Improved anti-glioblastoma efficacy by IL-13Rα2 mediated copolymer nanoparticles loaded with paclitaxel

Glioma presents one of the most malignant brain tumors, and the therapeutic effect is often limited due to the existence of brain tumor barrier. Based on interleukin-13 receptor α2 (IL-13Rα2) over-expression on glioma cell, it was demonstrated to be a potential receptor for glioma targeting. In this study, Pep-1-conjugated PEGylated nanoparticles loaded with paclitaxel (Pep-NP-PTX) were developed as a targeting drug delivery system for glioma treatment. The Pep-NP-PTX presented satisfactory size of 95.78 nm with narrow size distribution. Compared with NP-PTX, Pep-NP-PTX exhibited significantly enhanced cellular uptake in C6 cells (p < 0.001). The in vitro anti-proliferation evaluation showed that the IC50 were 146 ng/ml and 349 ng/ml of Pep-NP-PTX and NP-PTX, respectively. The in vivo fluorescent image results indicated that Pep-NP had higher specificity and efficiency in intracranial tumor accumulation. Following intravenous administration, Pep-NP-PTX could enhance the distribution of PTX in vivo glioma section, 1.98, 1.91 and 1.53-fold over that of NP-PTX group after 0.5, 1 and 4 h, respectively. Pep-NP-PTX could improve the anti-glioma efficacy with a median survival time of 32 days, which was significantly longer than that of PTX-NP (23 days) and Taxol® (22 days). In conclusion, Pep-NP-PTX is a potential targeting drug delivery system for glioma treatment.


Results
Characterization of the Pep-NP-PTX. The nanoparticles were prepared via an emulsion/solvent evaporation method. The mean size of NP-PTX were 91.23 ± 1.56 nm (Fig. 1C), after modified with Pep-1 peptide, the size slightly increased to 95.78 ± 2.37 nm with the same narrow size distribution of Pep-NP-PTX (Fig. 1D). TEM photographs showed that NP-PTX and Pep-NP-PTX were generally spherical and of regular size (Fig. 1A,B), consistent with the size distribution. Due to the mean diameters less than 100 nm, such nanoparticles may accumulate more readily in tumor due to the EPR effect. Zeta potential of NP-PTX and Pep-NP-PTX were − 35.2 ± 2.15 mV and − 34.5 ± 1.74 mV, respectively, indicating that the surfaces of NP and Pep-NP were both strong negative charged ( Table 1). The result suggested that the constructed nanoparticles had a good stability.
In vitro PTX release. The   the same controlled release behavior. As showed in Fig. 2, 65.23% of PTX in NP and 66.43% of PTX in Pep-NP were released in PBS (pH 7.4), while 70.28% of PTX in NP and 76.94% of PTX in Pep-NP were released in PBS (pH 5.0) after 72 h. There was no significant difference about the release pattern between NP-PTX and Pep-NP-PTX in both pH 7.0 and pH 5.0. Therefore, the modification of Pep-1 peptide did not change the release pattern of PTX from the nanoparticles. Cellular uptake of PTX-loaded Pep-NP. The quantitative cellular uptake of PTX-loaded Pep-NP in C6 cells which was shown concentration-dependent model was presented in Fig. 3. From the results, it could be seen that the cellular uptake of Pep-NP group was 1.55, 1.65, 1.42 and 1.58 folds higher than that of NP group, and 3.39, 3.06, 3.11 and 3.03 folds higher than that of Taxol ® group at 37 °C at the PTX concentration of 10, 20, 40 and 60 μ g/mL, respectively.
Cell cytotoxicity. The in vitro cell cytotoxicity of different formulations was evaluated on C6 cells by using MTT method. As shown in Fig. 4A, the cytotoxicity of Cremophor EL was obviously higher than that of nanoparticles when the incubation concentration was more than 10 μ g/mL. However, blank NP and Pep-NP did not show obvious cytotoxicity at concentrations ranged from 0.1 μ g/mL to 1000 μ g/mL. These results suggested that blank NP and Pep-NP were not toxic to C6 cells probably due to the biocompatibility of the block polymers.
In vivo imaging. The in vivo glioma-targeting efficiency of NP and Pep-NP was also investigated in intracranial C6 glioma-bearing nude mice. Compared with NP group, the fluorescence intensity of Pep-NP group in the glioma site was much stronger than NP group at any time post-injection (Fig. 5A). After 24 h post-injection, the fluorescence of tissues (heart, liver, spleen, lung, kidney and brain) was also visualized under the in vitro imaging system. As shown in Fig. 5B, intensive fluorescence signal was observed in liver and spleen, indicating that most of the nanoparticles were nonspecifically taken up and eliminated by mononuclear phagocyte system (MPS). However, ex vivo evaluation of brains showed an obvious glioma site accumulation of the nanoparticles (Fig. 5C). The fluorescence of Pep-NP group was much stronger than that of NP group in the glioma section, indicating that Pep-1 peptide could facilitate the enrichment of nanoparticles in the glioma via IL-13Rα 2 mediated endocytosis.
Brain biodistribution. Brain biodistribution of PTX following intravenous administration of Taxol ® , NP-PTX and Pep-NP-PTX were assessed in intracranial C6 glioma-bearing mice. The amount of PTX in the normal brain and glioma section was determined with HPLC-mass spectrometry. There was    The anti-glioma efficacy was also evaluated by measuring the survival time of the intracranial glioma-bearing mice treated with the different PTX formulations ( Fig. 8 and Table 3). The medium survival time of Pep-NP-PTX group was 32 days, suggesting that Pep-NP-PTX significantly prolonged animal survival time when compared with Saline (17 days), Taxol ® (22 days) and NP-PTX (23 days).
These results indicated that Pep-1 conjugated PEG-PLGA nanoparticles constructed in this study showed a desirable therapeutic effect, which could offer a potential drug delivery system for glioma treatment.
In vivo safety evaluation. The systemic toxicity of blank Pep-NP was evaluated in healthy mice.
Compared with the saline group, no deaths and obvious body weight loss were observed in all test groups during the study period (Fig. 9). The tissue sections of heart, liver, spleen, lung, kidney and brain stained with H&E showed no any apparent change in cellular structure and no necrosis, congestion or hydropic degeneration was observed compared with the saline group (Fig. 10). Moreover, there was no significant difference about the serum aspartate transaminase (AST), alanine transaminase (ALT), urea nitrogen (BUN) and creatinine levels between Pep-NP and saline group ( Table 4), indicating that no   **p < 0.01 significantly higher than that of Taxol ® , ###p < 0.001 significantly higher than that of NP-PTX.  inflammatory reactions occurred in these tissues. Taken together, our results exhibited that intravenous successive administration of 100 mg/kg Pep-NP for 6 days did not cause systemic toxicity.

Discussion
Treatment of GBM, the most frequent primary central nervous system tumor, is one of the most challenging problems 18 . Surgical resection generally fails to control progression of the glioma, and recurrence is practically inevitable 19 , due to its diffuse invasion of the surrounding normal tissue. In addition, the currently available chemotherapy is less than optimal for glioma treatment, mainly owing to the delivery problems. Most of the therapeutic drugs can't be delivered to the glioma directly for the existence of BTB. It acts as a physical and biological barrier to protect the glioma from chemotherapeutic agents to ensure an optimal environment for glioma. In recent years, the emphasis for treatment of glioma has been the application of receptor-mediated transcytosis for the targeting delivery of drug into the glioma. It provides an opportunity for active targeting to overcome the poor permeation of BTB, particularly when the receptor is up-regulated under the diseased condition. Therefore, receptor-mediated transcytosis is also known as the molecular Trojan horse approach. ANG1005 20 , a conjugation complex of PTX with a receptor-targeting peptide (Angiopep-2), exhibited an efficient therapeutic strategy for increasing the potency of PTX for GBM 21 and breast cancer 22 . Although this approach was originally applied to molecular or macromolecular cargos, it has now been extended to nanoparticulate drug delivery system with the same level of success [23][24][25][26] .
Among these receptors, IL-13Rα 2 has attracted increasing interest for its potential role in tumor-specific therapeutics. It has been found to be significantly up-regulated in a number of human tumors, including glioblastomas 27,28 and ovarian carcinomas 29 . IL-13 has the ability to form a second complex with its high-affinity receptor, IL-13Rα 2. However, the use of endogenous ligands as a target vector may compete for binding sites due to the very high concentration of endogenous ligands in the circulation. Pep-1, isolated by a cyclic disulphide-constrained heptapeptide phages display library, showed high specificity to IL-13Rα 2 surprisingly 30 . In the previous study, we developed a glioma targeting system by courmarin-6-labeled PEG-PLGA copolymer nanoparticles modified with Pep-1 17 . The Pep-NP exhibited enhanced uptake by C6 glioma cells in vitro and accumulation in glioma-bearing brain in vivo through IL-13Rα 2 mediated endocytosis.
In this study, we constructed a targeting nanoparticle drug delivery system by conjugating with Pep-1, which transported PTX for glioma treatment. It is well known that the rapid proliferation of cancer cells leads to a leaky vasculature in the tumor rendering it more permeable, which is better known as the EPR effect 31 . The targeting nanoparticles were prepared by emulsion/evaporation method, and Pep-1 peptide was functionalized to nanoparticle via a maleimide-thiol coupling reaction. Both of the developed NP and Pep-NP were less than 100 nm. These nanoparticles might be suitable for tumor drug delivery as this size range was reported to exhibit a prolonged blood circulation and a relatively low rate of mononuclear phagocyte system (MPS) uptake 32 .
In addition, the resulting NP and Pep-NP showed a similar size distribution and zeta potential, indicating that the nanoparticles might have similar systemic pharmacokinetic behavior (Fig. 6B).
In vitro PTX release study showed similar biphases release pattern of the nanoparticles formulations. In the first 6 h, a burst release was obtained. Ten hours later, a mild, sustained and under controlled release was presented. The initial faster release was believed to be derived from the agents that located at the outer layer of the particles while the later slower one from that incorporated into the nanoparticle core and released in a prolonged way along with the erosion or degradation of the matrix 33,34 . No From in vitro cell cytotoxicity study, blank NP and Pep-NP did not show obvious cytotoxicity at all concentrations points, whereas the cytotoxicity of Cremophor EL was higher than that of nanoparticles at concentrations ranged from 10 μ g/mL to 1000 μ g/mL. This showed that PEG-PLGA block copolymer nanoparticles were one of the better drug delivery carrier with good biocompatibility.
All the PTX formulations blocked the C6 proliferation in a concentration-dependent manner (Fig. 4B). In vitro cell uptake study results showed that the concentration of PTX in Pep-NP group was much higher than Taxol ® group (p < 0.001) and NP group (p < 0.001) at each concentration point (Fig. 3). The elevated association of Pep-NP also resulted in stronger anti-proliferative following encapsulation of PTX in C6 cells. Hence, the IC 50 of Pep-NP-PTX was 2.39 times lower than that of NP-PTX, which we believed was contributed by the enhanced cellular association of the nanoparticles following the modification with Pep-1. Although the IC 50 of Taxol ® was lower than Pep-NP-PTX, it may be due to the combined effects of PTX and Cremophor EL. Therefore, we believed that the enhanced cytotoxicity of Pep-NP-PTX mainly attributed to the enhanced cellular uptake facilitated by Pep-1 and IL-13Rα 2 interaction.
Based on the biodistribution results in previous study 17 , in vivo NIR imaging investigation was performed to further evaluate the glioma targeting efficacy of Pep-NP. In fact, during the glioma occurrence and development, the blood-brain barrier endothelial cells may be affected and blood-brain barrier tight junction's ultrastructure can be destroyed to some extent. The higher the level of brain glioma, the greater degree of damage of blood-brain barrier tight junction integrity occured [35][36][37] . In our study, the intracranial tumor xenograft model was established by inoculation of C6 14 days, which reached the most advanced stage glioma. Therefore, blood-brain barrier is not the main obstacle for Pep-NP-PTX penetration into glioma in our used model. As shown in Fig. 5C, NP exhibited a modest tumor accumulation after 24 h due to the EPR effect. But Pep-NP group exhibited much stronger fluorescence intensity in the glioma site at all-time points when compared with that of unmodified NP. Although ex vivo liver and spleen exhibited intensive fluorescence, ex vivo brain imaging in Pep-NP group showed stronger fluorescence signal in glioma site and was in good consistent with the in vivo imaging result. The results indicated that the modification of Pep-1 enhanced nanoparticle accumulation in the glioma section through IL-13Rα 2 mediated endocytosis than unmodified NP which depended on limited EPR effects to get into tumor site.
Biodistribution study in glioma-bearing mice showed that PTX concentration detected in the glioma section at all-time points followed the order: Pep-NP-PTX > NP-PTX > Taxol ® (Fig. 6A).    that Pep-NP-PTX exhibited a desirable glioma biodistribution profile with significantly increased PTX delivery in vivo glioma region through IL-13Rα 2 mediated endocytosis. For evaluating the anti-tumor efficacy in vivo, the glioma-bearing mice were treated with saline, Taxol ® , NP-PTX and Pep-NP-PTX every two days for four injections at PTX dose 10 mg/kg. The tumor size of the PTX-treated groups was all notably smaller than that of the saline group. In addition, Pep-NP-PTX showed the strongest ability to inhibit tumor growth (IRT = 73.4%), in comparison with Taxol ® (p < 0.01) and NP-PTX (p < 0.001). Moreover, an obvious prolonged survival was achieved in those glioma-bearing mice treated with Pep-NP-PTX, the medium survival time 32 days, significantly higher than NP (p < 0.05), Taxol ® (p < 0.05) and saline (p < 0.05). No significant difference was found between the NP with Taxol ® and saline groups. These results offered the robust evidence for Pep-1 modified nanoparticles mediated targeted therapeutic benefits of glioma. It is well known that most of the intravenously injected nanoparticles are taken up and eliminated by mononuclear phagocyte system (MPS), including liver and kidney tissue 38 . Thus, an increase in biochemical parameters including AST, ALT, blood urea nitrogen and creatinine could reflect acute inflammation in liver and kidney. In our study, there were no such inflammatory reactions caused by Pep-NP, indicating that intravenous successive administration of Pep-NP did not cause acute toxicity. Further studies will be performed to illuminate the long-term toxic effects of the Pep-NP.

Conclusions
In this study, we proposed PEG-PLGA nanoparticles modified with Pep-1 and loaded with PTX as an effective drug delivery system through IL-13Rα 2 mediated endocytosis in mediating PTX transport for the treatment of GBM. Compared with NP-PTX, Pep-NP-PTX (p < 0.001) exhibited an enhanced uptake in C6 cells, thus inducing a strengthened anti-proliferation effect with an IC 50 of 146 ng/ml. It also exhibited a favorable brain biodistribution with increased PTX delivery in the glioma site. More importantly, compared with that of Taxol ® and NP-PTX, Pep-NP-PTX showed desirable anti-glioma efficacy with significantly enhanced the survival of glioma-bearing mice. Systemic safety tests showed no acute toxicity to hematological system, heart, liver, spleen, lung, kidney and brain in mice after intravenous administration of blank Pep-NP per day for six days. Taken together, these results indicated that IL-13Rα 2 could be exploited as a prospective receptor and PEG-PLGA nanoparticle modified with the high affinity and specificity ligand Pep-1 was a potential targeting drug delivery system for glioma treatment via improving the penetration across BTB.

Cell line. The C6 cell line was obtained from Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cell line was cultured in RPMI 1640 medium, supplemented with 10% FBS, 1% penicillin and 100 mg/mL streptomycin sulfate. Cells were cultured in incubators maintained at 37 °C with 5% CO 2 . All experiments were performed in the logarithmic phase of cell growth.

Animals.
Male ICR mice (male, 4-5 weeks, 18 ± 2 g) and male Balb/c mice (male, 4-5 weeks, 18 ± 2 g) were supplied by Department of Experimental Animals, Nanjing Medical University (Nanjing, China) and maintained under standard housing conditions. All animal experiments were performed in accordance with protocols evaluated and approved by the ethics committee of Nanjing Medical University.

Preparation of Pep-NP-PTX.
Pep-1 conjugated PEG-PLGA (Pep-PEG-PLGA) was synthetized by our previously-described method 17 . Pep-1 conjugated nanoparticle was prepared through emulsion/ solvent evaporation method. Briefly, 19 mg MePEG-PLGA copolymer, 2 mg Pep-PEG-PLGA and 1 mg PTX were dissolved in 1 mL ethyl acetate, which was then added into 2 mL 1% (w/v) poloxamer 188 aqueous solution. The mixture was sonicated using a probe sonicator (Xin Zhi Biotechnology Co., Ltd., China) for 5 min at 190 W output. The primary emulsion formed was added drop-wisely into 10 mL 0.5% (w/v) poloxamer 188 aqueous solution under moderate stirring. Ethyl acetate was evaporated at 40 °C using rotary evaporator. The resultant bluish solution was filtrated through 0.45 μ m and 0.22 μ m cellulose acetate filter membrane to remove the aggregates. The nanoparticles were concentrated by ultrafiltration (10 kd MWCO Millipore, USA) and washed twice to remove excessive emulsifier. Finally, the Pep-NP-PTX suspension was kept at 4 °C for further use. The preparation of DiR-labeled nanoparticles was prepared with the same procedure as NP except 0.2 mg DiR were dissolved in the ethyl acetate solution.
Particle size and zeta potential of Pep-NP-PTX. The morphology of Pep-NP-PTX was investigated using transmission electron microscope (TEM). The particle size and zeta potential of the nanoparticles were determined by dynamic light scattering (DLS) (Zs90, Malvern, U.K.).

Encapsulation efficiency and loading capacity of Pep-NP-PTX.
The amount of PTX encapsulated in the nanoparticles was measured by HPLC method (LC-10AT, SHIMADZU, Japan). A reverse phase C-18 Ultrasphere ODS column (150× 4.6 mm, 5 mm, Thermo Fisher, USA) was used. The mobile phase consisted of acetonitrile and water (47:53, v/v). The wavelength of detection was 227 nm. The flow rate was set at 1.0 mL/min, and the column was maintained at 30 °C. The retention time of PTX was about 7.9 min. The calibration curve was linear in the range of 0.1-100 μ g/mL with a correlation coefficient of R 2 = 0.9998.
To determine the EE and LC of Pep-NP-PTX, the concentration of PTX in samples was dissolved in acetonitrile and analyzed by HPLC as described. The EE% and LC% were calculated as indicated below (n = 3). Pep-NP-PTX were added into wells at the PTX concentration ranged from 10 to 60 μ g/ml, respectively. At the end of the incubation period, the samples were removed from the wells and the cell monolayers were washed with cold PBS. The cells were lysed by 400 μ L of 1% TritonX-100 per well for 10 min. An aliquot of the cell lysate from each well was used to determine the total cell protein content using the BCA protein assay. The concentration of PTX in samples was determined by HPLC as described above.
Cell cytotoxicity. The cell cytotoxicity of Pep-NP-PTX on C6 cells was evaluated by using the MTT assay. C6 cells were seeded into 96-well plates at the density of 5000 cells/well and incubated at 37 °C in a 5% CO 2 atmosphere. 24 h later, Taxol ® , NP-PTX and Pep-NP-PTX were added into wells at the PTX concentration ranged from 0.01 to 20 μ g/ml, respectively. After 72 h incubation, 20 μ L MTT was added into each well and incubated for 4 h. Then the unreacted dye was removed and 200 μ l of DMSO was added to each well to dissolve the dark blue crystal. Finally, the optical density was measured by microplate reader at wavelength of 490 nm. Cells without exposure to the PTX formulations were used as control.
The cell cytotoxicity of blank nanoparticles was evaluated also by the same me-thod with the concentration ranged from 0.1 to 1000 μ g/ml.
In vivo imaging. The intracranial tumor xenograft model was established by inoculation of C6 cells (5.0 × 10 5 cells suspended in 5 μ L PBS) into the the right striatum (1.8 mm lateral to the bregma and 3 mm of depth) of male Balb/c mice. The release of Dir from NPs is very low, indicating that Dir could be accurate fluorescence probes for NP behavior in vivo, and the fluorescence signals detected in organs well represented the distribution of the NPs 40 . After cultured for 14 days, the glioma-bearing mice were injected with Dir-loaded NP and Pep-NP at the dose of 0.8 mg/kg Dir, the concentration of Dir was measured by fluorescent spectrophotometer.
The fluorescent images were acquired at 3 and 24 h after injection using an in vivo imaging system (Caliper, USA). After 24 h post-injection, the mice were sacrificed and the fluorescence of tissues (including heart, liver, spleen, lung, kidney and brain) were visualized under the in vitro imaging system. Biodistribution of Pep-NP-PTX in intracranial glioma-bearing mice. Forty five glioma-bearing ICR mice were randomly divided into three groups and intravenously injected with Taxol ® , NP-PTX and Pep-NP-PTX at the dose of 8 mg/kg PTX, respectively. At different time points (0.5, 1 and 4 h, n = 5 at each time point) after injection, brain and glioma of the mice were collected. The tissues were homogenized in 0.9% sodium chloride solution with 1% TritonX-100 after the weight measurement. The supernatant was obtained after centrifugation.
For the determination of PTX, 100 μ L supernatant of tissue homogenates (or 100 μ L plasma) were mixed with 100 μ L methanol containing 100 ng/mL docetaxel (internal standard). Then 1 mL ether was added to extract the PTX and docetaxel. After dried the organic phase under N 2 , the residue was dissolved with 200 μ L 80% methanol solution and analyzed by HPLC-mass spectrometry (Agilent 1200 LC-MS, USA).
In vivo anti-glioma efficacy. In vivo tumor growth inhibition was performed to evaluate the anti-glioma efficacy of the constructed formulations. Glioma-bearing ICR mice model was established as described above. Two days after the implantation, the mice were randomly divided into four groups (3 mice per group) and intravenously administered with saline, Taxol ® , NP-PTX and Pep-NP-PTX at the dose of 10 mg/kg PTX, respectively. The treatment was repeated every other day for four injections. Fourteen days later, the mice were sacrificed with tumor collected and tumor volume was calculated with the formula: π /6 × larger diameter × (smaller diameter) 2,41 .
The anti-tumor efficacy of the formulations was also evaluated on intracranial glioma-bearing mice by measuring their survival time. Two days after C6 cells injection, the ICR mice were randomly divided into 4 groups (8 mice per group): saline group, Taxol ® group, NP-PTX group and Pep-NP-PTX group.
Each group was intravenously administered at the dose of 10 mg/kg PTX. The treatment was repeated every other day for four injections. The survival data were presented as Kaplan-Meier plots and analyzed with a log-rank test.
In vivo safety evaluation. Ten male ICR mice were randomly divided into two groups (n = 5). Each group received an intravenous injection of blank Pep-NP (100 mg/kg) or saline per day for six injections. The body weight was monitored each day. Blood sample and tissues (heart, liver, spleen, lung, kidney and brain) were collected at 24 h after the last administration for hematologic and histochemistry analysis. The AST, ALT, BUN and creatinine levels were assayed using Hitachi 7080 Chemistry Analyzer (Hitachi Ltd., Japan). The tissues were fixed with paraformaldehyde for 24 h and embedded in paraffin. Each brain was cut into 5 μ m by parafin section, processed for routine hematoxylin and eosin (H&E) staining, and then visualized under fluorescent microscope (Imager A1, Zeiss, Germany).