iRGD-Decorated Polymeric Nanoparticles for the Efficient Delivery of

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iRGD-decorated polymeric nanoparticles for the efficient delivery of vandetanib to hepatocellular carcinoma: preparation and, in vitro and in vivo evaluation Jianguo Wang, Hangxiang Wang, Jie Li, Zhikun Liu, Hai-Yang Xie, Xuyong Wei, Di Lu, Runzhou Zhuang, Xiao Xu, and Shusen Zheng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03166 • Publication Date (Web): 06 Jul 2016 Downloaded from http://pubs.acs.org on July 18, 2016

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ACS Applied Materials & Interfaces

iRGD-decorated polymeric nanoparticles for the efficient delivery of vandetanib to hepatocellular carcinoma: preparation and, in vitro and in vivo evaluation Jianguo Wanga,, Hangxiang Wanga*, Jie Lia, Zhikun Liua, Haiyang Xiea, Xuyong Weia, Di Lua, Runzhou Zhuanga, Xiao Xua,* and Shusen Zhenga,*

a

The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003,

PR China; Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health,, Hangzhou, 310003, PR China;Key Laboratory of Organ Transplantation, Zhejiang Province, Hangzhou 310003, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, 310003, PR China

* Corresponding author: H.W. (email: [email protected]), X. X. (email: [email protected]) and S. Z. (email: [email protected])

Keywords: cancer nanomedicine; self-assembly; molecularly targeted agents; hepatocellular carcinoma; targeted delivery;

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Abstract: Molecularly targeted agents that are designed to target specific lesions have been proven effective as clinical cancer therapies; however, most currently available therapeutic agents are poorly water-soluble and require oral administration, thereby resulting in low bioavailability and a high risk of side effects due to dose intensification. The rational engineering of systemically injectable medicines that encapsulate such therapeutic payloads may revolutionize anticancer therapies and remains an under-explored area of drug development. Here, the injectable delivery of a nanomedicine complexed with an oral multitargeted kinase inhibitor, vandetanib (vanib), was explored using polymeric nanoparticles (NPs) to achieve the selective accumulation of drug payloads within tumor lesions. To demonstrate this concept, we used biodegradable amphiphilic block copolymer poly (ethylene glycol)-block-poly(D, L-lactic acid) (PEG-PLA) to nanoprecipitate this potent agent to form water-soluble NPs that are suitable for intravenous administration. NP-vanib induced cytotoxic activity by inhibiting the angiogenetic events mediated by VEGFR and EGFR kinases in tested cancer cells and inhibited the growth, tube formation and metastasis of HUVECs. The intravenously injection of NP-vanib into mice bearing HCC BEL-7402 xenografts more effectively inhibited the tumor than the oral administration of vanib. In addition, due to the modular design of these NPs, the drug-loaded particles can easily be decorated with iRGD, a tumor-homing and -penetrating peptide motif, which further improved the in vivo performance of these vanib-loaded NPs. Our results demonstrate that reformulating targeted therapeutic agents in NPs permits their systemic administration and thus significantly improves the potency of currently available, orally-delivered agents.


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1. Introduction Cancers have evolved a diverse set of signaling pathways to promote malignant states, e.g., uncontrolled proliferation, angiogenesis, the suppression of differentiation, and invasion 1. Therefore, molecularly targeted agents that are designed to target specific lesions have been widely used as clinical cancer therapies 2. In general, these therapeutics are highly selective and exhibit therapeutic benefits over conventional cytotoxic agents in various cancers, as evidence by clinical data 3. However, the inefficient delivery of these drugs at appropriate doses to the tumor site(s) of interest due to complex in vivo environments significantly limits their application. Moreover, most of these agents are often poorly water-soluble or incompatible with pharmaceutically acceptable excipients. Thus, they are routinely orally administered, which often results in low bioavailability and/or increased toxicity 4, 5. Therefore, novel efficient delivery technologies that can overcome the biological barriers posed by oral administration, enhance the bioavailability of drugs, and reduce the incidence of severe side effects are desirable. Numerous current studies have been focused on the discovery of new therapeutic targets derived from cancerous signaling pathways and new therapeutic agents designed to inhibit specific pathways 6. In addition, considerable progress has been made for the nanoformulation of conventional chemotherapeutics over past decades. However, few efforts have been devoted to reformulate these molecularly targeted therapeutic agents into appropriate delivery vehicles for maximizing the in vivo therapeutic efficacy. Packaging clinically approved therapeutics into nanoscale delivery platforms is likely to revolutionize anticancer therapies, because numerous studies have demonstrated that nanomaterials with diameters less than 200 nm can preferentially accumulate within solid tumors through the enhanced permeability and retention (EPR) effect 7, 8. The drug payloads encapsulated in biodegradable and biocompatible polymeric nanoparticles (NPs) are conferred with several favorable properties, such as increased aqueous solubility and chemical stability, 3 ACS Paragon Plus Environment


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sustained and tunable drug release profiles, and improved pharmacokinetics 9-11. Moreover, other valuable functionalities (e.g., tumor targeting ligands) can easily be integrated due to the modular particle surface 12. Recently, this NP technology promise as a strategy for the utilization of abandoned or clinically unavailable compounds, such as wortmannin and 7ethyl-10-hydroxycamptothecin (SN38), which failed to be clinically translated due to delivery challenges 13, 14. Despite the advantages of NP-mediated delivery of conventional cytotoxic agents and a growing interest in their clinical applications, the use of nanoparticulate platforms to encapsulate and deliver molecularly targeted agents remains in its infancy in oncology 15. Accordingly, we hypothesized here that assembling these therapeutics within appropriate nanocarriers changes their dosage route to expand their therapeutic windows. To examine this possibility, we selected vandetanib (vanib) as a model drug. Vanib is an oral angiogenesis inhibitor, primarily inhibiting several tyrosine kinases of cell receptors, including endothelial growth factor receptor (EGFR), vascular EGFR (VEGFR) and RETtyrosine kinase 16. Therefore, vanib was clinically approved to manage medullary thyroid cancer by the US Food and Drug Administration (FDA). Currently, vanib is under investigation for treatment of various tumors, including lung, prostate and ovarian cancer 17-19. Hepatocellular carcinoma (HCC) is characterized by highly vascular tumors that require VEGFR for tumor angiogenesis. Previous studies indicated that vanib may represent a potential treatment regimen for the systemic therapy of patients with advanced HCC 20-22. Positive progression-free survival (PFS) and overall survival (OS) trend was observed upon treating vanib; however, the therapeutic effect compared with placebo in phase II study was not statistically significant 20. This could be attributable to the low bioavailability due to oral administration and dose reduction induced by adverse side effects. Because this agent is water insoluble, we therefore envision that nanoparticulate delivery vehicles could significantly enhance solubility of vanib, thus making it applicable for systemic injection. In addition, 4 ACS Paragon Plus Environment

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tumor-specific ligands could further increase the accumulation of nanodrugs in desirable tumor lesions, while effectively reducing the occurrence of adverse side effects. In this article, we report a new systemically injectable and clinically viable nanomedicine that is characterized by the sustained release of encapsulated vanib, which targets the tumor for selective cancer therapy. Taking advantage of the intrinsic hydrophobicity of vanib, we hypothesized that this molecule can assemble in the hydrophobic core of appropriate amphiphilic block copolymers, e.g., poly (ethylene glycol)-block-poly(D,L-lactic acid) (PEGPLA), the latter of which has been clinically approved by the FDA 23. Several unique advantages can be obtained using PEG-PLA copolymers as follows: i) excellent biodegradability and biocompatibility; ii) prolonged systemic circulation after intravenous injection; iii) low critical micelle concentration (CMC), promising them as ideal drug delivery carriers24, 25. Additionally, to ensure tumor-specific delivery, we conjugated a small cyclic peptide motif, iRGD (CRGDK/RGPD/EC) 26, to the surface of the particle via maleimidethiol coupling (Figure 1). These highly water-soluble nanoformulations of vanib (termed NPvanib and iNP-vanib, respectively) could be intravenously injected, which circumvented low bioavailability associated with oral administration. We carefully examined the in vitro cytotoxicity of these NPs against several HCC cell lines and their ability to inhibit angiogenesis. To establish the potential of this NP fabrication to be translated to the clinic, we evaluated the antitumor efficacy in mouse models of human HCC. Our nanoparticle-mediated delivery platforms provide a simple, broadly applicable strategy to effectively enhance the potency and safety of molecularly targeted agents that have previously been limited to oral administration.

2. Materials and Methods 2.1. Materials, cell culture, and animals



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Poly (ethylene glycol)-block-poly(D, L-lactic acid) (PEG5k-PLA16k) and maleimidefunctionalized PEG-PLA (Mal-PEG8k-PLA16k) were obtained from Advanced Polymer Materials Inc. (Montreal, Canada). Vandetanib (ZD6474) was purchased from Selleck (USA), and its purity was 99.7%. VEGF was purchased from PeproTech. BEL-7402, Hep-G2, HuH-7 and HCC-LM3 cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). BEL-7402 cells were cultured in RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). Hep-G2, HuH-7 and HCC-LM3 cells were cultured in Earle’s minimum essential medium (MEM) containing 10% FBS and 1% nonessential amino acids. Human umbilical endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC), and cultured in DMEM containing 10% FBS. All cells were maintained at 37 °C in 5% CO2. Balb/C nude mice (Male, average body weight 18 g, 5 weeks old) were purchased from Beijing Huafukang Biological Technology Co. Ltd. (HFK Bioscience, Beijing). The mice were maintained at the animal facility of the Zhejiang Academy of Medical. All animal husbandry and procedures were approved by the Animal Care and Use Committee of Zhejiang University. 2.2. Preparation of vanib-loaded nanoparticles

Vanib-loaded NPs were prepared via the nanoprecipitation method 14, 27. Briefly, vanib (0.5 mg/ml) and PEG5k-PLA16k (10 mg/ml) were first dissolved in acetone and together dropped into water. The resulting solution was then stirred for 2 h to allow the acetone solvent to evaporate. Syringe filter (0.45 µm, Millipore, USA) was used to remove non-encapsulated drug aggregates. The NPs were concentrated using an Amicon Ultra-4 centrifugal filter (MWCO 10 kDa; Millipore) to construct vanib-loaded NPs (termed NP-Vanib). To conjugate the tumor-penetrating ligand, iRGD, to the surface of particles, the iRGD motif, which contains a reactive cysteine residue, was incubated with maleimide-functionalized PEG8k6 ACS Paragon Plus Environment

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PLA16k in DMSO to generate iRGD-PEG-PLA. Subsequently, the iRGD-PEG-PLA was coassembled with PEG-PLA at a ratio of 1/10 (w/w) to construct vanib-loaded, targeting NPs (termed iNP-Vanib). Given the success of nanoprecipitation for vanib, we next characterized the morphology of these paticles by dynamic light scattering (DLS) analysis and transmission electron microscopy (TEM). The particle size distribution and polydispersitye index (PDI) of the vanib-loaded NPs were measured using a Malvern Nano-ZS 90 laser particle size analyzer (Malvern, USA). Samples for TEM were stained with 1% uranyl acetate and observed using TECNAL 10 (Philips) at an acceleration voltage of 80 kV.

2.3 Encapsulation efficiency (EE) and drug loading (DL)

The drug encapsulation efficiency (EE) was determined by high-performance liquid chromatography (HPLC) analysis. After careful preparation of vanib-loaded NPs or iNPs using the nanoprecipitation method, the solutions were centrifuged at 2500 rpm for 30 min using ultra-filter (Amicon Ultra, MWCO 10 kDa, Millipore). The filtrate was collected and the amounts of free drugs were determined at 220 nm using analytic HPLC. All experiments were repeated three times, and all data were expressed as the mean ± SD. The EE values were calculated by Eqs. 1, and the percentages of drug loading (DL) were calculated using Eqs. 2 EE=WFed−Wfree/WFed×100%

(1)

DL=WFed−Wfree/Wtotal×100%

(2)

Where WFed, Wfree, and Wtotal represent the total amount of drug fed for encapsulation, the amount of free drug in the filtrate, and the total amount of the drugs fed for encapsulation and the materials used for NP fabrication, respectively. 2.4. In vitro drug release of vanib from polymeric NPs



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The vanib release from NPs was investigated in vitro using a dialysis-diffusion method. Briefly, 10 mL of the vanib-loaded NPs (0.1 mg/ml, vanib equivalent dose) were placed into the dialysis bag (molecular weight cutoff: 3 kDa) and suspended in releasing medium (PBS buffer containing 0.2% Tween 80; pH 7.4 and pH 4.6, respectively). The end-sealed dialysis bag was incubated against 50 mL of release medium at 37°C with gentle shaking (100 rpm). At predetermined time intervals (0.5, 1, 2, 4, 8, 12, 24, 36, and 48 h), the release media (1 mL) were collected and fresh media (1 mL) were supplemented. The content of the released vanib was determined using HPLC. The chromatographic conditions were used as follows: a gradient elution method of 20-80% acetonitrile/water in 0-16 min was applied at a flow rate of 1 mL/min at room temperature. During assays, an aliquot of 50 µL of each sample was injected into the analytic HPLC on a Hitachi Chromaster 5000 system equipped with a C18 reverse-phase column (5 µm, 250 mm × 4.6 mm, YMC Co., Ltd., Kyoto, Japan). UV detection was performed at 220 nm. 2.5. Cytotoxicity, apoptosis and cell cycle analysis of vanib-loaded NPs

The cell viability was measured using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. HCC cells were seeded in 96-well plates (5000 cells/well; 100 µl media) and incubated overnight. Subsequently, the adherent cells were treated with different concentrations of free vanib, NPVanib or iNP-Vanib (0.1, 0.5, 1, 2, 4, 8, 10, 20, or 50 µM). After 48 h treatment, each well was added with 10 µl of CCK-8 solution, and the absorbance was then measured at 450 nm after 2 h of incubation. All experiments were performed in triplicate. The half maximal inhibitory concentration (IC50) was calculated using the GraphPad prism 5.0 software. The ability of vanib-loaded NPs to induce apoptosis was assessed by flow cytometry (FCM). BEL-7402 cells were treated with free vanib, NP-Vanib or iNP-Vanib (10 µM) for 48 h. The cells were then harvested and washed twice with cold PBS. 100 µL of binding buffer 8 ACS Paragon Plus Environment

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was added to prepare cell suspension. Subsequently, 5 µl of FITC AnnexinV and 5 µl of propidium iodide were added (BD, USA), which was then gently mixed and incubated for 15 minutes in the dark. After then, 400 µl of binding buffer was added while gently mixing. The apoptotic rate of HCC cells were quantified by a flow cytometer (BD Biosciences, San Jose, CA, USA). To investigate the effect of vanib-loaded NPs on the cell cycle, we performed flow cytometry analysis. BEL-7402 cells (3×105 cells/well) were seeded in 6-well plates, cultured overnight, and then treated with drugs. After 48 h of incubation, cells were harvested and fixed with 70% cold ethanol overnight. The cell pellets were centrifuged (1500 rpm, 5 min, 4 °C) and then washed twice with PBS. Subsequently, the cells were resuspended in 500 µL of propidium iodide (Sigma-Aldrich) and incubated at room temperature for 30 min in the dark. Finally, a flow cytometer was used to analyze the cell cycle phase distributions. The resultant raw data were subjected to a ModFit analysis to determine the percentages of cells in different stage of mitosis. 2.6. Transwell invasion and tube formation assay of HUVECs

Transwell assay was used to assess the effect of the invasion of HUVECs in vitro. In brief, 50 µL of Matrigel (Cat No. 354234, BD Biosciences) was placed into the Transwell plate (Millipore) and allowed to polymerize for 30 min at 37 °C. 700 µL of DMEM medium supplemented with 10% FBS and 10 ng/ml VEGF was added to the bottom chambers, and 200 µL of DMEM medium (without FBS) containing HUVECs (4×104 cells) was added into the top chambers. Subsequently, the top chambers were treated with free vanib, NP-Vanib and iNP-Vanib, and the plates incubated at 37 °C for 24 h. Cotton swabs were used to remove the Matrigel and cells remaining in the upper chamber. The cells on the bottom surface of the membrane were fixed with methanol for 10 min, and stained with 0.5% crystal violet for 15 min. The invading cells on the membrane were washed with distilled water and photographed 9 ACS Paragon Plus Environment


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under optical microscope. The cells were counted in at least three random microscopic fields (magnification, ×100). All experiments were repeated three times. To evaluate the anti-angiogenetic activity using free vanib, NP-Vanib or iNP-Vanib, the formation of HUVEC capillary-like structures on matrigel was observed and photographed under microscope. Briefly, 50 µL matrigel was added to each well of 96-well plate and incubated at 37 °C for 45 min to allow for polymerization. Onto Matrigel bed were then seeded with 1.5×104 cells of HUVEC and incubated with various concentrations (1, 5 and 10 µmol/L) of free vanib, NP-Vanib or iNP-Vanib in DMEM for 4 h. DMSO (

iRGD-Decorated Polymeric Nanoparticles for the Efficient Delivery of

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