␣2-mediated endocytosis, Biomaterials 35 (2014) 5897–5907. [27] B.Y. Wang, L.Y. Lv, Z. Wang, Y. Jiang, W. Lv, X. Liu, Z.Y. Wang, Y. Zhao, H.L. Xin, Q.W. Xu, Improved anti-glioblastoma efficacy by IL-13R␣2 mediated copolymer nanoparticles loaded with paclitaxel, Sci. Rep. 5 (2015) 16589. [28] Y. Zhao, Y. Jiang, W. Lv, Z.Y. Wang, L.Y. Lv, B.Y. Wang, X. Liu, Y. Liu, Q.Y. Hu, W.J. Sun, Q.W. Xu, H.L. Xin, Z. Gu, Dual targeted nanocarrier for brain ischemic stroke treatment, J. Control. Release 233 (2016) 64–71. [29] J.J. Zhao, B. Zhang, S. Shen, J. Chen, Q.Z. Zhang, X.G. Jiang, Z.Q. Pang, CREKA peptide-conjugated dendrimer nanoparticles for glioblastoma multiforme delivery, J. Coll. Interface Sci. 450 (2015) 396–403. [30] S. Dar Li, L. Huang, Pharmacokinetics and biodistribution of nanoparticles, Mol. Pharm. 5 (2008) 496–504. [31] T.S. Levchenko, R. Rammohan, A.N. Lukyanov, K.R. Whiteman, V.P. Torchilin, Liposome clearance in mice: the effect of a separate and combined presence of surface charge and polymer coating, Int. J. Pharm. 240 (2002) 95–102. [32] T. Stylianopoulos, M.Z. Poh, N. Insin, M.G. Bawendi, D. Fukumura, L.L. Munn, R.K. Jain, Diffusion of particles in the extracellular matrix: the effect of repulsive electrostatic interactions, Biophys. J. 99 (2010) 1342–1349. [33] O. Lieleg, R.M. Baumgartel, A.R. Bausch, Selective filtering of particles by the extracellular matrix: an electrostatic bandpass, Biophys. J. 97 (2009) 1569–1577. [34] M.J. Ernsting, M. Murakami, A. Roy, S.D. Li, Factors controlling the pharmacokinetics: biodistribution and intratumoral penetration of nanoparticles, J. Control. Release 172 (2013) 782–794. [35] H. Arima, K. Motoyama, T. Higashi, Potential use of polyamidoamine dendrimer conjugates with cyclodextrins as novel carriers for siRNA, Pharmaceuticals (Basel) 5 (2011) 61–78. [36] B. Klajnert, M. Bryszewska, Dendrimers: properties and applications, Acta Biochim. Pol. 48 (2001) 199–208.