Biomaterials 48 (2015) 84e96

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Dual-modified liposomes with a two-photon-sensitive cell penetrating peptide and NGR ligand for siRNA targeting delivery Yang Yang a, YanFang Yang a, XiangYang Xie a, b, ZhiYuan Wang a, Wei Gong a, Hui Zhang a, Ying Li a, FangLin Yu a, ZhiPing Li a, XingGuo Mei a, * a b

Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, China Wuhan General Hospital of Guangzhou Military Command, Wuhan 430070, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 September 2014 Accepted 20 January 2015 Available online 11 February 2015

Tumor-oriented nanocarrier drug delivery approaches with photosensitivity have drawn considerable attention over the years. However, due to its low penetrability and ability to harm tissues, the use of UV light for triggered nanocarrier release in in vivo applications has been limited. Compared with UV light, near-infrared (NIR) light deeply penetrates tissues and is less damaging to cells. In this study, we devised and tested a strategy for functional siRNA delivery to cells by loading siRNA into cationic liposomes bearing a photolabile-caged cell-penetrating peptide (pcCPP) and asparagine-glycine-arginine peptide (NGR) molecules attached to the liposome surface (pcCPP/NGR-LP). Here, the positive charges of the lysine residues on the CPP were temporarily caged by the photosensitive group (PG), neutralizing its charges and thereby forming a pcCPP. This event subsequently led to conditional NIR light-dependent cell-penetrating functionality. After administration, the pcCPP/NRG-LP was inactivated in the circulatory system as it could not penetrate the tumor cell membrane. The NGR moiety selectively bound to CD13-positive tumors, which facilitated the active accumulation of pcCPP/NGR-LP in tumor tissues. Then, upon illumination using NIR light at the tumor site, the PG was uncaged, the interaction of the CPP with the cell membrane was restored and the activated dual-modified liposomes exhibited enhanced tumor cellular uptake and selectivity due to the synergistic effect of CPP-mediated cellular entry and NGRmediated endocytosis. Subsequent research demonstrated that the pcCPP/NGR-LP showed good physicochemical properties, effective cellular uptake, endosomal escape and significant gene silencing in HT1080 cells in vitro. Additionally, after systemic administration in mice, pcCPP/NGR-LP accumulated in the tumor, augmented c-myc silencing and delayed tumor progression. In conclusion, the combined application of these pcCPP and NGR modifications may provide a reasonable approach for the selectively targeted delivery of siRNA. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Near-infrared (NIR) two-photon photolysis NGR-targeted Cell-penetrating peptides Small interfering RNA

1. Introduction The efficient passage of macromolecular therapeutics, such as small interfering RNA (siRNA), through the plasma membrane remains a major challenge for drug delivery. To address this need, a new approach employing “cell-penetrating peptides (CPPs)” for macromolecular payload delivery seems promising. They can facilitate the entry of different covalently attached bulky cargo into cells without requiring specific receptors [1]. However, the cationic

* Corresponding author. E-mail address: [email protected] (X. Mei). http://dx.doi.org/10.1016/j.biomaterials.2015.01.030 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

nature of CPP sequences, and thus lack of cell specificity, limits their use for drug delivery applications [2]. With the focus of solving this dilemma on conventional CPP, several targeting strategies have been introduced to build ‘off-on’ switches to CPP activity based on sensitivity to external triggers (e.g., light, ultrasound and temperature) and endogenous triggers (e.g., enzymatic activity and pH) [3]. Because reliance on the endogenously triggered release mechanisms may have disadvantages, mainly due to the large inter-individual variability in the expression level of enzymes or intratumoral pH [4], it would be favorable to develop a common triggered methodology that is independent of the conditions of the tumor microenvironment. Among the various external triggers, light is especially attractive, as it can be remotely applied with extremely high spatial and

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temporal precision [5]. To date, a variety of photoresponsive modalities have been utilized to control the activity of CPP [6]. In one example, we reported a nanostructured lipid carrier (NLC) modified with photoresponsive CPP (pCPP-NLC) [7]. The cationic residues of CPP were temporarily masked with UV one-photon excitation (1PE)-responsive group to minimize non-specific cellular uptake. Upon UV light illumination, the group was cleaved, the CPP regained its activity and facilitated rapid intracellular delivery of NLC into cells. In another example, Hansen et al. developed a constrained and UV-activatable cell-penetrating liposomal system with a PEGylated lipid-CPP-lipid anchored loop structure (“n” shape) on its surface [8]. Upon the target cells and with application of UV irradiation, the linker was cleaved and the loop opened, transforming into CPP-PEGylated liposomes with revived cell penetration ability. However, due to its low short wavelength (280e400 nm), UV light exhibits limited penetration depth in biological tissues and can possibly cause tissue damage, which hampers the application of UV light for the treatment of large or internal tumors. Thus, the use of UV light for adjustable CPP activity in in vivo applications may be limited. Near-infrared (NIR) two-photon photolysis can overcome the aforementioned problems because NIR, which has a wavelength longer than that of UV, can penetrate up to 10 cm into living tissue and causes minimal tissue damage at the site of application [9]. Over the past few decades, NIR light-triggered photodynamic therapy (PDT) has emerged as an alternative treatment approach to chemotherapy and radiotherapy to treat cancer in the clinic [10]. Inspired by these results, in our study, we attempted to build ‘NIR light off-on’ switches to CPP activity based on a NIR two-photon excitation (2PE)-responsive group, which can be cleaved by NIR two-photon absorption. NIR 2PE-responsive groups, such as o-nitrobenzyl derivatives, have been extensively studied to date [11,12]. Some studies reported that the 4,5-dimethoxy-2-nitrobenzyl group can be excited by two-photon NIR, and it has recently been reported to be applied as an NIR 2PE-excitation-responsive caged compound to control the function of living cells [13e16]. Because of its stability and ease of preparation, we attempted to introduce this photosensitive group (PG) into the side chains of lysine in the sequence of CPP (CGRRMKWKK), which is a novel CPP that enhances the delivery of molecules across biological barriers to achieve intracellular access [17]. The functional molecules (designed as photolabile-caged CPP,

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pcCPP) would lead to conditional NIR light-dependent cell-penetrating functionality. Although pcCPP could improve the targeting delivery to some extent, modifications on the surface of the nanocarrier using active targeting ligands were required to elicit cell surface binding and receptor-mediated endocytosis. Among the various types of active targeting moieties, an Asparagine-Glycine-Arginine (NGR) peptide motif was selected, as NGR was confirmed to be very efficient in targeting highly expressed CD13 to tumor cells [18]. In this study, the pcCPP and NGR were conjugated with DSPE-PEG2000-MAL or DSPE-PEG5000-MAL, respectively, and they were mixed with other adjuvants to prepare cationic liposomes (pcCPP/NGR-LP) for loading siRNA. Because of the specificity of siRNA-mediated gene knockdown, delivery of siRNA can be used as a sensitive method to assess the ability of pcCPP/NGR-LP to deliver macromolecules intracellularly. The working scheme of these dual-modified liposomes is shown in Fig. 1. According to the navigation effects of NGR, the pcCPP/NGRLP is accumulated in tumor sites. The penetration effect of the CPP is hindered in circulation, but upon arriving at the targeted tissue, the uncaging of the pcCPP is triggered by NIR illumination in the tumor site, causing the nonfunctional pcCPP to be converted to an activated CPP. The peptide then regains sufficient positive charges and its cell transduction activity is subsequently restored. Finally, with the aid of activated CPP, the siRNA-loaded liposome will entrance into target cells efficiently. Here, we describe the physicochemical and biological characterization of the pcCPP/NGR-LP for siRNA based cancer therapy at the cellular level, and the in vivo tumor therapy efficiency of the pcCPP/NGR-LP was also explored. 2. Materials and methods 2.1. Materials 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethyleneglycol) (ammonium salt) (DSPE-mPEG2000) and 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-maleimide(polyethylene glycol) (DSPEPEG2000-Mal or DSPE-PEG5000-Mal) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). The NGR (CYGGRGNG) was custom-synthesized by Shanghai GL Biochem Co, Ltd. (Shanghai, China). Soybean phosphatidylcholine (SPC) and cholesterol (Chol) were purchased from Lipoid GmbH (Mannheim, Germany). 3b[N(N0 , N0 -dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Lyophilized c-myc siRNA against cmyc mRNA (50 -AACGUUAGCUUCACCAACAUU-30 ), negative control siRNA (siN.C.) and FAM-labeled negative control siRNA (FAM-siRNA) (antisense strand, 50 -ACGUGACACGUUCGGAGAATT-30 ) were obtained from GenePharma (Shanghai, China). All

Fig. 1. Schematic illustration of pcCPP/NRG-LP. Dual-modified liposomes are retained in the tumor due to the active targeting effect by the NGR ligand. In the dark, pcCPP/NRG-LP is inactive, as it cannot penetrate the tumor cell membrane. However, upon illumination using NIR light at the tumor site, the photosensitive group (PG) is released, the interaction of the CPP with the cell membrane is restored and the activated liposomes rapidly enter the cells.

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chemicals were of reagent grade and were obtained from SigmaeAldrich, unless otherwise stated.

streptomycin. The cells were maintained in a 37  C humidified incubator in a 5% CO2 atmosphere.

2.2. Synthesis of the pcCPP

2.8. In vitro cellular uptake

CPP (CGRRMKWKK) was synthesized by a standard solid phase Fmoc-protocol using Rink amide resin (0.44 mmol/g; Nankai Hecheng, Tianjin, China) on a CEM Liberty peptide synthesizer (CEM, Matthews, North Carolina, USA). The aminotermini of amino acids were acetylated and the carboxyl-termini were amidated. The amino acids used for the synthesis were: Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Met-OH, Fmoc-Lys(Mtt)-OH and Fmoc-Trp-OH. The Trt, Pbf and Mtt represented trityl, 2,2,4,6,7-pentamethyldihydro benzofuran-6-sufonyl and 4-methyl trityl groups, respectively. After synthesis of the fully protected peptide, the Mtt groups (orthogonally protecting group) were selectively removed with 1% trifluoroacetic acid (TFA) in dry dichloro methane (DCM), repeating 10 times for 2 min each time. The free ε-amine of the lysine side chain was reacted with 1(bromomethyl)-4,5-dimethoxy-2-nitrobenzene (photosensitive group, PG) (4 mol equivalents relative to the resin loading) using K2CO3 (2.5 eq.) in dry DCM for 12 h. The pcCPP, equipped with the PGs at the desired positions, were cleaved from the resin and globally deprotected with Reagent K (TFA/mcresol/water/ethanedithiol at a ratio of 85:10:2.5:2.5).

For the in vitro cellular uptake analysis, HT-1080 or MCF-7 cells were seeded in 6-well plates at a density of 2  105 cells per well overnight. After the attachment period, the cells were rinsed with PBS and incubated with free FAM-siRNA or different liposomal formulations containing FAM-siRNA. pcCPP/NGR-LP (pcCPP-LP) was pretreated with or without NIR illumination (l ¼ 740 nm, 3.48  1012 J cm2) for 30 min prior to their addition to the cells (NIR-pretreated pcCPP-LP or nonpretreated pcCPP-LP). The concentration of FAM-siRNA was 75 nM. After incubation with the liposomal formulations for 4 h at 37  C, the cells were trypsinized, washed twice with cold PBS, and then immediately analyzed using a flow cytometer (BD FACSCalibur, USA).

2.3. Photo triggered uncaging of pcCPP A solution containing the pcCPP (400 mM) was prepared in MOPS buffer at pH 7.4 and was illuminated using a NIR pulse laser (l ¼ 740 nm, 3.48  1012 photon s1). Aliquots (50 mL) were removed at various time points and immediately frozen in the dark until analysis via reverse-phase HPLC (Agilent 1211, Agilent Technologies, USA). For HPLC separations, An Akasil C18 column (4.6 mm  250 mm, 5 mm, Agela Technologies, flow rate 1 mL/min) was employed, and eluting products were detected by UV at 220 nm. A solvent system consisting of 0.1% aqueous solution of TFA (v/v, solvent A) and 0.1% TFA in acetonitrile (v/v, solvent B) was used for HPLC elution [16]. 2.4. Synthesis of functional conjugates CPP or pcCPP were conjugated with DSPE-PEG2000-Mal (1:1 M ratio) in DMF contained triethylamine (TEA, 5 eq.) at room temperature (20e25  C) for 24 h under stirring [19]. The reaction mixture was dialyzed (molecular weight cutoff (MWCO) 3.5 kDa) in distilled water for 48 h to remove the DMF and unreacted peptides. The final solution was lyophilized and stored at 20  C until further use. DSPE-PEG5000NGR were synthesized as our previously report [20]. The conjugations were confirmed by determining the molecular weight of the resulting DSPE-PEG2000-CPP, DSPE-PEG2000-pcCPP or DSPE-PEG5000-NGR via MALDI-TOF MS.

2.9. In vitro confocal imaging Following the culturing of HT-1080 (2  105 cells per well) for 24 h on a Petri dish, free FAM-siRNA or different liposomal formulations containing FAM-siRNA was introduced. Among these samples, pcCPP/NGR-LP (pcCPP-LP) was pretreated with or without NIR light illumination, as mentioned above. The final concentration of each FAM-siRNA was 225 nM. Following treatment for 4 h, the medium was removed and discarded and the cells were washed with cold PBS (0.1 M, pH 7.4) three times, followed by fixation using 4% paraformaldehyde for 20 min. Nuclear staining was performed using Hoechst 33258 for 10 min at ambient temperature. The fluorescent images of the cells were analyzed via confocal laser scanning microscopy (CLSM) (UltraVIEW Vox, PerkinElmer, USA). 2.10. Evaluation of intracellular trafficking and endosomal escape To track the internalization and endosomal release of liposomal FAM-siRNA, following a 4 h incubation with NIR-pretreated pcCPP/NGR-LP containing FAMsiRNA (pcCPP/NGR-LP was pretreated with NIR light illumination, as mentioned above), HT-1080 cells were washed three times with cold PBS and cultured in complete medium for an additional 0.5 h or 2 h. Endosome/lysosome labeling was performed for 0.5 h by LysoTracker Red (Invitrogen/Molecular Probes, CA, USA) at a concentration of 500 nM. Subsequently, the cells were rinsed with cold PBS and were then analyzed by CLSM. 2.11. In vitro siRNA transfection and analysis of gene expression

The mean diameter and zeta-potential of each formulation was determined in three serial measurements using a Malvern Zetasizer Nano ZS90 instrument (Malvern Instruments Ltd., U.K.). The morphology of pcCPP/NGR-LP was observed via transmission electron microscopy (TEM, HITACHI, H-7650, Japan) and atomic force microscopy (AFM, NanoWizarc, JPK Ltd., Germany). And serum stability of siRNA in its aqueous solution and pcCPP/NGR-LP was evaluated using agarose gel electrophoresis.

HT-1080 cells were seeded at a density of 2.0  106 cells/well in a 35-mm dish. After 24 h of culture in a humidified atmosphere of 5% CO2 at 37  C, the medium was exchanged with fresh serum-free medium containing various siRNA-loaded samples. Among these samples, pcCPP/NGR-LP (pcCPP-LP) was pretreated with or without NIR light illumination, as mentioned above. The final concentration of siRNA (c-myc siRNA or siN.C.) used in the experiment was 100 nM. The cells were incubated for 48 h (for mRNA assays) or 72 h (for protein quantification) at 37  C. Subsequently, c-myc mRNA and protein were evaluated using qRT-PCR (quantitative real-time polymerase chain reaction) and Western blot analysis, respectively. For the qRT-PCR assessment, the method has been reported by Xiang et al. [22], with minor modifications. Briefly, the analysis was performed on the IQ5 real-time PCR detection system (Bio-Rad), and the relative gene expression was quantified by the 2DDCt method using the IQ5 Optical System Software version 2.0 (Bio-Rad). The primers used for PCR amplification were: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward: 50 -GGGTGTGAACCATGAGAAGT-30 ; GAPDH reverse: 50 GACTGTGGTCATGAGTCCT-30 ; c-myc forward: 50 -GGCTATTCTGCCCATTTGGGGAC-30 ; c-myc reverse: 50 -GGCAGCAGCTCGAATTTCTTC-3’. The cycling procedure was as follows: 95  C for 15 min followed by 40 cycles at 95  C for 10 s and 61  C for 30 s. Specificity was verified by melt curve analysis and agarose gel electrophoresis. In Western blotting experiments, transfected cells were lysed in lysis buffer (CelLytic M Cell Lysis Reagent) for 30 min on ice, and the supernatant was collected after centrifugation at 12,000 rpm. Cell lysates were separated on a 10% acrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked for 1 h in 5% skim milk and then incubated with polyclonal antibody against c-myc overnight. Membranes were washed in PBST (PBS with 0.1% Tween 20) three times and then incubated for 1 h with a secondary antibody. The membranes were washed four times and then developed by an enhanced chemiluminescence system according to the manufacturer's instructions (PerkinElmer, Waltham, MA).

2.7. Cell culture

2.12. Cell apoptosis assay

Human fibrosarcoma cells (HT-1080 cells) and human breast adenocarcinoma cells (MCF-7 cells) were used to evaluate the targeting effect of the constructed liposomes, as these two cell lines have previously been used as CD 13 positive and CD 13 negative cell models, respectively [21]. HT-1080 and MCF-7 cells purchased from the Cell Resource Centre of IBMS (Beijing, China) were maintained in culture medium consisting of modified eagle's medium (MEM) and dulbecco's modified eagle's medium (DMEM) supplemented with 10% FBS, 100 IU/mL penicillin, and 100 mg/mL

HT-1080 cells were plated on 25 cm2 tissue culture flasks at 2.0  106 cells per flask in 6 mL of complete MEM medium. After a 24 h culture, the cells were washed with PBS and exposed to fresh serum-free medium containing siRNA-loaded liposomes. Among these liposomes, pcCPP/NGR-LP (pcCPP-LP) was pretreated with or without NIR light illumination, as mentioned above. The final concentration of siRNA (c-myc siRNA or siN.C.) used in the experiment was 100 nM. Following 6 h of incubation, the medium was replaced with complete medium for an additional 72 h at

2.5. Preparation of liposomes To prepare the non-modified liposome (N-LP) and NGR-modified liposomes (NGR-LP), dry lipid film composed of SPC, Chol, DC-chol, DSPE-mPEG2000, or DSPEPEG5000-NGR (only for NGR-LP), was hydrated with a siRNA (siN.C., c-myc siRNA or FAM-siRNA) aquenous solution. To control for the size, the lipid dispersion was extruded 11 times through 200-nm polycarbonate membrane filters using a mini extruder (Avanti, Canada) at 40  C. To prepare the CPP-modified liposomes (CPP-LP), pcCPP-modified liposomes (pcCPP-LP) and pcCPP/NGR-co-modified liposomes (pcCPP/NGR-LP), the post-insertion technique was used. For the pcCPP/NGR-LP preparations, the NGR-LP was first heated to 50  C for 30 min, then cooled to room temperature, and then transferred onto a dry film formed by drying the conjugate of DSPE-PEG2000-pcCPP in methanol. The system was incubated at 37  C incubation for another 2 h. For the CPP-LP or pcCPP-LP preparation, dry film of DSPEPEG2000-CPP or DSPE-PEG2000-pcCPP were hydrated with preformed N-LP at the above conditions. 2.6. Characterization of liposomes

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37  C. Subsequently, the cells were collected and stained with the Annexin V-FITC apoptosis detection kit (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer's instructions and were immediately analyzed using the FACScan flow cytometer with 10,000 events collected. 2.13. Animal model Female BALB/c nude mice (weighing 18e22 g) were purchased from Vital River Laboratories (Beijing, China). A xenograft tumor model was produced via subcutaneous injection of HT-1080 cells as described in our previous report [20]. All procedures involving animal housing and treatment were approved by the Animal Care and Use Ethics Committee of the Academy of Military Medical Sciences. 2.14. In vivo imaging When the tumor volume reached approximately 200 mm3, the HT-1080 xenografted mice were injected in the tail vein with 200 mL of 5% glucose (control), free Cy5-siRNA or different liposomal formulations containing Cy5-siRNA at 1.2 mg/kg. Four hours after injection, the group cured with pcCPP/NGR-LP (pcCPP-LP) was irradiated with or without an NIR laser (l ¼ 740 nm, 3.48  1012 photon s1) for 45 min (1 min interval after 2 min irradiation) [23]. Subsequently, in vivo fluorescence imaging was performed with a IVIS® Lumina II in vivo imaging system (IVIS® Lumina II In Vivo Imaging System, Caliper life sciences, USA) at the indicated times (6 h and 24 h after injection). After in vivo imaging, the mice were sacrificed by cervical dislocation, and the tumor and major organs, including heart, liver, spleen, lung, kidney, were excised and imaged. 2.15. In vivo antitumor efficacy After the tumors had grown to approximately 50 mm3, the HT-1080 xenografted mice were randomly divided into eight groups (n ¼ 6) and treated with 5% glucose (control), free c-myc siRNA, various liposomal formulations carrying c-myc siRNA or pcCPP/NGR-LP carrying siN.C. by intravenous injection once every other day for a total of 10 d at a concentration of 1.2 mg/kg siRNA. Four hours after administration, the group cured with pcCPP/NGR-LP (pcCPP-LP) was irradiated with or without an NIR laser, as described above. The body weight and tumor size were measured daily. The estimated tumor volume was calculated using the formula: volume (mm3) ¼ (length  width2)/2. On the 18th day, the animals were sacrificed and the tumors were weighed and photographed. 2.16. Detection of c-myc expression in tumor tissues For the analysis of c-myc expression in vivo, tumor tissues were excised 24 h after the last administration. Tumor fragments were processed for total mRNA or protein extraction followed by qRT-PCR and Western blot assays, respectively. To analyze the c-myc mRNA, the extracted mRNA samples were individually normalized to the same 260 nm absorbance value and detected by qRT-PCR as described in a previous report [22]. To assess the c-myc protein expression, the selected tumor tissues were homogenized in 1 mL of NP-40 Lysis Buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, sodium pyrophosphate, b-glycerophosphate, sodium orthovanadate, sodium fluoride, EDTA and leupeptin) (Beyotime, China) with PMSF (1 mM). The lysates were incubated on ice for a total of 30 min and with vortexing every 5 min. Following the purification and quantification, the protein expression was determined by Western blot analyses as described above. 2.17. Statistical analysis The data are presented as the means ± standard deviation (SD). The difference between any two groups was determined via ANOVA. P < 0.05 was considered to be statistically significant.

3. Results and discussion 3.1. Synthesis of the pcCPP

Fig. 2. Synthetic procedure for pcCPP (A). MALDI-TOF mass spectra of pcCPP (B).

The NIR light-triggered uncaging of pcCPP used for the preparation of liposomes is the primary step for the success of the dualmodified liposomal siRNA delivery system (pcCPP/NGR-LP) developed in this study. The pcCPP includes two units: CPP (CGRRMKWKK) and the photosensitive group (4,5-dimethoxy-2nitrobenzyl group). The synthetic procedure for pcCPP is illustrated in Fig. 2A. In this system, the positive charges of the lysine residues on the CPP were temporarily masked by PG, forming the pcCPP. The crude products were precipitated with cold diethylether, lyophilized and purified by preparative reverse phase, high-performance liquid chromatography (PrepLC 4000, Waters) to more than 95% purity. The molecular weight of the peptides was confirmed by MALDI-TOF MS (Autoflex III; Bruker Daltonics Inc.,

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Billerica, Massachusetts, USA). pcCPP expected mass: 1776.8 Da, observed mass: 1777.2 Da (Fig. 2B). 3.2. NIR-induced uncaging of pcCPP Prior to the conjugation of the pcCPP to the PEGylated lipid, the rate of pcCPP uncaging was characterized by exposing its solution in buffer to NIR light illumination (l ¼ 740 nm, 3.48  1012 photon s1). The possible process whereby CPP is generated photochemically from pcCPP is represented in Fig. 3A. Once illuminated with the NIR light, the PG was removed and the CPP was exposed [14]. Aliquots were removed at various time points, and the molar fractions of caged and free CPP were calculated based on the HPLC chromatograms. Consequently, two newly formed peaks appeared in the HPLC chromatograph at approximately 7.9 min (same retention time as CPP) and 4.7 min (Fig. 3C), which is different from the tested pcCPP sample (approximately 11.1 min) (Fig. 3B). The results indicated the occurrence of specific uncaging, as predicted. As illustrated in Fig. 3D, with an increase in irradiation time, there was an improvement in the degree of uncaging. When the time was 30 min, approximately 84.82 ± 3.36% of the initial intact pcCPP construct was uncaged, which was available for cell binding and penetration. Thus, further experiments were performed using 30 min of light illumination. Furthermore, the amount of pcCPP did not decrease in the dark. It was illustrated that pcCPP could be present and stable until coming into contact with NIR. 3.3. Synthesis of functional conjugates The pcCPP/NGR-LP was developed by the modification of two synthesized functional materials, DSPE-PEG2000-pcCPP and DSPEPEG5000-NGR. DSPE-PEG2000-pcCPP, DSPE-PEG2000-CPP and DSPEPEG5000-NGR were synthesized as shown in Fig. 4. Here, DSPEPEG2000-CPP was the cleavage product of DSPE-PEG2000-pcCPP,

which was used to compare the penetration ability before and after activation of DSPE-PEG2000-pcCPP. The pcCPP, CPP and NGR were terminated with cysteine to introduce free sulfhydryl (-SH), and these materials were conjugated to DSPE-PEG2000-Mal or DSPEPEG5000-Mal via the sulfhydrylemaleimide reaction, which enabled pcCPP, CPP or NGR to be conjugated at a specific site (-SH) (Fig. 4A). The MALDI-TOF MS results confirmed the successful formation of DSPE-PEG2000-pcCPP, DSPE-PEG2000-CPP and DSPE-PEG5000-NGR, with the observed mass-charge ratios of approximately 4670.3 (Fig. 4B, marked by an arrow), 3986.0 (Fig. 4C, marked by arrow) and 6335.6 (Fig. 4D, marked by arrow), which was equal to the theoretical mass-charge ratios of 4670.0, 3987.0 and 6336.0, respectively. The final product was then used for preparing targeted liposomes in experiments.

3.4. Preparation and characterization of liposomes The physicochemical properties of the four distinct liposome formulations are summarized in Table 1. The siRNA encapsulation efficiency of all liposomes was about 90% (see Supplementary information). The zeta-potential of CPP-LP (17.07 ± 0.43 mV) was more positive than that of any other groups, owing to the cationic charge of the CPP. Considering the comparison between the pcCPP and CPP inserting liposomes, the zeta-potential of pcCPP-LP (11.05 ± 0.59 mV) was less than that of CPP-LP, suggesting that the positive charges of the lys side chains of the CPP were effectively shielded by the PG. Positive NGR peptides on the surface of NGR-LP (13.83 ± 0.41 mV) and pcCPP/NGR-LP (12.11 ± 0.35 mV) increased the zeta potential of liposomes. Moreover, after pretreating with NIR illumination, pcCPP-LP and pcCPP/NGR-LP showed a value of 14.55 ± 0.48 mV and 16.16 ± 0.28 mV, respectively. As shown in Fig. 5A and B, TEM and AFM images of pcCPP/NGRLP demonstrated that the particle sizes were close to the values measured using the laser particle analyzer (156.03 ± 0.12 nm)

Fig. 3. Principle of NIR irradiation reaction of pcCPP (A). HPLC profiles before (B) and after (C) NIR irradiation. HPLC analysis of the uncaging of pcCPP by NIR irradiation (D). The responses of pcCPP was plotted versus time following the illumination of NIR light (l ¼ 740 nm, 3.48  1012 photon s1) or kept in the dark at 37  C.

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Fig. 4. Principle of the synthesis of DSPE-PEG2000-pcCPP/CPP/NGR (A). MALDI-TOF mass spectra of DSPE-PEG2000-pcCPP (B), DSPE-PEG2000-CPP (C) and DSPE-PEG5000-NGR (D).

(Fig. 5C). As shown in Fig. 5D, pure siRNA was fully degraded after 8 h. In contrast, siRNA in pcCPP/NGR-LP did not fully degrade even after 24 h. 3.5. Analysis of cellular uptake and endosomal escape According to the design strategy, when pcCPP is triggered by NIR light and activated to CPP, the cellular uptake of the siRNA-loaded liposome is expected to enhance due to the CPP penetration effect. To verify this hypothesis, a CD 13-negative MCF-7 cell line was used to investigate the uptake of NIR-pretreated pcCPP/NGR-LP. As shown in Fig. 6A, NIR-pretreated samples (NIR-pretreated pcCPP-LP or NIR-pretreated pcCPP/NGR-LP) exhibited stronger intracellular fluorescence than non-pretreated samples (non-pretreated pcCPPLP or non-pretreated pcCPP/NGR-LP), which revealed the contribution of photo-triggered uncaging to the pcCPP in the pcCPP-LP or pcCPPNGR-LP to cellular uptake. On the contrary, the internalization of N-LP and CPP-LP on MCF-7 cells was not significantly influenced by the illumination of NIR light, where very similar

fluorescence intensities were exhibited. This suggests that NIR light can trigger pcCPP effectively, and the liposomes then commit an advanced translocation via the exposed CPP. HT-1080 cells were selected as the CD13-positive cell type used to investigate the effect of NGR. The competitive binding of nonpretreated pcCPP/NGR-LP and NGR-LP to HT-1080 cells was performed by adding excess free NGR (1 mg/mL) to the media prior to the introduction of these liposomes. As shown in Fig. 6B, the results suggest that the cellular uptake of non-pretreated pcCPP/NGR-LP and NGR-LP in the presence of excess free NGR in HT-1080 cells was suppressed significantly, becoming almost equivalent to that of NLP. The results indicated that when the expression level of CD 13 on the cell surface was lower, non-pretreated pcCPP/NGR-LP and NGRLP could not efficiently recognize and bind with the target cell via the NGR motif, and thus, the uptake efficiency of non-pretreated pcCPP/NGR-LP and NGR-LP was not ideal. To investigate systematically what effects various liposomal formulations might have on the cellular internalization of siRNA, we individually incubated various liposomes with HT-1080 cells. As

Table 1 Characteristics of the liposomes. Sample ID

Liposomes components (mol ratio of total lipid)

Diameter (nm)

N-LP NGR-LP CPP-LP pcCPP-LP pcCPP-LP (NIR-pretreated) pcCPP/NGR-LP

SPC/Chol/DC-chol/DSPE-PEG2000 (48.5:8.1:40.4:3.0) SPC/Chol/DC-chol/DSPE-PEG2000/DSPE-PEG5000-NGR (48.0:8.0:40.0:3.0:1.0) SPC/Chol/DC-chol/DSPE-PEG2000/DSPE-PEG2000-CPP (47.1:7.8:39.2:2.9:2.9) SPC/Chol/DC-chol/DSPE-PEG2000/DSPE-PEG2000-pcCPP (47.1:7.8:39.2:2.9:2.9) SPC/Chol/DC-chol/DSPE-PEG2000/DSPE-PEG2000-pcCPP (47.1:7.8:39.2:2.9:2.9) SPC/Chol/DC-chol/DSPE-PEG2000/DSPE-PEG5000-NGR/DSPE-PEG2000-pcCPP (46.6:7.8:38.8:2.9:1.0:2.9) SPC/Chol/DC-chol/DSPE-PEG2000/DSPE-PEG5000-NGR/DSPE-PEG2000-pcCPP (46.6:7.8:38.8:2.9:1.0:2.9)

145.58 149.45 153.68 154.89 155.66 156.03

pcCPP/NGR-LP (NIRpretreated)

The data are expressed as the mean ± SD for three different preparations (n ¼ 3).

± ± ± ± ± ±

Polydispersity index 0.11 0.16 0.10 0.10 0.10 0.12

0.08 0.09 0.08 0.10 0.11 0.11

± ± ± ± ± ±

0.01 0.02 0.01 0.01 0.01 0.02

153.23 ± 0.11 0.10 ± 0.01

Zeta potential (mV) 10.54 13.83 17.07 11.05 14.55 12.11

± ± ± ± ± ±

0.63 0.41 0.43 0.59 0.48 0.35

16.16 ± 0.28

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Fig. 5. Physicochemical characterization of pcCPP/NGR-LP. Morphological appearance of pcCPP/NGR-LP based on TEM (A) and AFM (B). Particle size distribution of pcCPP/NGR-LP (C). siRNA serum stability assay (D).

shown in Fig. 6C, a significantly increased mean fluorescence intensity was detected in the cells treated with NGR-LP and nonpretreated pcCPP/NGR-LP compared with those treated with NLP, suggesting a role for NGR. Following pretreatment with NIR light illumination, NIR-pretreated pcCPP-LP and NIR-pretreated pcCPP/ NGR-LP displayed a significant improvement in cellular uptake, suggesting the activation of pcCPP by NIR light. In addition, among all of the liposomal formulations, NIR-pretreated pcCPP/NGR-LP displayed the greatest improvement in the cellular uptake of FAMsiRNA. However, the internalization of non-pretreated pcCPP/NGRLP was less than that of CPP-L or NGR-L. Moreover, the uptake efficiency of non-pretreated pcCPP-LP was not ideal. The mean fluorescence intensity of the formulation declined to a level similar to that of N-LP. Taken together, these results indicated that when pcCPP was cleaved by NIR light and activated to form the CPP and the cellular uptake of pcCPP/NGR-LP was enhanced due to the CPPmediated cell penetration and NGR-mediated endocytosis. Thus, the synergistic effect of the two ligands on cellular uptake was clear. Consistent with these findings, CLSM analysis also confirmed the significant synergetic effect of pcCPP/NGR-LP on cellular uptake in HT-1080 cells. As shown in Fig. 6D, some of the liposomes (NGRLP, CPP-LP, NIR-pretreated pcCPP-LP and non-pretreated pcCPP/ NGR-LP, NIR-pretreated pcCPP/NGR-LP) exhibited increased intracellular distribution compared with N-LP, which could stem from the effects of their NGR or CPP ligands. However, among all of the tested formulations, the NIR-pretreated pcCPP/NGR-LP exhibited

the maximum intracellular fluorescence intensity. Cancer cell recognition using dual modifications was greatly enhanced compared with that of the mono-modified and non-modified liposomes. The results could be a consequence of the synergism between NGR mediation and photo activation and are consistent with the data from the flow cytometry analysis. Efficient uptake of siRNA does not necessarily result in an efficient gene silencing effect because endocytosed siRNA needs to escape from the endosome to produce its effects in the cellular cytoplasm. As shown in Fig. 6E, for NIR-pretreated pcCPP/NGR-LP carrying FAM-siRNA, a relatively high frequency of colocalization spots (yellow) of the green (FAM-siRNA) and red (Lysotracker Red) fluorescence in the cytoplasm was found after incubation for 0.5 h, indicating that the majority of liposomal siRNA was within endolysosomes in the early phase of uptake (0.5 h). However, the green fluorescence was partially separated from the red fluorescence over time (2 h), pointing to the successful escape of liposomal FAMsiRNA. This triggered escape is because cationic lipids can form ion pairs with the anionic lipids in the endosome membrane and thus destabilize the endosomal membrane by excluding the surface-bound water [24,25]. 3.6. In vitro gene silencing and cell apoptosis assay c-myc is one of the most highly amplified, downstream oncogenes that promote cell growth, proliferation, invasion, expansion and angiogenesis. There have been many reports on the therapeutic

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Fig. 6. Cellular uptake of pcCPP/NGR-LP into MCF-7 cells with or without NIR illumination (A). Cellular uptake of non-pretreated pcCPP/NGR-LP into HT-1080 cells with or without free NGR (B). Cellular uptake of various formulations into HT-1080 cells (C). Confocal laser scanning microscopy (CLSM) analysis of the uptake of various formulations by HT-1080 cells (D). Intracellular trafficking of FAM-siRNA in HT-1080 cells undergoing 0.5 h or 2 h of routine culture following 4 h of incubation with NIR-pretreated pcCPP/NGR-LP (E). Hoechst 33258 for nuclei staining (blue), FAM-siRNA fluorescence (green) and LysoTracker Red for endosomes/lysosomes (red) were recorded. The data are presented as the means ± SD (n ¼ 3). * indicates P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 7. The level of c-myc mRNA determined by qRT-PCR (A). c-myc protein expression determined by Western blot analysis (B). Cell apoptosis following exposure to different formulations (C). Early apoptotic cells are shown in the lower right quadrant, and late apoptotic cells are shown in the upper right quadrant. The data are presented as the means ± SD (n ¼ 3). *indicates P < 0.05.

application of c-myc siRNA for the treatment of fibrosarcoma [26]. Accordingly, we studied the gene as a therapeutic target against fibrosarcoma and evaluated the downregulation of the target gene by siRNA-loaded liposomes. 3.6.1. qRT-PCR analysis As exhibited in Fig. 7 A, siN.C.-loaded NIR-pretreated pcCPP/ NGR-LP did not downregulate c-myc expression, whereas the cells exposed to various liposomes carrying c-myc siRNA demonstrated reduced c-myc gene expression, indicating sequencespecific gene silencing. NGR-LP expressed more powerful gene silencing than N-LP, which correlated with the facilitated internalization of siRNA, suggesting NGR-mediated cellular uptake of the NGR-modified vector as demonstrated in the cellular uptake study (Fig. 6B). In comparison with cells treated with nonpretreated pcCPP-LP and non-pretreated pcCPP/NGR-LP, much lower levels of c-myc mRNA were detected in the cells incubated with NIR-pretreated pcCPP-LP and NIR-pretreated pcCPP/NGR-LP. This was attributed to the improved entry of siRNA into the cells caused by effective uncaging of the pcCPP with NIR illumination. Additionally, compared with other groups, c-myc siRNA-loaded NIR-pretreated pcCPP/NGR-LP exhibited the greatest silencing effect in HT-1080 cells, which was consistent with the data presented regarding cellular uptake (Fig. 6C). 3.6.2. Western blot analysis To investigate whether this reduction in c-myc mRNA was followed by a decrease of c-myc protein, we conducted Western blot analyses in HT-1080 cells. As exhibited in Fig. 7B, in contrast with the control group, the delivery of c-myc siRNA by the liposomes exclusively knocked down c-myc protein expression. However, neither siN.C.-loaded NIR-pretreated pcCPP/NGR-LP nor free c-myc siRNA had an effect on c-myc protein expression, indicating that no

non-specific gene silencing took place and that free siRNA could not readily translocate across the cell membrane due to their large size and highly negative charge [27]. Furthermore, c-myc siRNA-loaded CPP-LP and NIR-pretreated pcCPP-LP presented an enhanced silencing response, which resulted from the beneficial intracellular trafficking mediated by the CPP. Due to the effect of NGR mediation, NGR-LP and non-pretreated pcCPP/NGR-LP exhibited markedly improved silencing of c-myc protein expression compared with the N-LP. In addition, similar to the mRNA expression profiles (Fig. 7A), the lowest c-myc protein expression was observed in cells treated with NIR-pretreated pcCPP/NGR-LP carrying c-myc siRNA. This likely results from the beneficial intracellular trafficking mediated by the CPP and NGR, providing further support for the anticipated specificity and sensitivity. 3.6.3. Cell apoptosis assay As shown in Fig. 7C, the exposure to every type of liposomeloaded c-myc siRNA resulted in an abundance of apoptotic cells (33%e61%). In contrast, only a slight effect was observed in the control cells and the siN.C.-loaded NIR-pretreated pcCPP/NGR-LP treated cells (3%), indicating that the apoptosis response originated from the downregulated expression of c-myc in HT-1080 cells. Approximately 33% of cells undergo apoptosis after transfection with c-myc siRNA-loaded N-LP; however, a higher percentage of cells (46%) displayed apoptosis after treatment with cmyc siRNA-loaded NGR-LP. In addition, the delivery of c-myc siRNAloaded NIR-pretreated pcCPP-LP and NIR-pretreated pcCPP/NGR-LP resulted in a higher percentage of apoptotic cells than nonpretreated pcCPP-LP and non-pretreated pcCPP/NGR-LP. As we expected, cells exposed to NIR-pretreated pcCPP/NGR-LP loaded cmyc siRNA presented the highest percentage, reaching 61%, of induced apoptosis, which was consistent with the c-myc protein expression assay described above (Fig. 7B).

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Fig. 8. Biodistribution of Cy5-siRNA contained in various liposomes in mice bearing HT-1080 tumor xenografts. Whole body imaging at different time points after systemic administration (A). Fluorescence detection of isolated main tissues and organs from mice at the end point of observation (B).

Overall, these results demonstrated that the combination of the pcCPP and NGR ligand introduced into the liposomal formulations markedly facilitated the RNAi-mediated gene silencing and growth inhibition. 3.7. In vivo distribution of siRNA The selective distribution and prolonged retention of siRNA formulated nanocarrier in tumors could potentially benefit the antitumor activity of this therapeutic nucleic acid in vivo. To verify this, in the present study, the in vivo biodistribution and tumor accumulation profiles of Cy5-siRNA-loaded liposomes were clearly visualized by monitoring the whole body fluorescence intensity in a subcutaneous HT-1080 xenograft-bearing nude mouse model. Based on whole body imaging (Fig. 8A), free siRNA was almost

eliminated from the body after 24 h, and tumor accumulation did not occur. In contrast, high accumulation of pcCPP/NGR-LP (with NIR), pcCPP-LP (with NIR) and NGR-LP were detected in the tumor during the entire period (24 h after injection). Moreover, the most intense distribution in tumors was displayed in the pcCPP/NGR-LPtreated (with NIR) mice and was further confirmed by the strongest fluorescence identified in isolated tumors (Fig. 8B). This phenomenon indicted that the introduction of NGR-induced tumor-targeting in Cy5-siRNA, and the incorporation of pcCPP further enhanced its accumulation in tumors. For Cy5-siRNA-loaded N-LP and pcCPP-LP (without NIR), the duration of fluorescence in tumors also reached 24 h; however, a decrease in fluorescence intensity was found. It was confirmed that the non-modified liposomes and unactivated liposomes resulted in relatively limited targeted delivery. The tumor fluorescence intensity in CPP-LP treated mice

Fig. 9. Antitumor activity (A) and body weight changes (B) in HT-1080 tumor-bearing mice after treatments with 5% glucose and various liposomes carrying c-myc siRNA or siN.C. Arrows represent drug administration. Relative tumor volume (%) ¼ tumor volume/primary tumor volume  100. Photographs of tumors at the end of treatment (C). The weights of dissected tumors at the end of treatment (D). The data are presented as the means ± SD (n ¼ 6). * indicates P < 0.05. Expression of c-myc mRNA (E) and protein (F) in tumors was detected 24 h after the last administration. * indicates P < 0.05 (n ¼ 3).

Y. Yang et al. / Biomaterials 48 (2015) 84e96

almost disappeared after 24 h. CPP-LP possessed a lower tumortargeting ability in vivo than other modified liposomes, which could be attributed to CPP's lack of selectivity, whereas the higher uptake of FAM-siRNA in CPP-LP treated cells (Fig. 6C) derives from the promoted entry of liposome into cells via CPP. Furthermore, twenty-four hours after administration, the mice were sacrificed and major organs and tumor tissues were isolated and observed (Fig. 8B). Obviously, the tumor accumulation was the highest for pcCPP/NGR-LP (with NIR). The results implied that the pcCPP/NGR-LP (with NIR) could efficiently target to solid tumors and decrease non-specific accumulation in normal organs, such as the heart, spleen and lungs. As seen in the images (Fig. 8B), all mice presented comparable fluorescence intensity in the kidneys, which was consistent with another recent study [28] and indicated that the in vivo fate of all siRNA-containing samples involved renal excretion. These initial data, resulting from in vivo imaging, provided substantial evidence that pcCPP/NGR-LP (with NIR) has the potential to accomplish specific targeting of siRNA to the tumor in vivo. 3.8. In vivo therapeutic efficacy To determine whether our dual-modified liposomal system displays antitumor activity in vivo, the effects of these liposomes on tumor growth inhibition in HT-1080 xenografted nude mice were investigated. As shown in Fig. 9A, treatment with the free c-myc siRNA did not show increased tumor growth inhibition in comparison with 5% glucose, likely due to the transitory retention in systemic circulation and poor permeability into tumor cells. In contrast, various formulations of c-myc siRNA showed a partial inhibition of tumor growth. One week after administration, the maximal reduction in tumor growth was noted in the group treated with c-myc siRNA-loaded pcCPP/NGR-LP (with NIR). This correlated with the above-mentioned data revealing the advantage of pcCPP/ NGR-LP (with NIR) over the other carriers we tested in apoptosis induction in vitro (Fig. 7C) and tumor-specific distribution in vivo (Fig. 8), indicating the combined processes of photo activation and NGR-mediated targeting. Compared with N-LP, exposure of c-myc siRNA to either NGR-LP or pcCPP-LP (with NIR) retarded tumor progression. This result revealed that the incorporation of either NGR or CPP into the carrier enhanced the antitumor therapeutic effects in vivo. However, CPPs are not cell or tissue specific. This drawback limited the in vivo antitumor efficacy of CPP-LP. In addition, the injection of 5% glucose and siN.C.-loaded pcCPP/NGRLP (with NIR) did not delay tumor growth, indicating that the antitumor effect of liposomes carrying siRNA was c-myc siRNA sequence-specific. These results indicate that the therapeutic efficacy of the c-myc siRNA-loaded pcCPP/NGR-LP (with NIR) is significantly superior to that of free and other formulations of cmyc siRNA in HT-1080 xenograft models. As shown in Fig. 9B, there was no significant change in the body weight of various groups of mice during the experimental period. These results suggest that there is negligible acute or severe toxicity related to the indicated treatment at the test dose. The excised tumors treated with the pcCPP/NGR-LP (with NIR) exhibited a much smaller size and weight, as shown in Fig. 9C and D. The synergistic activity of the pcCPP and NGR modifications was evident in the results. To evaluate whether reduced tumor growth, as exhibited above, was associated with c-myc gene silencing in tumor cells, c-myc expression at the mRNA (Fig. 9E) and protein (Fig. 9F) levels in the tumors was assayed by qRT-PCR and Western blot analyses, respectively. As shown in Fig. 9E and F, the expression of both mRNA and c-myc protein, in either the free siRNA group or siN.C.-

95

loaded pcCPP/NGR-LP (with NIR) group, showed no alterations compared with the 5% glucose-treated tumors. Conversely, pronounced inhibition was noted in the c-myc siRNA-loaded pcCPP/ NGR-LP (with NIR) group at both the level of mRNA and protein. Taken together, these results were consistent with the antitumor therapy mentioned above, supporting the direct causality between delayed tumor progression and the silenced c-myc gene. Recently, a lot of non-viral carriers have been developed by many groups in order to delivery the siRNA effectively into the cells both in vitro and in vivo. Similarly, Huang's group developed a PEGylated LPD (liposome-polycation-DNA) nanoparticle for delivery of c-myc siRNA into HT-1080 tumors in mice by modification with NGR [29]. However, such mono-active targeting may sometimes suffer from certain limitations due to low degrees or heterogeneity in receptor expression among different tumor cells, or differences in ligands-receptor affinity and receptor-mediated internalization rates [30]. In contrast, our dual-modified design is intended to improve the selective delivery to cancer cells and to reduce intrinsic toxicity to healthy cells. However, the disadvantage is that the externally applied trigger is restricted to superficial tissues, although deep-seated tissue may be reached with the aid of laparoscopy in the clinic. 4. Conclusion In this study, we developed a payload delivery vehicle consisting of liposomes that display enhanced specificity for tumors and efficient cellular uptake by exploiting the synergistic activities of two different ligands: pcCPP and NGR. The research data indicated that pcCPP/NGR-LP is expected to be a promising delivery system for oncotherapy. In addition, the dual-modified liposomes might be seen as alternatives to photodynamic therapy in which NIR light is used to locally generate cytotoxic reactive oxygen species at the tumor site that cause cancer cell death, but not to induce intracellular delivery of macromolecular therapeutics. In the future study, we will continue to improve in vivo study including survival analysis, and further explore the application of pcCPP/NGR-LP in oncotherapy. Acknowledgments We are grateful for the financial support from the National Natural Science Foundation of China (Grant No. 81202466 and 81402874) and the Important National Science & Technology Specific Projects (Grant No. 2012ZX09301003-001-009) of China. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.01.030. References
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