Multi-targeting Peptide-Functionalized Nanoparticles Recognized

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Multi-targeting peptide-functionalized nanoparticles recognized vasculogenic mimicry, tumor neovasculature and glioma cells for enhanced anti-glioma therapy Xingye Feng, Jianhui Yao , Xiaoling Gao, Yixian Jing , Ting Kang , Di Jiang , Tianze Jiang , Jingxian Feng , Qianqian Zhu , Xinguo Jiang, and Jun Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09934 • Publication Date (Web): 30 Nov 2015 Downloaded from http://pubs.acs.org on December 13, 2015

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ACS Applied Materials & Interfaces

Multi-targeting peptide-functionalized nanoparticles recognized vasculogenic mimicry, tumor neovasculature and glioma cells for enhanced anti-glioma therapy Xingye Feng,a Jianhui Yao,a Xiaoling Gao,b Yixian Jing,a Ting Kang,a Di Jiang,a Tianze Jiang,a Jingxian Feng,a Qianqian Zhu,a Xinguo Jiang,a and Jun Chen*,a a

Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy,

Fudan University, 826 Zhangheng Road, Shanghai, 201203, PR China b

Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiaotong

University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, PR China Corresponding Author's E-mail: [email protected]

Abstract: Chemotherapy failure of glioma, the most aggressive and devastating cancer, might ascribed to the physiologic barriers of the tumor mainly including heterogeneous tumor perfusion and vascular permeability, which result in a limited penetration of chemotherapeutics. Besides, the vasculogenic mimicry (VM) channels, which is highly resistant to the antiangiogenic therapy and serves as a complement of angiogenesis, was abound in glioma and always associated with tumor recurrence. In order to enhance the therapy effect of anti-glioma, we developed a PEG-PLA-based nano-drug delivery system (nanoparticles, NP) in this study and modified on its surface with CK peptide, which was composed of a human sonic hedgehog (SHH) targeting peptide (CVNHPAFAC) and a KDR targeting peptide (K237) through a GYG linker, for facilitating an efficient VM channels, tumor neovasculature and glioma cells multi-targeting delivery of paclitaxel. In vitro cellular assay showed that CK-NP-PTX not only exhibited the strongest anti-proliferation effect on U87MG cells and HUVEC cells, but also resulted in the most efficient destruction of VM channels when compared with CVNHPAFAC-NP, K237-NP and the unmodified ones. Besides, CK-NP accumulated more selectively at the glioma site as demonstrated by iIn vivo and ex vivo imaging. As expected, the glioma bearing mice treated with CK-NP-PTX 1

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achieved the longest median survival time compared with CVNHPAFAC-NP-PTX and K237-NP-PTX. These findings indicated that the multi-targeting therapy mediated by CK peptide might provide a promising way for glioblastoma therapy. Keywords: human sonic hedgehog; KDR receptors; tumor-homing peptide; vasculogenic mimicry channels; glioblastoma; multi-targeting therapy; nanoparticle

1. Introduction Treatment of glioblastoma multiforme (GBM), the most common and deadly cancer with a median survival of less than 18 months, remains to be a challenge in the oncology.1-3 Currently many strategies of anti-glioma have focused on the angiogenesis which derive from preexisting vessels to meet the requirement of oxygen and nutrition demanded by tumor for its continuous aggress and progress.4, 5 In spite of the fact that glioma described with the distinct feature of highly vascularization, the anti-angiogenesis, however, has been supposed to have some inevitable detrimental aspects such as the reactive resistance mediated largely by the tumor microenvironment as well as accelerating tumor cell invasion and tumor metastasis mainly activated by the hypoxic response.6-8 To solve these problems above, ligand based nanoparticles-mediated tumor cells and vascular endothelial cells dual targeting therapy was developed and represented as a relatively efficient means for glioma therapy.9 Nonetheless, the effectiveness of such method was compromised by the existing vasculogenic mimicry (VM) channels which provided an alternative microcirculation pathway for tumor.10 During the proliferation, partial tumor cells would be lack of indispensable oxygen and nutrition, to acclimatize itself to the tumor environment, the residual aggressive or invasive cancer cells are capable of form highly patterned vascular channels.11 Apart from stimulating the formation of neovascularization, the VM channels, formed by tumor cells without participation of endothelial, were also generated as a complement of angiogenesis in many cancers including glioma.12,

13

VM channels are highly

drug-resistant to conventional anti-angiogenic agents and thus closely associated with tumor recurrence and leads to increasing patient mortality.14-16 Therefore, besides 2


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tumor neovasculature and glioma cells, VM channels would be another important target for anti-glioma therapy. K237 (HTMYYHHYQHHL-NH2) peptide, isolated from phage display, was designed to bind to KDR (VEGFR-2) receptors which are highly expressed in tumor neovasculature.17 As VEGF-KDR interaction plays a pivotal role in the proliferation of cultured primary human umbilical vein endothelial cells (HUVEC), hence the peptide exerted a moderate activity of anti–angiogenesis

17, 18

Because of this distinct

features, application of K237 peptide was emerged as an effective method for destruction of tumor neovascular.19 In addition, it has been demonstrated that KDR receptors exist in VM channels and are served as progenitors for VM channels formation.20 This indicated that K237 peptide might be used as a novel tactic for simultaneous destruction of angiogenesis consisting of endothelial cells and VM channels composing of tumor cells. CVNHPAFAC-NH2 peptide, a tumor-homing peptide which showed a great similarity with a region from the human SCUBE2 protein, selected via the phage-display technology, was capable of binding to human sonic hedgehog.21 As generally accepted that sonic hedgehog (SHH) signaling pathway is important in regulating responses of cells such as cellular proliferation, survival, growth and other organism.22, 23 Aberrant activation of the pathway of SHH/Gli1 was considered closely associated with various tumorigenesis including glioblastoma, pancreas, melanoma, colorectal, and prostate carcinomas.22,

24

Importantly, the sonic hedgehog was

abundant in the tumor microenvironment and always involved in the tumor angiogenesis and overexpressed in tumor cells of glioblastoma.25, 26 In the work of finding of CVNHPAFAC-NH2, the peptide was further coupled to K237 peptide via a GYG linker to form a new sequence CVNHPAFACGYGHTMYYHHYQHHL-NH2 (CK),21 which has been demonstrated to be able to bind to SHH and VEGFR-2 simultaneously.21 Therefore, this CK peptide was hypothesized to be able to serve as an efficient targeting ligand to enhance the therapeutic effect of glioma by simultaneously delivering chemotherapeutics to VM channels, endothelial cells and glioma cells. 3



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In the present study, to justify the above hypothesis, PEG-PLA was used to develop a drug delivery system (DDS) and be loaded with paclitaxel for tumor therapy, and the prepared NP-PTX was modified on its surface with CK peptide for precise drug delivery. To evaluate the cellular targeting ability and endocytosis mechanism of CK-NP, HUVEC cells (always used as the model of neovascular endothelial cells) and U87MG cells were used in the in vitro experiments. DiR, a near infrared dye, were encapsulated into nanoparticles to study the bio-distribution of CK-NP and quantitative evaluate its tumor targeting effect compared with unmodified NPs. Anti-tumor effect of CK-NP-PTX was finally evaluated in the nude mice bearing intracranial U87MG glioma. Besides, the toxicity of DDS prepared in this study was also analyzed in the normal mice. 2. Results and discussion VM channels was widely exists in many malignant tumors including melanoma, lung

cancer,

hepatocellular

carcinoma,

mesothelial

sarcomas,

alveolar

rhabdomyosarcomas and also glioma.27-31 Importantly, completely different from angiogenesis, VM channels are tumor cell-constituted meshwork which is independent of endothelial cells and always associated with poor prognosis in patients with glioma.31, 32 In order to achieve an efficient therapy of glioma, a PTX loaded nanoparticles was prepared in this study and modified on its surface with CK peptide for simultaneous targeting deliver chemotherapeutics to glioma cells, angiogenesis and VM channels. It was demonstrated that the favorable stability, optimized size and targeting moieties facilitated selected accumulation of the PTX loaded nanoparticles at the tumor site, which finally resulted in an efficient anti-VM formation, killing of glioma cells and endothelial cells of angiogenesis. 2.1 Characterization of nanoparticles Since physicochemical properties of nanoparticles are the critical factors affecting the pharmacokinetics (PK), biodistribution and intratumoral penetration of nanoparticles.33 Therefore, controlling the particle size, zeta potential is of significance to prolong the nanoparticulate blood circulation. In our study, the average hydrodynamic diameter of NPs was examined and exhibited the size of approximate 4


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above 100 nm, which is optimal for utilizing the enhanced permeability and retention (EPR) effect.34 Meanwhile, the Zeta-potential, EE and LC of NPs were also investigated, results showed in Table 1 indicating that there was no obvious effect after peptide modified nanoparticles, this might ascribed to the targeting moieties on the surface of NPs.35 Importantly, after modification of peptides (CVNHPAFAC, K237 and CK), the size and other properties of nanoparticles showed a negligible change indicating that the modification of peptide exerted unconspicuous effect on the nanoparticles. The TEM images showed a spherical shape and uniform size distribution of NP-PTX, CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX. Besides, all of the nanoparticles displayed a core-shell structure and an undetectable phenomenon of aggregation (Fig 1A, B, C and D). The results of XPS analysis showed that nitrogen on the surface of CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX were 0.74%, 0.69% and 0.71%, respectively, while undetectable signal on the unmodified NPs. The conjugation efficiency of CVNHPAFAC peptide, K237 peptide and CK peptide were found to be 55.07%, 61.19% and 57.37%, respectively. The modification density of CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX were rather close (696, 739 and 683 molecules conjugated to each NP, respectively). The stability of nanoparticles is one of the key factors to determine the distribution of chemotherapeutics and finally affect the toxicity of PTX loaded NPs.36, 37 Hence , the stabilities of NP-PTX and the peptide-functioned ones were both studied with using DMEM containing 10% FBS as the medium. Results showed that the size of various nanoparticles did not change obviously within the determined days (Fig 1E).

Table 1. Characterization of different nanoparticles (Data represent with the mean 5



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value). Nanoparticles NP-PTX CVNHPAFAC-NP-PTX K237-NP-PTX CK-NP-PTX

size(nm)

PDI

ZP (mV)

EE (%)

LC (%)

105.76 116.95 115.89 117.36

0.092 0.157 0.148 0.172

-33.35 -27.59 -28.87 -26.72

51.17 49.36 50.23 48.72

1.53 1.42 1.47 1.36

Size: Mean hydrodynamic diameter; PDI: Polydispersity index; ZP: Zeta potntial; EE: Encapsulation efficiency; LC: Loading capacity.

Figure 1. Characterization of NPs and peptide functionalized NPs. Particle size distribution and TEM (transmission electron microscope) image of NP-PTX (A), CVNHPAFAC-NP (B), K237-NP-PTX (C) and CK-NP-PTX (D). (E) Stability study of NPs in DMEM with 10% FBS. (F) PTX release profiles from NP-PTX, CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX in PBS (pH 7.4) with 0.5% Tween-80 and PBS (pH 7.4) containing 10% rat plasma at 37ºC. The bar is 200 nm. 2.2 Cumulative release of PTX The PTX release from nanoparticles was evaluated in vitro under different conditions (PBS containing 0.5% tween 80 or 10% rat plasma, pH 7.4). At 6


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predetermined time points (0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 48, 72, 96 h), the release sample was analyzed and cumulative release rate curve was profiled. Results showed in Fig 1F demonstrated clearly that all the PTX formulations prepared in this study exhibited a controlled-release behavior and displayed a similar trend under the same condition, indicating that after modification of peptides, the release pattern of PTX did not change obviously. The PTX release from the nanoparticles in the media containing rat plasma (84.36%,

83.69%, 81.99%, and 80.76% for NP-PTX,

K237-NP-PTX, CVNHPAFAC-NP-PTX and CK-NP-PTX, respectively) is slightly faster than that in the Tween 80-contained media (71.02%, 68.66%, 70.69%, and 70.76% for NP-PTX, K237-NP-PTX, CVNHPAFAC-NP-PTX and CK-NP-PTX, respectively), which might contributed to the enhanced matrix erosion in plasma.38 2.3 Immunofluorescent staining of SHH The expression of SHH in glioma slides was detected through staining with Alexa555®-conjugated goat anti-rabbit IgG secondly antibodies. As shown in Fig 2, the glioma slide displayed a higher immunoreactivity of SHH which is in accordance with previously study,26, 39 such results suggested an active SHH/GLI1 signaling in glioma.

Figure 2. Immunofluorescence staining for sonic hedgehog (SHH) in the glioma tissue. Blue represents nuclei stained with DAPI. Red represents SHH receptors visualized by Alexa555®-conjugated goat anti-rabbit IgG secondly antibodies. (Left: original 7



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magnification 20 ×. Right: original magnification 40 ×) 2.4 Cellular selectivity As shown in Fig 3A, the fluorescent signals detected in HUVEC cells under the fluorescent microscopy indicated a peptide-mediated means of cellular uptake of nanoparticles, and the CK-NP exhibited much higher accumulation in cells compared with CVNHPAFAC-NP and K237-NP, which confirming that the coupled peptide had an incremental effect. For the U87MG cells, only CVNHPAFAC-NP and CK-NP displayed an enhanced effect, but not the K237-NP. It was ascribed to K237 peptide which was selected as tumor angiogenesis rather than tumor cells targeting peptide. Quantitative results in the Fig 3B further confirmed the peptide-dependent cellular association of nanoparticles with a 2.38, 3.05 and 3.81 folds higher in HUVEC cells and 2.34, 1.21 and 2.56 folds higher in U87MG cells when compared with that of NP for CVNHPAFAC-NP, K237-NP and CK-NP, respectively.

8


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Figure 3. Qualitative (A) and quantitative (B) analysis of cellular association of coumarin-6-labeled NPs and peptide functioned NPs in vitro. In image A: (a) Fluorescence of nanoparticles in HUVEC cells. (b) Bright field image of HUVEC cells. (c) Fluorescence of nanoparticles in U87 cells. (d) Bright field image of U87MG cells. In image B: (a) Quantitative study of cellular uptake of nanoparticles in HUVEC cells. (b) Quantitative study of cellular uptake of nanoparticles in U87MG cells. Green: coumarin-6 labeled nanoparticles. Original magnification: 20 ×. Data represented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001 significantly higher than the cellular association of unmodified NP. 2.5 Cellular endocytosis mechanism and subcellular location 9



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Endocytosis plays a key role in the process of cellular association of nanoparticles and it involves at least four basic mechanisms: caveolae-mediated endocytosis, clathrin-mediated

endocytosis,

micropinocytosis

and

clathrin

and

caveolae-independent endocytosis.40, 41 As shown in Fig 4A and B, cellular uptake of CK-NP in HUVEC cells and U87MG cells was both energy-dependent, Golgi apparatus and endosomes-mediated, as it was significantly reduced by NaN3, BFA and monensin. In HUVEC cells, the association of CK-NP was also restricted by chlorpromazine and M-β-CD, indicating that the endocytosis was also clathrin and lipid raft –mediated while caveolae and microtubule were involved in the process of U87MG uptake of CK-NP as the accumulation of nanoparticles decreased obviously after cells treated with colchicines and cyto-D. Importantly, after pretreated with CK peptide, both cells exhibited an extremely lower uptake of CK-NP compared with that of the non-inhibited control group, indicating that modification of CK peptide contributed to an enhanced effect of cellular association of nanoparticles in both HUVEC cells and U87MG cells. In contrast, when pretreated with K237 peptide and CVNHPAFAC peptide, respectively, HUVEC cells did not exhibited a decreased cellular uptake. This was mainly because CK peptide was composed of the two kinds of peptides. But in U87MG cells, CVNHPAFAC peptide significantly restricted the cellular association of CK-NP while K237 peptide did not, indicating that U87MG cells were positive for expression of SHH but not VEGFR-2. For the colocation assay, results shown in Fig 5A and Fig 6A demonstrated that after a 0.5 h incubation period, most nanoparticles coexisted with endosomes indicating that cellular uptake of NP and CK-NP was endosomes associated. Efficient endosomal escape is required for CK-NP to effectively kill tumor cells and endothelial cells. After 2 h incubation period, it was found that most CK-NP but not NP distributed in cytoplasm of HUVEC cells (Fig 5B) and U87MG cells (Fig 6B).

10


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Figure 4. Cellular uptake of coumarin-6 labeled CK-NP in the presence of various endocytosis inhibitors in HUVEC cells (A) and U87MG cells (B). Data represented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 significantly different with that of control.

Fig 5. Intracellular localization of coumarin-6 labeled NP and CK-NP incubated with HUVEC cells for 0.5 h (A) and 2 h (B). Blue represents nuclei stained with DAPI. 11



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Green represents particles that tracked by coumarin-6, red represents endosomes labeled by Lysotracker Red.

Fig 6. Intracellular localization of coumarin-6 labeled NP and CK-NP incubated with U87 cells for 0.5 h (A) and 2 h (B). Blue represents nuclei stained with DAPI. Green represents particles that tracked by coumarin-6, red represents endosomes labeled by Lysotracker Red. 2.6 MTT assay Given that elevated uptake of nanoparticles could resulted in higher sensitivity of cells to chemotherapeutics,42 anti-proliferation assay was performed to evaluate the effect of the various peptide-functionalized PTX-loaded nanoparticles. Cell viability of HUVEC cells and U87MG cells was profiled in the Fig 7A and B, and the IC50 value was also calculated to intuitionistic evaluate the anti-proliferation effect of the PTX formulations. Results showed that HUVEC cells exhibited the highest sensitivity to CK-NP-PTX with the IC50 value of 47.84 ng/ml, lower than that to 12


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CVNHPAFAC-NP-PTX (88.30 ng/ml), K237-NP-PTX (69.23 ng/ml), NP-PTX (163.1 ng/ml) and Taxol® (206.0 ng/ml). In the case of U87MG cells, IC50 value of 61.44 ng/ml, 66.23 ng/ml, 119.9 ng/ml, 128.0 ng/ml, and 163.5 ng/ml were obtained for CK-NP-PTX,

CVNHPAFAC-NP-PTX,

K237-NP-PTX,

NP-PTX

and

Taxol,

respectively.

Figure 7. Cellular viability of HUVEC cells (A) and U87 cells (B) after treated with various PTX formulations (Taxol®, NP-PTX, CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX) for 48 h at 37 ºC. 2.7 Inhibition of VM channels formation The branches of capillary-like tube in Fig 8A represented the formation of VM channels. When compared with the non-PTX control, the cells treated with K237-NP-PTX and CK-NP-PTX displayed higher degree of VM channels damage while a negligible destruction was observed following the Taxol® and NP-PTX treatments. Importantly, a certain degree of tube formation inhibition was also induced by CVNHPAFAC-NP-PTX. Such results were further demonstrated through quantitative calculation (Fig 8B).

13



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Figure 8. In vitro evaluation of destruction of VM channels after treated with Taxol®, NP-PTX, CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX. The PTX free group was used as control. (A) Qualitative detection under a phase-contrast microscopy and photographed at 40 magnification. (B) Quantitative analysis via the Image J® 1.46 version program. 2.8 In vivo imaging Considering that multiple factors affected the tumor targeting efficiency of nanoparticulate drug delivery, for example the macrophages of the reticuloendothelial system would captured the stealth nanoparticles and finally squelch the therapy effect,43 the evaluation of tumor targeting effect of nanoparticles in vivo was performed here. Results showed that peptide-functioned NP exhibited a more selective accumulation at tumor site when compared with the unmodified NP (Fig 9A). More importantly, the mice treated with DiR-labeled CK-NP demonstrated the highest aggregation at the tumor site, indicating that CK peptide achieved the targeting effect of both CVNHPAFAC and K237 peptide after the coupling. Such results were further 14


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confirmed by ex-vivo imaging and semi-quantitative fluorescence density analysis as shown in Fig 9B, C and D.

Figure 9. (A) In vivo distribution of DiR labeled NP (a), CVNHPAFAC-NP (b), K237-NP (c) and CK-NP (d) in U87MG tumor bearing mice at 2 h, 6 h, 12 h and 24 h. (B) Ex-vivo imaging of dissected organs after 24 h post-injection. (C) and (D) Semi-quantitative analysis of the fluorescent intensity of DiR labeled nanoparticles in different organs and tumors. 2.9 Distribution of nanoparticles in tumor Many treatments of glioma were compromised by the limited tumor penetration and vascular permeability of chemo-agents.44 Therefore, an effective treatment required the ability to access the inside avascular region.45 In the evaluation of tumor distribution of nanoparticles, as shown in Fig 10, the fluorescence intensity of NP exhibited the weakest signal and only a few of nanoparticles accumulated around of tumor blood vessels. The KDR targeting peptide K237-functioned nanoparticles 15



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exhibited a stronger vascular accumulation, CVNHPAFAC-NP not only aggregated around vascular but also limit distributed into the interior of tumor, while CK-NP exhibited the strongest fluorescence intensity not only around the neovasculature but also tumor parenchyma. Such results indicated that the combinative peptide, CK peptide, possessed the highest tumor targeting effect which was superior to any one of the two peptides.

Figure 10. Distribution of nanoparticles in tumors 3 h after administration. Blue: DAPI stained cell nuclei. Green: Coumarin-6-labeled nanoparticles. Red: CD31 stained blood vessels. 2.10 Anti-VM channels effect in vivo As shown in Fig 11, compared with that of the control group which treated with saline,

the

VM

channels

of

glioma

tissue

of

the

16


mice

treated

with

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CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX exhibited a different degree of fracture. Obviously, the mice treated with CK-NP-PTX displayed the strongest VM channels destruction, while the group treated with NP-PTX displayed a negligible destruction of VM channels. Importantly, after i.v. injected with K237-NP-PTX, the VM channels of the mice glioma brain exhibited a relatively obvious destruction, indicating that VEGFR-2 played a pivotal role in formation of VM channels. Meanwhile, CVNHPAFAC-NP-PTX also exhibited a feeblish anti-VM channels effect due to the fact that VM channels were formed by glioma cells which was positive for SHH expression.

Fig 11. Evaluating the destroying effect of various PTX formulations on VM channels 17



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in tumor site after treated with saline, Taxol®, NP-PTX, CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX, respectively. (A) Qualitative detection under a phase-contrast microscopy and photographed at 40 magnification. (B) Quantitative analysis via the Image J® 1.46 version program. 2.11 Anti-glioma efficacy of nanoparticles in glioma-bearing mice As shown in Fig 12A, the body weight of mice treated with saline, Taxol® and NP-TX exhibited a downward trend. In contrast, a rising trend was observed in the mice treated with CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX. The detection of survival time showed that the mice treated with CK-NP-PTX exhibited the longest medium survival time (57 days), while those treated with saline, Taxol® and NP-PTX, CVNHPAFAC-NP-PTX and K237-NP-PTX achieved the medium survival time of 17, 20, 27, 41 and 37 days, respectively (Fig 12B). H&E staining was applied to examine the histopathologic changes in glioma after treated with various PTX formulations.46, 47 As shown in Fig 11C, glioma treated with CK-NP-PTX

exhibited

the

most

significant

apoptosis

of

tumor

cells.

CVNHPAFAC-NP-PTX displayed a slightly higher toxicity to tumor cells when compared with K237-NP-PTX, indicating that consistent with previously study,48, 49 dual targeting therapy was superior to glioma cells or endothelial cells therapy only to some extent. For the evaluation of toxicity to normal tissues, it was founded that there were no significant microscopic changes of the brain and main organs in normal mice at the current therapeutic dosage of PTX (Fig 13), indicating that the prepared PTX-loaded nanoparticles leaded to unconspicuous toxicity.

18


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Figure 12. Antitumor efficacy of Taxol®, NP-PTX, CooP-NP-PTX (PTX dose of 5 mg/kg) and saline, respectively. (A) Changes in body weights of mice after treatment. (B) Kaplan-Meier survival curve of tumor bearing mice. (C) H&E staining of glioma tissue sections after treated with various PTX formulations including Taxol®, NP-PTX, CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX. The saline group was used as control.

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Fig 13. Histological analysis of brain, heart, liver, spleen, lung and kidney obtained from normal nude mice after treated with saline, NP-PTX, CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX, respectively. 3. Conclusion To construct a nanoparticulate DDS which can simultaneous targeted deliver chemotherapeutics to glioma cells, angiogenesis and VM channels of glioma, a core-shell nanoparticles formed with the blend of MPEG-PLA and HOOC-PEG-PLA were prepared in this study via the emulsion/solvent evaporation method and finally decorated on its surface with a tumor homing peptide, CK peptide, which could selectively bind to KDR receptors and SHH receptors. For the therapy of glioma, PTX was selected as the model drug and the resulted CK-NP-PTX exhibited an average particle size of 117.36 nm with a uniformly spherical shape observed under TEM. The confirmation of peptide conjugation was performed through the XPS analysis. In vitro cellular uptake experiments demonstrated that the enhanced cellular uptake of 20


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nanoparticles was exactly mediated by CK peptide and in vivo tumor targeting assay showed that CK-NP was highly selective accumulated at the tumor site compared with unmodified NP. In addition, CK-NP-PTX exhibited the highest anti-VM effect both in vitro and in vivo, and the glioma bearing-mice treated with CK-NP-PTX achieved the longest survival. Importantly, the experiment results of CK-NP-PTX in vitro and in vivo was both superior to CVNHPAFAC-NP-PTX and K237-NP-PTX. Collectively, the CK-NP-PTX prepared in this study hold great potential in the multi-targeting therapy of glioma. 4. Experimental section 4.1 Materials The CVNHPAFAC-NH2 peptide, K237 peptide (HTMYYHHYQHHL-NH2) and CK peptide (CVNHPAFACGYGHTMYYHHYQHHL-NH2) were synthesized by China Peptides Co., Ltd (Shanghai, China). Methoxy poly (ethylene glycol)

3000-poly

(lactic

acid) 34000 (MPEG-PLA) were kindly provided by East China University of Science and Technology. R-carboxyl-poly (ethylene glycol) – poly (lactic acid) (COOH-PEG-PLA, Mw 33,400 Da) block copolymers were synthesized via ring-opening polymerization of lactide and initiated by HOOC-PEG-OH as described elsewhere.50 Coumarin-6, DiR (1, 1’-dioctadecyl -3, 3, 3’, 3’-tetramethyl indotricarbocyanine Iodide) was provided by Biotium (Hayward, CA). DAPI (4, 6-diamidino-2-phenylindole) was purchased from Molecular

Probes

(Eugene,

OR,

USA),

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxy-succinimide (NHS), Hoechst 33258 and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). PTX was obtained from Xi’an Sanjiang Biological Engineering Co. Ltd (Xi’an, China) and Taxol® was from Bristol-Myers Squibb Company. Alexa Fluor 594 anti-mouse CD31 antibody was purchased from Biolegend and growth factor-reduced Matrigel matrix was purchased from BD Bioscience (San Diego, CA, USA). All the other solvents were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) and were of analytical or chromatographic grade. Dulbecco’s Modified Eagle Medium (DMEM) (high glucose) cell culture medium,

certified fetal bovine serum (FBS), 21



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penicillin/streptomycin stock solutions and 0.25% Trypsin-EDTA were all obtained from Invitrogen Co, USA. 4.2 Cells and animals Primary human umbilical vein endothelial cells (HUVEC) were purchased from Cascade Biologics (USA) ， U87MG glioblastoma cells were obtained from Cell Institute of Chinese Academy of Sciences (Shanghai, China). Both of those cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37ºC in a 5% CO2/95% air humidified environment incubator (Thermo HERA cell, USA). Specific pathogen-free male BALB/c nude mice (20 ± 2 g) were purchased from the BK Lab Animal Ltd (Shanghai, China) and maintained at 25 ± 1ºC with free access to food and water. All the animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Fudan University (Shanghai, China). 4.3 Establishment of mice model Glioma bearing mice model was established via the intracranial injection method as described previously.50 Briefly, trypsinized U87MG cells (5 × 105 cells/5 µl in pH 7.4 PBS) were injected into the right corpus striata of nude mice by using a stereotaxic apparatus. Then all the mice were raised in standard condition for two weeks and applied for subsequent experiments. 4.4 Preparation of peptide-functioned NP and unmodified NP Nanoparticles was prepared via the emulsion/solvent evaporation method as described previously.51 Briefly, the blend of MPEG-PLA (22.5 mg), HOOC-PEG-PLA (2.5 mg) and PTX (0.25 mg) were dissolved in 1 ml of dichloromethane, 2 ml of 1% sodium cholate aqueous solution were added, and the mixture was subjected to ultrasonication (2.4 min, 280 W) using probe sonicator (Ningbo Scientz Biotechnology Co. Ltd., China) under the condition of ice bath. After dispersed with 8 ml of 0.5% sodium cholate aqueous solution, the dichloromethane were evaporated by a ZXB98 rotavapor (Shanghai Institute of Organic Chemistry, China), then the obtained aqueous phase was subjected to centrifugation (15,000 rpm and 45 min) via 22


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a TJ-25 centrifuge (Beckman Counter, USA). Finally, the unmodified NP-PTX was collected and persevered at 4℃ for further use. K237-NP-PTX, CVNHPAFAC-NP-PTX and CK-NP-PTX were prepared via the EDC/NHS technique as reported previously.52 Briefly, excess EDC (200 mM) and NHS (100 mM) were used to activate the carboxyl of NP-PTX in deionised water at room temperature and stir gently for 30 min. After discarded the residual EDC and NHS through centrifugation at 14,500 rpm for 45 min, the collected activated NP-PTX was resuspended with PBS (pH 7.4) and reacted for 6 h with K237 peptide, CVNHPAFAC peptide and CK peptide (at the molar ratio of carboxyl to peptide 1:1), respectively. The obtained peptide-functionalized NP-PTX solution was centrifuged at 15,000 rpm for 1 h to remove the unconjugated peptide in supernatant. Coumrin-6and DiR-labled nanoparticles were prepared via the similar procedure except that PTX was replaced with coumrin-6 and DiR, respectively. 4.5 Characterization of nanoparticles Dynamic light scattering detector (Zetasizer, Nano-ZS, Malvern, UK) was used to determine the particle size and zeta potential (ZP) of nanoparticles. The morphology of nanoparticle was examined under the transmission electron microscope (TEM, H-600; Hitachi, Japan) after negative staining with sodium phosphortungstate solution. Conjugation of peptide to the surface of nanoparticles was verified through X-ray photoelectron spectroscopy (XPS) analysis via a RBD upgraded PHI-5000C ESCA system (Perkin Elmer) after lyophilized using an ALPHA 2-4 Freeze Dryer (0.070 Mbar Vakuum, -80ºC, Martin Christ, Germany). The drug encapsulation efficiency (EE) and loading capacity (LC) of PTX in the nanoparticles were also determined. The NP samples were dissolved in acetonitrile and subjected to the high performance liquid chromatography (HPLC) analysis as described previously,53 and finally calculated as indicated below (n = 3). EE (%) =

Amount of PTX in the nanopartic les × 100% Total amount of PTX added

LC(%) =

Amount of PTX in nanoparticles ×100% nanoparticles weight 23



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The amount of CVNHPAFAC peptide, K237 peptide and CK peptide conjugated on the surface of nanoparticles was quantified by the BCA protein assay as described previously,54 and the peptide conjugation efficiency (CE %) was finally calculated according to the formula as follows: CE (%) =

Amount of peptide conjugated on the surface of nanoparticle × 100% Total amount of peptide added

To calculate the peptide surface density (S), the number of peptide molecules was divided by the average number (N) of nanoparticles according to a method described previously. 51The number (N) of the nanoparticles was calculated as follows: N=

6 × m (m: nanoparticle weight; D: number-based mean nanoparticle π× D3 ×ρ

diameter; ρ: nanoparticle weight per volume unit, estimated to be 1.1 g/cm3). We further studied the stability of NP formulations in the media of DMEM containing 10% FBS after seven-day incubations. The size of NPs was measured carefully at predetermined time points.

4.6 In vitro release of PTX loaded nanoparticles In vitro PTX release behavior of nanoparticles was determined via the method of dialysis using phosphate buffer solution (PBS, pH 7.4) containing 0.5% (V/V) tween-80 or 10% (V/V) rat plasma as the release midia.55 Briefly, 1 ml of PTX formulation with the concentration of PTX adjusted to 100 µg/ml was introduced into a dialysis bag (MWCO 8000 Da; Green Bird Inc, Shanghai, China) and incubated in 30 ml of release media at 37ºC at the shaking speed of 120 rpm for 96 h. Then, 0.2 ml of release sample was withdrawn at the predetermined time points and HPLC analysis was used to analyze the concentration of PTX.33

4.7 Immunofluorescence staining of SHH Slides of glioma were prepared via the method reported previously.33 Briefly, glioma-bearing mice were sacrificed with the brains harvested, fixed in 4% formaldehyde and then dehydrated in graded sucrose solution. Then the brains were imbedded in OCT (Sakura, Torrance, CA, USA), frozen at 80ºC and sectioned at 10 µm. For the evaluation of SHH expression, slides were immersed in 0.2 % Triton 24


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X-100 for 5 minutes at room temperature. Then the primary rabbit anti-SHH antibodies were introduced followed by the staining with Alexa555®-conjugated goat anti-rabbit IgG secondly antibodies. Cell nuclei were visualized by DAPI and the expression of SHH was observed under a Zeiss LSM 510 confocal microscope.

4.8 In vitro tumor cells and endothelia cells targeting assay To evaluate the targeting effect of peptide-modified NP on glioma cells and endothelial cells in vitro, cellular uptake examination was performed with coumanrin-6 acting as the fluorescent probe. HUVEC cells and U87MG cells were seeded in 24-well plate at the density of 5 × 104 cells per well. After incubated at 37 ºC and 5% CO2 for 24 h, each well was replaced with 1 ml serum-free fresh medium containing 200 µg coumarin-6 labeled nanoparticles. One hour later, the nanoparticles were discarded and the cells were washed twice with cold PBS (pH 7.4). Then the cells were fixed with 4% formaldehyde for 15 min and observed under the fluorescent microscopy (Leica DMI4000 B, Germany). For quantitative analysis, 5 × 103 HUVEC cells or U87MG cells were seeded in each well of 96-well plate and cultured for 24 h under standard condition. Then, 200 µg/ml coumarin-6-labeled nanoparticles were added into the wells and incubated for one hour. After that, the cells were washed twice with cold PBS to terminate the cellular association of nanoparticles and then fixed with 4% formaldehyde for 15 min. To visualize the nucleus, cells were subjected to 2 µg/ml Hochest 33258 staining for 10 min. Finally, a KineticScan® HCS Reader (version 3.1, Cellomics Inc., Pittsburgh, PA, USA) was used for the quantitative analysis of the cellular uptake of the nanoparticles.

4.9 Cellular endocytosis mechanism and subcellular location For revealing the mechanism of the cellular uptake of nanoparticles, various endocytosis inhibitors (including chlorpromazine, colchicines, cyto-D, BFA, filipin, NaN3, methyl-β-cyclodextrin (M-β-CD), monensin, nocodazole, CVNHPAFAC peptide, K237 peptide and CK peptide) were introduced. HUVEC cells and U87MG cells were seeded in 96-well plates at the density of 5 × 103 cells per well and incubated for twenty-four hours, then the mentioned endocytosis inhibitors and 25



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Page 26 of 36

peptides were added at an appropriate concentration as reported previously.52 One hour later, the cells were washed twice with PBS and incubated with 200 µg/ml coumarin-6-labeled CK-NP for another one hour. After that, cells were washed twice with PBS, fixed in 4% formaldehyde, and then treated with Hochest 33258 for nuclei staining before the quantitative observation. As endosomes were always involved in the endocytosis process, a colocation assay was further performed to determine the subcellular fate of CK-NP. Briefly, HUVEC cells and U87MG cells were seeded in the glass-bottom dishes at a density of 1 × 104 per dish. After 24 h incubation, cells were treated with 200 µg/ml coumarin-6-labeled NPs and CK-NPs for 2 h or 6 h. Then 50 nmol/l Lysotracker Red was introduced and incubated with cells for 30 min. After fixing the cells with 4% formaldehyde and staining the nuclei with DAPI for 10 min, the images were photographed via a confocal microscope (TCS SP5, Leica, Germany).

4.10 MTT assay MTT assay was used to investigate the cytotoxicity of the peptide-functioned nanoparticles. Both HUVEC cells and U87MG cells were seeded in 96-well plates at the density of 5 × 103 per well when the cells were in the logarithmic growth phase. After attached for 24 h, the cells were exposed to different PTX formulations (Taxol®, NP-PTX,

CVNHPAFAC-NP-PTX,

K237-NP-PTX

and CK-NP-PTX) at the

concentration of PTX ranged from 1 ng/ml to 1 × 103 ng/ml and cultured for 48 h. Thereafter, 20 µl MTT (5 mg/ml) was added and interacted with cells for 4 h followed by dissolved the formative formazan crystals through the addition of 150 µl DMSO. Finally, the cell viability was evaluated via a microplate reader (Thermo Multiskan MK3, USA) and IC50 values were calculated.

4.11 VM channels formation To

evaluate

the

ability

of

CVNHPAFAC-NP-PTX,

K237-NP-PTX

and

CK-NP-PTX to destroy the formation of VM channel in vitro, a Matrigel-based tube formation assay was performed as described previously.56 Prechilled 48-well plate was plated with 150 µl growth factor-reduced Matrigel and incubated for 45 min at 37 ºC. After Matrigel was polymerized, U87MG cells (1 × 104) were trypsinized and 26


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resuspended

with

serum-free

medium

containing

Taxol®,

NP-PTX,

CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX, respectively, and then seeded onto the Matrigel. The medium containing no paclitaxel formulation was used as the control. Eight hours later, the plate was subjected to a phase-contrast microscopy and photographed at 40 magnification (n = 3). Then the images were subjected to quantitative analysis via the Image J® 1.46 version program.

4.12 In vivo imaging Glioma bearing nude mice was used to evaluate the tumor-targeting efficacy of the peptide-functionalized nanoparticles in vivo. DiR-loaded NP, CVNHPAFAC-NP, K237-NP and CK-NP were injected intravenously at the dosage of 1 mg/kg of DiR. Then the biodistribution of nanoparticles was observed through an In Vivo IVIS spectrum imaging system (PerkinElmer, USA) at 2 h, 6 h, 12 h and 24 h, respectively. The mice were sacrificed at 24 h after injection with organs (including brain, heart, liver, spleen, lung and kidney) collected and imaged with the fluorescence intensity of each of organs semi-quantitative analyzed.

4.13 Penetration of nanoparticles into tumor To investigate the distribution of nanoparticles in glioma tissue, twelve glioma bearing mice were randomly divided into four groups (n=3) and intravenously injected

coumarin-6-labeled

NP,

CVNHPAFAC-NP,

K237-NP

and

CK-NP,

respectively. After three hours, the mice were sacrificed with the brain slides prepared as described above. For analyzing the localization of nanoparticles at the tumor site, Alexa Fluor 594 anti-mouse CD31 antibody was introduced to stain the tumor neovascular as reported previously,51 and then all the slides were subjected to confocal microscopy analysis (LSM710, Leica, Germany) after staining the nucleus with DAPI.

4.14 Anti-VM channels effect in vivo Eighteen glioma-bearing mice were randomly divided into six group and treated with Saline, Taxol®, NP-PTX, CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX, respectively, at day 0, 2, 4 and 6 post inoculation. For the evaluation of

in vivo destroying effect on VM channels, the mice above were sacrificed after the last 27



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intravenous injection with the tumor-bearing brains harvested. After embedded in paraffin, the brain slides were prepared and double stained with anti-CD34 and PAS as reported previously.57 Finally, the images were collected and subjected to statistical analysis (n = 3).

4.15 Anti-glioma efficacy of nanoparticles in glioma-bearing mice For the evaluation of anti-tumor effect in vivo, the mice were i.v. injected with Taxol®, NP-PTX, CVNHPAFAC-NP-PTX, K237-NP-PTX and CK-NP-PTX (dosing at 5mg/kg), respectively, at 0, 2, 4 and 6 days, with the group received saline was used as control. Then the survival time and relative body weight change of mice in each group were monitored and analyzed. To further detect the apoptosis of cells in glioma tissues induced by the PTX formulations, eighteen glioma-bearing mice were randomly divided into six groups and treated with various PTX formulations and saline. One week later, the mice were heart perfused and the brains were harvested, fixed with 4% Paraformaldehyde, then embedded in paraffin for further H&E staining. To evaluate the toxicity of the PTX loaded nanoparticles prepared in this study to normal organs, eighteen normal nude mice were randomly divided into six groups and treated with Taxol®, NP-PTX,

CVNHPAFAC-NP-PTX, K237-NP-PTX and

CK-NP-PTX (dosing at 5 mg/kg), respectively, at 0, 2, 4 and 6 days. The mice i.v. injected with saline were used as control. After that, mice were sacrificed with brains and major organs (including heart, liver, spleen, lung and kidney) harvested. Then the obtained tissues were embedded in paraffin followed by sectioning and H&E staining.

4.16 Statistical Analysis All the data expressed as comparison among multiple groups was performed by one-way ANOVA analysis followed by Bonferroni tests. The IC50 values were calculated via the GraphPad Prism® 5.0 version program. Statistical significance was defined as p < 0.05.

Acknowledgment This work was supported by National Key Basic Research Program (2013CB932500), National Natural Science Foundation of China (81373353), Grants 28


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from Shanghai Science and Technology Committee (13NM1400500) and Program for New Century Excellent Talents in University (NCET-12-0130).

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Shape Effects of Filaments Versus Spherical Particles in Flow and Drug Delivery. Nat. Nanotechnol 2007, 2, 249–255. (38) Guo, J.; Gao, X.; Su, L.; Xia, H.; Gu, G.; Pang, Z.; Jiang, X.; Yao, L.; Chen, J.; Chen,

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Multi-targeting Peptide-Functionalized Nanoparticles Recognized

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