Enhanced Antiglioma Efficacy of Ultrahigh Loading Capacity

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Enhanced anti-glioma efficacy of ultrahigh loading capacity paclitaxel prodrug conjugate self-assembled targeted nanoparticles Yan Jiang, Xiuzhen Wang, Wei Lv, Xin Liu, Hongjuan Zhang, Mingwan Zhang, Xinrui Li, Hongliang Xin, and Qunwei Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13805 • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 16, 2016

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ACS Applied Materials & Interfaces

Enhanced anti-glioma efficacy of ultrahigh loading capacity paclitaxel prodrug conjugate self-assembled targeted nanoparticles Yan Jiang1#, Xiuzhen Wang2#, Wei Lv1, Xin Liu1, Hongjuan Zhang2, Mingwan Zhang1, Xinrui Li1, Hongliang Xin1*, Qunwei Xu1*

1. Department of Pharmaceutics, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China 2. Department of Medicinal Chemistry, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China

#

These authors contributed equally to this manuscript.

Corresponding author: Hongliang

Xin

(Tel.:

+86-25-86868476,

Fax:

+86-25-86868467,

E-mail:

+86-25-86868467,

E-mail:

[email protected]). Qunwei

Xu

(Tel.:

+86-25-86868468,

Fax:

[email protected]);

1

ACS Paragon Plus Environment


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ABSTRACT Glioblastoma multiforme (GBM) presents one of the most lethal brain tumor with a dismal prognosis. And nano-drug delivery system (nano-DDS) have raised a lot of concern, while the conventional nanoformulations addressed many limitations, especially the low drug loading capacity and poor stability in vivo. Herein, we proposed PTX prodrug (PTX-SS-C18) conjugate self-assembled nanoparticles (PSNPs) functionalized with Pep-1, glioma homing peptide, to overcome the Blood Brain Tumor Barrier (BBTB) via interleukin 13 receptor α2 (IL-13Rα2) mediated endocytosis for targeting GMB. This nanocarrier was with ultrahigh drug loading capacity (56.03%) and redox-sensitivity to the up-expression of glutathione in glioma tumors. And compared with PEG-PSNPs, Pep-PSNPs could significantly enhance cellular uptake in U87MG cells via IL-13Rα2 mediated endocytosis. Enhanced cytotoxicity of Pep-PSNPs against U87MG cells and BCEC cells pretreated with glutathione monoester (GSH-OEt) confirmed that this nanosystem was sensitive to reduction environment, and there was significant difference between targeting and non-targeting

groups

in

MTT

assay.

Real-time

fluorescence

image

of

intracranialU87MG glioma-bearing mice revealed that Pep-PSNPs could more efficiently accumulate at tumor site and improve the penetration. Furthermore, the ex vivo fluorescence imaging and corresponding semi-quantitative results displayed that the glioma fluorescence intensity of Pep-PSNPs group was 1.74-fold higher than that of non-targeting group. Pep-PSNPs exhibited remarkable anti-glioblastoma efficacy with an extended median survival time. In conclusion, Pep-PSNPs had a promising

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perspective as a targeting drug delivery system of PTX for glioma treatment.

Keywords: Paclitaxel; Prodrug; Self-assembled nanoparticles; Redox-responsive; Pep-1 peptide; Targeting

1. Introduction Glioblastoma multiforme (GBM) is commonly recognized as a frequent and fatal tumor of the brain with a poor prognosis, a median survival of less than 18 months and a 5-year survival rate of less than 3%.1-3 In order to improve the glioma therapeutic effects, nano-drug delivery system (nano-DDS) to overcome blood–brain barrier (BBB) and blood–brain tumor barrier (BBTB) have experienced tremendous development in recent years. Nevertheless, there also exist some challenges in conventional nano-DDS. Firstly, the nanocarriers suffer low drug loading capacity and encapsulation efficiency4,5 and lack of active targeting.6 Additionally, the poor stability of nanosystems in vivo leads to the undesired drug release.7 Therefore, it is imperative to propose a glioma microenvironment responsive nano-DDS with high drug loading capacity, good stability and active targeting effect. Receptor-mediated endocytosis remains the most important targeted mechanism for drug delivery across the BBTB.8 A specific ligand peptide of the over-expressed interleukin-13 receptor α2 (IL-13Rα2) in GBM, Pep-1, can specially bind to IL-13Rα2 with high affinity and mediate the transport of nanoparticles across the BBTB and homing to glioma.9,10 In our previous studies, it was confirmed that

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polymer nanoparticles modified with Pep-1 could target brain glioma precisely, exhibiting the potential for glioma targeting treatment.11-13 On the other hand, in the last few years, drug self-delivery systems (DSDSs), concerning that active drugs show nanoscale characteristic to realize intracellular delivery by themselves without the aid of nanocarriers have quickly attracted research attentions for cancer therapy.14 For example, prodrug self-delivery systems, disulfide-induced drug conjugates (two hydrophobic compounds connection via disulfide bond as a linkage) were prone to spontaneously self-assemble into nanoparticles with good stability.15,16 Additionally, peptide self-delivered nano-drug, the therapeutic peptide agent was proposed to self-assemble into nanoparticles to enhance the cell apoptosis.17 In particular, this kind of nanoparticles showed a range of advantages. Firstly, the conjugates were prone to self-assemble into nanoparticles in the absence of any surfactant, resulting in an ultrahigh drug loading. Secondly, the nanoparticles showed excellent biocompatibility and safety due to no addition of supplementary materials. Thirdly, the simple method of preparation, good stability and pharmaceutical properties provided convenience to further effective clinical translational research. Finally, disulfide bond not only can transform hydrophobic prodrugs into suitable nanomaterials for molecular self-assembly but also represents a key structure dependent of the redox-responsive tumor microenvironment. It has been demonstrated that disulfide bond is stable under physiological conditions but can be rapidly cleaved in tumor tissues in response to reductive molecules, especially glutathione (GSH),18,19 and the sensitivity of the linkage is conducive to the rapid

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release of drugs so as to improve the antitumor activity. Moreover, the GSH can reach a thousands of times higher concentration in tumor cytosol (1−11 mM) than in the normal cells (1−2 µM).20,21 According to the advantages of this nanosystem, especially the redox-responsive mechanism of dithioether, we herein used the disulfide bond PTX prodrug (PTX-SS-C18) conjugate as materials to develop an ultrahigh drug loading and stable nanocarrier as glioma targeted drug delivery system. Therefore, in this study, Pep-1 was utilized to promote the penetration of PTX into glioma site. As shown in Figure 1, Pep-1 conjugated PEGylated PTX-SS-C18 conjugate self-assembled nanoparticles (Pep-PSNPs) had remarkably advantages as followed: 1) The simple method of preparation made it easy to popularize and apply; 2) The drug loading was significantly increased; 3) It was stable under physiological conditions, which was favorable for avoiding drug leakage and reducing the toxicity of off-target effect. 4) Pep-1 peptide could target to the brain glioma and cross the BBTB via IL-13Rα2 mediated endocytosis, and then release PTX under the circumstances of high GSH level in glioma cells to enhance the anti-glioma efficacy.

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Figure 1. Design of Pep-1 conjugated PEGylated PTX-SS-C18 conjugate self-assembled nanoparticles (Pep-PSNPs) for glioma treatment.

2. Materials and methods 2.1. Materials Paclitaxel was purchased from Zelang Medical Technology Co., Ltd. (Nanjing, China). Dithiodiglycolic acid, 1-Octadecanol, Glutathione and Glutathione reduced ethyl ester were obtained from Aladdin Reagent Database Inc. (Shanghai, China). Pep-1 (CGEMGWVRC) peptides were synthesized by GL Biochem Co., Ltd (Shanghai,

China),

purified

by

HPLC

and

verified

for

purity≥98%.

Mal-PEG2000-DSPE and MeO-PEG2000-DSPE were purchased from Laysan Bio Co.,

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USA. Coumarin-6 and DiR were provided by Sigma-Aldrich (St. Louis, MO, USA). MTT, BCA kit and TritonX-100 were supplied by Beyotime Biotechnology Co., Ltd. (Nantong, China). Penicillin-streptomycin, DMEM medium, fetal bovine serum and trypsin solution were purchased from Gibco BRL (Gaithersberg, MD, USA). All 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). 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 Prof. Jun Chen of 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. 2.3. Preparation of PEG-PSNPs and Pep -PSNPs The procedure of synthesis of PTX-SS-C18 and Pep-PEG-DSPE was shown in supporting information. PSNPs were prepared by the method of ethanol injection. Briefly, PTX-SS-C18 (5 mg) was dissolved in anhydrous ethanol (500 µL) and then injected into the distilled water (10 mL) under mechanical stirring (650 rpm) at room

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temperature for 5 min. The residual ethanol was further removed with a rotary evaporator, and then the nanoparticles were extruded through 450 and 220 nm nmpolycarbonate membranes, respectively. PEG-PSNPs and Pep-PSNPs were produced by mixing PSNPs with MeO-PEG2000-DSPE or Pep-PEG-DSPE in water (MeO-PEG2000-DSPE or Pep-PEG-DSPE/PSNPs = 20/100, w/w). For coumarin-6 and DiR-labeled nanoparticles, both coumarin-6 and DiR were co-dissolved with PTX-SS-C18 in anhydrous ethanol during nanoparticle preparation. 2.4. Characterization of PEG-PSNPs and Pep -PSNPs The morphology of nanoparticles was observed using transmission electronic microscopy (TEM) (JEOL USA, Wilmington, DE, USA). The particle size and zeta potential were investigated using dynamic light scattering (DLS) (Zs90, Malvern, U.K.). 2.5. In vitro reduction-triggered release profiles of PTX from PSNPs The release behaviors of PTX from PSNPs were investigated using ultrafiltration centrifugation method in PBS (0.04M, pH 7.4) with 1 µM GSH or HAc-NaAc buffer (0.04 M, pH 5.0) with 10 mM GSH containing 0.5% (w/v) Tween-80 at 37°C. In brief, PEG-PSNPs and Pep-PSNP (containing 30 µg of PTX) were dispersed into 4 mL of mediator solution and every sample was gently shaken at an appropriate speed. And then, the solutions were transferred to centriprep centrifugal filter units (MWCO = 30K Da) at various time points, centrifuging at 4000 rpm for 10 min to get free PTX. The concentration of PTX released from nanoparticles was monitored by HPLC. 2.6. Intracellular uptake assay

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For qualitative analysis, fluorescent microscopy (Imager A1, Zeiss, Germany) was used to evaluate intracellular distribution of coumarin-6 labeled PSNPs. U87MG cells were seeded into a 24-well plate at a density of 5×104 cells/well for 24 h. Afterwards, the cells were incubated with various formulations containing coumarin-6 at different concentrations (5, 10 and 30 ng/mL) for an additional 1 h at 37 ºC and 4 ºC, respectively. And then, the cells were washed three times with pre-cooled PBS and fixed with 4% formaldehyde for 15 min, and viewed using fluorescent microscopy. For quantitative experiment, U87MG cells were seeded into a 24-well plate at a density of 1×105 cells/well for 24 h incubation, and then treated with various formulations containing PTX at different concentrations (10, 20, 50 and 100 µg/mL). After incubation for 1 h at 37 ºC and 4 ºC respectively, the media were replaced with pre-cooled PBS for washing three times. Then, the cells were lysed by 400 µL of 1% TritonX-100 per well for 10 min. After that, an aliquot of the cell lysate from each well was used to determine the total cell protein content using the BCA protein assay and the PTX concentration of the remainder cell lysates was measured by HPLC. For the competition assay, Pep-1 was added to the wells in advance at a concentration of 200 µg/ml. After incubation at 37 ºC for 30 min, the peptide was replaced with media containing the coumarin-6-labeled Pep-PSNPs or Pep-PSNPs, followed by abovementioned steps for both qualitative and quantitative assay. 2.7. In vitro cytotoxicity assay The antitumor activity of PSNPs was determined by the MTT assay with U87MG and BCEC cells. In brief, U87MG and BCEC cells were seeded into a 96-well plate at

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the density of 5×103 cells per well. After incubation for 24h, the media were removed and replaced with several formulations as the PTX concentrations ranged from 0.01 µg/mL to 10 µg/mL, respectively, and the cells were incubated for another 48 h. Subsequently, MTT stock solution (20 µL, 5 mg/mL in PBS) was added to each well, and the plates were further incubated for 4 h at 37 °C in the dark. Afterwards, cells were dissolved in 200 µL of DMSO. Finally, the absorbance was detected at 490 nm using a microplate reader (Thermo Multiskan MK3, USA). In order to investigate the sensitiveness of PSNPs to reduction environment, U87MG and BCEC cells were pretreated with or without 10 mM glutathione monoester (GSH-OEt) for 2 h at 37 °C. After that, cells were washed with PBS to remove the GSH-OEt and incubated with various formulations containing PTX at different concentrations (1µg/mL and 5µg/mL) for further 48h, followed by abovementioned steps. 2.8. Confocal microscope U87MG cells were seeded onto 14-mm2 glass cover slips that were placed in 6-well plate at a density of 1×105 cells/well, allowing for attachment for 24 h. Afterwards, cells were incubated with coumarin-6-labeled Pep-PSNPs for 1 h or 2 h at 37°C followed by treatment with organelle-selective dyes. Cells were exposed to75 nM Lysotracker Red for 30 min and 10 mM Hoechst 33342 for 10 min, respectively. Subsequently, the media were replaced with pre-cooled PBS for washing three times, and then analyzed using confocal laser scanning microscopy (Leica TCS SP2, Germany).

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2.9. In vivo imaging analysis The brain tumor targeting efficiency in vivo of Pep-PSNPs was evaluated with real-time fluorescence imaging analysis. Intracranial U87MG tumor-bearing mice were established as described previously.13 Tumor-bearing mice were tail intravenous injected with 100 µL of DiR-labeled PEG-PSNPs and Pep-PSNPs after implantation for 18 days. At 4, 12, 24 and 36 h post injection, the mice were anesthetized and imaged with an in vivo imaging system (Caliper, USA). To compare the distributions of PSNPs in organs and tumor site, the mice were sacrificed at 36 h and their organs were dissected and subjected to ex vivo fluorescence imaging. 2.10. In vivo anti-glioma efficacy Intracranial glioma bearing mice model was established to evaluate the in vivo anti-glioma efficacy of Pep-PSNPs. The mice were randomly divided into five groups (8 mice per group) as follows: physiological Saline, Taxol®, PTX-SS-C18, PEG-PSNPs and Pep-PSNPs. At the 2, 4, 6 and 8 days after glioma cells inoculation, the mice were administered with corresponding formulations via tail vein at a dose of 10 mg/kg PTX, respectively. Kaplan-Meier survival curves were plotted for every group. 2.11. Statistical analysis All the results were expressed as mean ± standard deviation (SD). One-way ANOVA was utilized for statistical evaluation. Statistical analysis was performed with SPSS 20.0 software. Differences were considered significant when *P

Enhanced Antiglioma Efficacy of Ultrahigh Loading Capacity

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