Dual-functional liposomes based on pH-responsive cell-penetrating peptide and hyaluronic acid for tumor-targeted anticancer drug delivery

Biomaterials 33 (2012) 9246e9258

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Dual-functional liposomes based on pH-responsive cell-penetrating peptide and hyaluronic acid for tumor-targeted anticancer drug delivery Tianyue Jiang a, Zhenhai Zhang b, Yinlong Zhang a, Huixia Lv a, *, Jianping Zhou a, *, Caocao Li a, Lulu Hou a, Qiang Zhang c a b c

State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210000, PR China State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 August 2012 Accepted 14 September 2012 Available online 29 September 2012

Dual-functional liposomes with pH-responsive cell-penetrating peptide (CPP) and active targeting hyaluronic acid (HA) were fabricated for tumor-targeted drug delivery. A series of synthetic tumor pH-triggered CPPs rich in arginines and histidines were screened by comparing tumor cellular uptake efficiency at pH 6.4 with at pH 7.4, and R6H4 (RRRRRRHHHH) was obtained with the optimal pH-response. To construct R6H4-modified liposomes (R6H4-L), stearyl R6H4 was anchored into liposomes due to hydrophobic interaction. HA was utilized to shield positive charge of R6H4-L to assemble HA-coated R6H4-L (HA-R6H4-L) by electrostatic effect for protecting the liposomes from the attack of plasma proteins. The rapid degradation of HA by hyaluronidase (HAase) was demonstrated by the viscosity and zeta potential detection, allowing the R6H4 exposure of HA-R6H4-L at HAase-rich tumor microenvironment as the protection by HA switches off and cell-penetrating ability of R6H4 turns on. After HAase treatment, paclitaxel-loaded HA-R6H4-L (PTX/HA-R6H4-L) presented a remarkably stronger cytotoxicity toward the hepatic cancer (HepG2) cells at pH 6.4 relative to at pH 7.4, and additionally coumarin 6-loaded HA-R6H4-L (C6/HA-R6H4-L) showed efficient intracellular trafficking including endosomal/lysosomal escape and cytoplasmic liberation by confocal laser scanning microscopy (CLSM). In vivo imaging suggested the reduced accumulation of near infrared dye 15 (NIRD15)-loaded HA-R6H4-L (NIRD/HA-R6H4-L) at the tumor site, when mice were pre-treated with an excess of free HA, indicating the active tumor targeting of HA. Indeed, PTX/HA-R6H4-L had the strongest antitumor efficacy against murine hepatic carcinoma (Heps) tumor xenograft models in vivo. These findings demonstrate the feasibility of using tumor pH-sensitive CPPs and active targeting HA to extend the applications of liposomal nanocarriers to efficient anticancer drug delivery. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: pH-response Cell-penetrating peptides Hyaluronic acid Liposomes Tumor-targeted drug delivery

1. Introduction Cell-penetrating peptides (CPPs), facilitating the cellular uptake of various cargos without causing any cellular injury, have been widely investigated in the fields of gene and drug delivery for cancer therapy [1e3]. Hereinto, octaarginine (R8), an arginine homopolymers derived from the basic domain of HIV-1 TAT protein, has been extensively used [4e6] as a result of its shortest possible peptide sequence still able to penetrate the plasma membrane [7]. Constant efforts have been devoted to exploit

* Corresponding authors. Tel.: þ86 25 83271102; fax: þ86 25 83301606. E-mail addresses: [email protected] (H. Lv), [email protected] (J. Zhou). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.09.027

a variety of CPPs based on the template of oligoarginine for improved cellular uptake efficiency [8e10]. However, CPPs with effective tumor targeting are still lacking and remain highly desirable, which present more accumulations in tumor cells but less in normal cells. In the light of this, the pH gradient between the tumor milieu and physiological environment [11] draws more attention to designing pH-responsive CPPs for tumor-targeted drug delivery, which can be used to conjugate drugs or modify nanocarriers. Histidine, a unique amino acid, is able to protonate for positive charge at the acidic tumor microenvironment instead of predominantly no charge at the physiological condition [12,13]. This feature of histidine has been used to design various pH-dependent peptides [13e15]. Zhang et al. replaced all the lysines of TK (AGYLLGKINLKKLAKL(Aib)KKIL-NH2) with histidines

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for a histidine-containing TH (AGYLLGHINLHHLAHL(Aib)HHILNH2), which has the capability of responding to tumor extracellular acidity so that it can be activated and subsequently enter cells more efficiently at pH 6.0 relative to at pH 7.4 [13]. Tu et al. substituted histidine for lysine and arginine residues, thereby producing membranolytic peptides with pH-dependent cytotoxicity [14]. On the other hand, histidine-containing the imidazole group has the proton sponge effect on endosomal/lysosomal escape, which provides a widespread range of applications for efficient intracellular delivery [16e18]. Unfortunately, recent studies have suggested that CPPs such as TAT on the surface of liposomes and micelles are susceptible to enzymatic cleavage by enzymes present in human plasma [19]. Additionally, for intravenous injection, positively charged nanoparticles, including cationic CPP-modified liposomes, cause severe toxicity, instability and a rapid clearance from the blood compartment, thereby limiting their applications in vivo [20e22]. To address this dilemma, surface PEGylation of CPP-modified nanoparticles (CPP-NPs) is regarded as a gold standard for improved safety, bioavailability and blood persistence [23e26], resulted from the reduced interactions between CPP-NPs and opsonin by the hydrophilic shell of poly(ethylene glycol) (PEG). Nevertheless, noting that the PEG shielding of positively charged CPP-NPs also impairs the cell-penetrating ability of CPPs [27]. PEG deshielding for CPPs exposure at the tumor site is a prerequisite for PEGylated CPP-NPs to be more effectively endocytosed into the cells [24e26,28]. On the other hand, physical coating of cationic NPs by anionic natural polymers, such as polyglutamic acid [29], polyacrylic acid [30], dextran [31], dependent upon the electrostatic absorptive interaction, is more convenient and highly appealing, compared with the complicated synthesis and purification of the functional PEG derivants. Hyaluronic acid (HA), a kind of natural acidic polysaccharide macromolecules, is generally regarded as non-toxic and biodegradable, which composes of N-acetylglucosamine and D-glucuronic acid disaccharide unit. HA with negative charge in neutral pH condition can be easily coated on the surface of cationic NPs [32e34]. In addition, overexpression of HA-binding receptors, such as CD44 and RHAMM, has been found on the cell surface of several malignant tumors [35e37], which brings about the broad applications of HA-based polymers in active tumor targeting for anticancer drugs [38e40]. Furthermore, compared with dextran, HA shows a weaker specificity to the liver receptors and lower liver accumulation [34], potentially contributing to enhanced accumulation of HA-coated NPs at the tumor site. More importantly, hyaluronidase (HAase) is widely distributed in the acidic tumor extracellular matrix, which plays a significant role in tumor growth, invasion and metastasis [41,42]. HA is able to be hydrolyzed by hyaluronidase (HAase), which allows the exposure of CPPs in HA-coated CPP-NPs (HA-CPP-NPs) to facilitate effective admission of NPs into the tumor cells. In order to combine the advantage of pH-responsive CPPs for efficient intracellular delivery, HA for improved blood persistence and both for tumor targeting, we developed dual-decorated liposomes for tumor-targeted drug delivery (Fig. 1). In blood, HA endues HA-coated pH-responsive CPP-modified liposomes (HA-CPP-L) with a strongly negative charge and a protection by HA hydrophilic shell away from the attack of plasma protein for enhanced stability and duration. HA-CPP-L exhibits a high accumulation at the tumor site by means of the enhanced permeability and retention (EPR) effect [43] and the affinity of HA with its binding receptors [35,36]. At the tumor milieu, HA-CPP-L disassembles to CPP-L as a result of the HA degradation by HAase for the exposure of the CPP on the liposomal surface. The exposed CPP has pH-response to the mildly acidic tumor microenvironment to

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increase the uptake of CPP-L into the cells. When CPP-L is internalized into endosomes and lysosomes, the imidazole group of histidine in CPP produces the proton sponge effect accompanied by membrane penetrating ability of CPP, leading to endosomal/lysosomal escape and cytoplasmic release for efficient intracellular trafficking. For proof of our idea, a series of synthetic CPPs rich in arginines and histidines were screened by means of the ratio of tumor cellular uptake on human hepatic carcinoma (HepG2) and human lung adenocarcinoma epithelial (A549) cells at pH 6.4 and at pH 7.4 to acquire R6H4, an optimal pH-responsive CPP, by using confocal laser scanning microscopy (CLSM) and flow cytometry, respectively. Stearyl R6H4 was synthesized and anchored into the liposomes, followed by HA coating to construct HA-coated R6H4modified liposomes (HA-R6H4-L). Hemolysis, plasma stability and buffering capacity of HA-R6H4-L were investigated. The in vitro cytotoxicity of paclitaxel-loaded HA-R6H4-L (PTX/HA-R6H4-L) and the intracellular delivery of coumarin 6-loaded HA-R6H4-L (C6/HAR6H4-L) on HepG2 cells were estimated by using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and CLSM, respectively. In vivo, the biodistribution and targetabilily of near infrared dye 15 (NIRD15)-loaded HA-R6H4-L (NIRD15/HA-R6H4-L) were explored in murine hepatic carcinoma (Heps) cell xenograft models by in vivo imaging, and additionally the antitumor efficacy of PTX/HA-R6H4-L against Heps tumor xenograft models was evaluated. 2. Materials and methods 2.1. Materials Octaarginine (R8), arginine- and histidine-rich CPPs (RH CPPs, RnHm), FITClabeled R8 (FITC-R8) and RnHm (FITC-RnHm), stearyl R8 (R8-C18) and R6H4 (R6H4-C18) were purchased from the GL Biochem Co., Ltd. (Shanghai, China). Characterization data of the used peptides with proper structures were shown in Table S1, such as molecular formula, sequence, molecular weight and purity. The purities of all synthetic CPPs were higher than 95%. Paclitaxel (PTX) was purchased from Chongqing Melian Pharmaceuticals Co., Ltd. (Chongqing, China). Soy phosphatidylcholine (SPC) was offered by Taiwei Pharmaceutical Co., Ltd. (Shanghai, China). Cholesterol (Chol) was provided by Huixing Biochemical Reagent Co., Ltd. (Shanghai, China). Sodium hyaluronic acid (HA, the molecular weight of 210 kDa) was obtained from Freda Biochem Co., Ltd. (Shandong, China). Coumarin 6 (C6) and hyaluronidase (HAase) were purchased from SigmaeAldrich Co. (Shanghai, China). Near infrared dye 15 (NIRD15) was provided by Huahai-Lanfan Chemical Technology Co., Ltd. (Liaoning, China). RPMI 1640 medium (1640, HycloneÒ), fetal bovine serum (FBS, HycloneÒ), penicillin-streptomycin solution (HycloneÒ), phosphate buffered saline (PBS, HycloneÒ) and MTT were provided by Sunshine Biotechnology Co., Ltd. (Nanjing, China). Trypsin (GibcoÒ) was purchased from Pufei Biotechnology Co., Ltd. (Shanghai, China). All other chemicals and reagents were analytical grade. 2.2. Cell culture HepG2 and A549 cells were obtained from the cell bank of Chinese Academy of Sciences. The cells were cultured in 1640 containing 10% (v:v) FBS, 100 U/mL penicillin and 100 mg/mL streptomycin in an incubator (Thermo Scientific, USA) at 37  C under an atmosphere of 5% CO2 and 90% relative humidity, and were subcultivated approximately every 3 days at 80% confluence using 0.25% (w:v) trypsin at a split ratio of 1:5. 2.3. Screening of pH-responsive RH CPPs to tumor microenvironment 2.3.1. Cytotoxicity of RH CPPs The antiproliferative effects of RH CPPs were evaluated by MTT assay. The test solutions were the culture medium (FBS free) containing various concentrations of RH CPPs. The cells (4  104 cells/well) were seeded in 96-well plates (Costar, USA) and cultured at 37  C for 24 h. After removing the medium, the cells were incubated with 200 mL of the test solution for 24 h at 37  C. Subsequently, 20 mL of MTT PBS solution (5 mg/mL) was added into each well, and then the cells were stained at 37  C for 4 h. The test solutions were removed, and MTT formazan crystals were dissolved by 150 mL of dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm by an ELISA reader (Thermo Scientific, USA). Relative cell viability (R) was calculated as follows. R(%) ¼ Atest/Acontrol  100%, where Atest and Acontrol were the

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Fig. 1. Schematic design of the dual-decorated liposomes (PTX/HA-R6H4-L) for tumor-targeted drug delivery. A) Degradation of HA by HAase. B) Disassembly of PTX/HA-R6H4-L to PTX/R6H4-L. C) Tumor pH-induced PTX/R6H4-L for enhanced cell penetration. a) Intravenous injection of liposomes. b) Accumulation of liposomes at the tumor site through passive and active targeting. c) Degradation of HA by HAase together with the exposure of R6H4 at HAase-rich tumor milieu. d) pH-Response of R6H4 to mildly acidic tumor microenvironment for improved cellular uptake. e) Internalization of liposomes into the tumor cells. f) Endosomal/lysosomal escape. g) Cytoplasmic release.

absorbance of the cells treated with the test solutions and the blank culture medium (FBS free) as a negative control, respectively. 2.3.2. Effect of arginine on pH-response of RH CPPs to tumor microenvironment To estimate the impact of arginine on pH-response of RH CPPs to tumor microenvironment, the cellular uptake of FITC-RnH3 (n ¼ 5, 6, 7, 8, 9) with the different amounts of arginine at pH 7.4 and pH 6.4 were investigated by using confocal laser scanning microscopy (CLSM) and flow cytometry, respectively. The test solutions were listed as follows: 1.5 mL of culture medium (FBS free) adjusted to pH 7.4 or pH 6.4 containing 10 mM FITC-RnH3. The cells (1  105 cells/well) were seeded in 35 mm glassbottomed dishes (Greiner, Germany) and cultured at 37  C for 48 h. After washed twice with PBS, the cells were incubated with the test solution for 1 h. The cells were washed twice with cold PBS and observed immediately without fixing by CLSM (Leica, Germany). Subsequently, the cells were trypsinized for 5 min to remove surface-bound peptides [44], and trypsinization was stopped by adding cold culture medium. After centrifugation at 1000 rpm for 5 min, the cells were washed thrice with PBS and analyzed by flow cytometry (BD FACSCanto, USA). Each sample was analyzed for 10,000 events. The pH-responsive ratio of FITC-RnH3 on the cells was calculated by the following equation. pH-responsive ratio ¼ (FpH 6.4 e F0)/(FpH 7.4 e F0), where FpH 6.4 and FpH 7.4 were the fluorescence intensities of the FITC-RnH3 at pH 6.4 and pH 7.4, respectively, and F0 was the fluorescence intensity of the blank cells as a control. 2.3.3. Effect of histidine on pH-response of RH CPPs to tumor microenvironment Similarly, the cellular uptake of FITC-R6Hm (m ¼ 3, 4, 5) with the different amounts of histidine at pH 7.4 and pH 6.4 were investigated by using CLSM and flow cytometry, respectively. The test solutions were prepared as follows: 1.5 mL of culture medium (FBS free) adjusted to pH 7.4 or 6.4 containing 10 mM FITC-R6Hm. The other experimental procedure was the same as that of arginine above. The pH-responsive ratio of FITC-R6Hm on the cells was calculated as well.

2.3.4. Cellular uptake kinetics of R6H4 with the optimal pH-response To identify the cellular uptake kinetics of the highly pH-responsive R6H4, regarded as a judgment of intracellular delivery efficiency, the cellular uptake of FITC-R6H4 at pH 7.4 and pH 6.4 was measured by flow cytometry at different time. The cells (1  105 cells/well) were seeded in 6-well plates (Costar, USA) and cultured at 37  C for 48 h. The cells were incubated with 1.5 mL of culture medium (FBS free) adjusted to pH 7.4 or pH 6.4 containing 10 mM FITC-R6H4. After incubation for prearranged time intervals (4, 30, 60, 120, 240 min), the cells were treated and the fluorescence intensity of FITC-R6H4 was analyzed by flow cytometry. 2.4. Preparation and characterization of liposomes Conventional liposomes (CL) were prepared by a thin film dispersion method followed by membrane extrusion. SPC and Chol (20:1, w:w) were dissolved in chloroform. PTX with 3.3% (w:w), C6 with 0.033% (w:w) and NIRD15 with 3.3% (w:w) of the total lipids weight were added into the liposomal compositions to prepare drug-loaded, fluorescence-labeled and near infrared fluorescence-labeled liposomes, respectively. A thin lipid film was formed after rotation vacuum evaporization at 40  C, followed by vacuum dry overnight to remove any traces of organic solvent, and then was hydrated in distilled water. Subsequently, the liposomes suspensions were dispersed by a probe-type ultrasonicator (Beidi-IIYJ, Beidi, China) in ice bath at 100 W, and extruded through polycarbonate membranes with the pore size of 450 nm and 220 nm successively. CPP-modified liposomes (R6H4-L, R8-L) were obtained by the same method above, except that the hydration medium was distilled water containing stearyl CPP (R6H4-C18, R8-C18) with 2.5 mol% of total lipids weight. HA-coated CPP-modified liposomes (HA-R6H4-L, HA-R8-L) were obtained by adding R6H4-L and R8-L into 1 mg/mL HA solution (1:2, v:v), respectively. The particle size and zeta potential of the liposomes were measured by a Dynamic Light Scattering Analyzer (Brookhaven, USA) and a ZetaPlus Zeta

T. Jiang et al. / Biomaterials 33 (2012) 9246e9258 Potential Analyzer (Brookhaven, USA), respectively. The entrapment efficiency (EE) was calculated by the following equations: EE ¼ W/W0  100%, where W and W0 are the amounts of drug in the liposomes after and before passing over the Sephadex G50 column, respectively. The morphology and size distribution of PTX-loaded HAR6H4-L (PTX/HA-R6H4-L) were observed by atomic force microscopy (AFM) (Nano Scope IIIa, Veeco, USA). 2.5. Hemolysis Hemolysis studies were carried out to evaluate the safety of the liposomes for application in vivo. The rat red blood cells (RBC) were harvested by centrifugating at 10,000  g for 10 min after the collection of the rat blood in heparin sodiumcontaining tubes. The plasma supernatant was discarded and 5% (w:v) glucose solution (GS) was added to wash RBC thrice. Finally, the RBC were resuspended in GS to a concentration of 2% (w:v). Liposomes and Cremophor EL were diluted with GS to the different concentrations. Subsequently, 2% RBC solutions were incubated with the equal volume of liposomes or Cremophor EL for 1 h in a 37  C water bath, followed by centrifugation at 10,000  g for 5 min. The intact and ruptured RBC were pelleted out to leave hemoglobin in the supernatant solution. Absorbance of hemoglobin was measured at the wavelength of 570 nm by an ELISA Reader (Thermo Scientific, USA). The observed hemolysis of RBC in glucose solutions and in 1% Triton X-100 was used as negative and positive control, respectively. 2.6. Plasma stability In order to demonstrate the impact of surface charge and the effect of HA on the plasma stability of the liposomes, variations in the particle size of CL, R6H4-L and HA-R6H4-L were assayed in the presence of the rat plasma. Liposomes were mixed with the equal volume of the rat plasma and incubated for prearranged time intervals (0.5, 1, 2, 4, 8 h) in a 37  C water bath. After incubation, 200 mL of the sample was diluted in 3 mL of distilled water and the particle size of the liposomes was measured by the Dynamic Light Scattering Analyzer.

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were estimated by MTT assay. The cells were seeded at a density of 1  104 cells per well in 96-well plates for 24 h. PTX/HA-R6H4-L, PTX/HA-R8-L and PTX/CL were pretreated with 2 mg/mL HAase for 30 min before their addition to cells. The cells were exposed to the culture medium (FBS free) adjusted to pH 7.4 or pH 6.4 containing various PTX concentrations of the different formulations for 24 h. With the same treatment described above, the absorbance was measured at 570 nm. 2.11. Animal and tumor xenograft models Male BALB/cA nude mice (18e20 g) and Kunming (KM) mice (20e25 g) were purchased from Model Animal Research Center of Nanjing University (Jiangsu, China) and College of Veterinary Medicine Yangzhou University (Jiangsu, China), respectively. All the animals were pathogen free and allowed to access food and water freely. The experiments were carried out in compliance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. To set up the tumor xenograft model, mice were subcutaneously inoculated in the armpit with mice hepatocellular carcinoma (Heps) cells (1  107 cells) suspended in PBS (20 mL). Tumor volume (V) was determined by measuring length (L) and width (W), and calculated as V ¼ L  W2/2. Tumor-bearing mice were used as the volumes of tumor reached around 100 mm3 at 8 days post-tumor inoculation. 2.12. In vivo imagining analysis NIRD15-loaded HA-R6H4-L (NIRD15/HA-R6H4-L) and R6H4-L (NIRD15/R6H4-L) were intravenously injected into the tumor-bearing nude mice at a dose of 5 mg/kg to investigate their biodistributions and tumor targeting efficacies in the live mice by in vivo detection. Additionally, to certify the tumor targeting of HA, the change in the accumulation at the tumor site of NIRD15/HA-R6H4-L, followed by intravenously pre-administrating HA solution was also evaluated. NIRF imaging experiments were performed at 4 h and 24 h post-injection using a Kodak In-Vivo Imaging System FX Pro (Kodak, USA) equipped with an excitation bandpass filter at 720 nm and an emission at 790 nm. Images were analyzed using the Kodak Molecular Imaging Software 5.X.

2.7. Exposure of R6H4 in HA-R6H4-L by degradation of HA 2.13. Antitumor efficacy To confirm the degradation of HA and the exposure of R6H4 on the surface of HA-R6H4-L by HAase, the change of the viscosity of HA solution and the zeta potential of HA-R6H4-L in the presence of HAase were investigated, respectively. HA solutions with the different concentrations (0.3 mg/mL, 0.67 mg/mL, 1.5 mg/mL, 3 mg/mL) and HA-R6H4-L at HA concentration of 0.67 mg/mL were mixed with the equal volume of 2 mg/mL HAase solution, respectively. After prearranged incubation time (0.5, 2, 4, 8 h) in a 37  C water bath, the viscosity of HA solution and the zeta potential of HA-R6H4-L were measured by a rotary viscometer (LVDV-III, Brookfield, USA) and the ZetaPlus Zeta Potential Analyzer, respectively. 2.8. Buffering capacity R6H4 different from R8 contains 40% histidines, which has the imidazole ring for the proton sponge effect. This effect of R6H4 and R6H4-L were judged by their buffering capacity. Acid titrations were performed by using 10 mL of liposomes with the lipid concentration of 3 mg/mL containing 2.5 mol% CPP, adjusted to pH 10 with 0.3 M NaOH. 40 mL aliquot of 0.02 M HCl was added into the liposome or CPP solution sequentially until the pH value declined to 4. The pH value of the solution was measured with a pH meter (pHS-25B, Dapu, China) after each addition of acid. The slope of the plot of pH value versus the amount of HCl indicates the buffering capacity. 2.9. Intracellular delivery In attempt to discern the effect of R6H4-L on endosomal/lysosomal escape for efficient intracellular trafficking, the double-labeling experiments were performed by using CLSM for observing the cytoplasmic distribution of HA-R6H4-L on A498 cells. The localization of C6-loaded HA-R6H4-L (C6/HA-R6H4-L), R8-L (C6/HA-R8L) and CL (C6/CL) was visualized by labeling the cells with the specific fluorescent probe, such as LysoTracker. The cells (1  105 cells/well) were seeded in 35 mm glassbottomed dishes (Greiner, Germany) and cultured at 37  C for 48 h. C6/HAR6H4-L, C6/HA-R8-L and C6/CL were pre-treated with 2 mg/mL HAase for 30 min before their addition to cells. The cells were incubated with these three C6 formulations at C6 concentration of 100 ng/mL, respectively. At prearranged time intervals (1, 3, 6 h), the cells were washed by cold PBS twice, and then stained with 50 nM LysoTracker Red (Invitrogen, USA) for 30 min at 37  C. The cells were washed by cold PBS twice and observed immediately without fixing by CLSM (Olympus, Japan). 2.10. In vitro cytotoxicity of liposomes In vitro antiproliferative activities of PTX-loaded HA-R6H4-L (PTX/HA-R6H4-L), HA-R8-L (PTX/HA-R8-L) and CL (PTX/CL) against HepG2 cells at pH 7.4 and pH 6.4

In vivo antitumor efficacy of the different PTX formulations was evaluated on Heps tumor xenograft models. Eighty-four Heps tumor-bearing mice were weighed and randomly divided into seven groups (n ¼ 12): 1) saline; 2) TaxolÒ (10 mg/kg); 3) PTX/CL (10 mg/kg); 4) PTX/R8-L (10 mg/kg); 5) PTX/R6H4-L (10 mg/ kg); 6) PTX/HA-R8-L (10 mg/kg); 7) PTX/HA-R6H4-L (10 mg/kg). The different PTX formulations were administrated via tail vein at Day 8, 10, 12, 14. Tumor size was measured every other day. At Day 16, two of mice in each group were sacrificed to prepare tumor sections. Tumor was extracted, washed with saline thrice and fixed in 10% formalin. Formalin-fixed tumors were embedded in paraffin blocks to prepare hematoxylin and eosin (HE) stained tumor sections, and then were visualized by optical microscope (Olympus, Japan). The survival rates were monitored throughout the study. 2.14. Statistical analysis Data are given as mean  standard deviation. Statistical significance was tested by two-tailed Student’s t-test or one-way ANOVA. Statistical significance was set at *P < 0.05, and extreme significance was set at **P < 0.01.

3. Results and discussion 3.1. Screening of pH-responsive RH CPPs to tumor microenvironment 3.1.1. Cytotoxicity of RH CPPs The cytotoxicity of a series of synthesized RH CPPs toward HepG2 and A549 cells were evaluated by using MTT assay. The cell viabilities of all the RH CPPs with the different concentrations, except R9H3 with the concentration of 100 mM, were higher than 80% in HepG2 (Fig. 2) and A549 cells (Fig. S1), indicating no significant cytotoxicity of RH CPPs toward both cell lines. Additionally, the cytotoxicity of RH CPPs rose concomitantly with the increase of arginine in RH CPPs, but remained consistent as the histidine in RH CPPs increased. It is suggested that the cytotoxicity of RH CPPs is positively related to the intensity of positive charge, which is mainly attributive to the introduction of more arginines into RH CPPs.

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Fig. 2. Cytotoxicity of RH CPPs and R8 at various concentrations against HepG2 cells.

3.1.2. Effect of arginine on pH-response of RH CPPs to tumor microenvironment To identify the role of arginine in tumor pH-response of RH CPPs, the cellular uptake of FITC-RnH3 (n ¼ 5, 6, 7, 8, 9) in HepG2 cells at pH 7.4 and pH 6.4 were evaluated by CLSM and flow cytometry, respectively. As shown in Fig. 3A, the fluorescence intensity of FITC-R8 as a control at pH 6.4 was comparable to that at pH 7.4, suggesting that R8 presents non-pH-dependent cellular uptake. Conversely, all of FITC-R5H3, FITC-R6H3 and FITC-R7H3 showed stronger fluorescence intensity at pH 6.4 relative to at pH 7.4. No significant difference was found in the fluorescence intensity of FITC-R8H3 or FITC-R9H3 at pH 6.4 and at pH 7.4, although they displayed extremely higher fluorescence intensity than FITC-R8 at both pH values. As shown in Fig. 3B, the mean fluorescence intensity of FITC-R8 was 981 at pH 7.4 and 872 at pH 6.4 with a pH-responsive ratio of

0.89, demonstrating that R8 has no tumor pH-dependent uptake consistent with the CLSM observations above. By comparison, the pH-responsive ratios of FITC-R5H3, FITC-R6H3, FITC-R7H3, FITCR8H3 and FITC-R9H3 were 3.36, 3.50, 1.67, 0.97 and 1.00, respectively, suggesting that the pH-responsive effect of RnH3 initially rises and subsequently reduces as the ratio of arginine in RnH3 increases. The comparable intensity of FITC-R8H3 or FITC-R9H3 at pH 7.4 and pH 6.4 reconfirmed their lack of pH-response, although the mean fluorescence intensities of FITC-R8H3 and FITC-R9H3 at pH 6.4 were 3.87-fold and 4.88-fold of that of FITC-R8, respectively. The similar tendency was also found in A549 cells by using CLSM (Fig. S2A) and flow cytometry (Fig. S2B), respectively. Generally, cationic CPPs can promote the cellular uptake by means of strong binding on the membrane surfaces with anionic phosphates, sulfates, and carboxylates of cellular components via electrostatic interaction [45]. However, cationic property is not everything for cell penetration. Neither nonahistidine (H9) nor nonalysine (K9) was capable of protein transduction compared with nonaarginine (R9) [46]. In our study, R5H3, obtained by replacing 3 arginines of R8 with histidines, had a lower cellular uptake than R8 at both pH values, demonstrating that arginine plays a more crucial role than histidine in cell-penetrating ability of RH CPPs. On the other hand, the length of CPPs was also a key point for cellular uptake [7,47,48]. Oligomers of 6e20 arginines residues had a first rise then descent trend of the cellular uptake with a maximum amount of around 15 [47]. Similarly, the cellular uptake of RnH3 stood a comitant increase with arginines from 5 to 9 regardless of the pH value. Both R8H3 and R9H3 showed extremely high cellular uptake at both pH values with non-pHresponse. The main reason is that more dominant arginines impair the effect of 3 histidines on modulating the protonating degree at the different pH values, thereby yielding no pHdependent uptake of RnH3 with more than 7 arginines. Accordingly, R6H3 had the strongest pH-dependent cellular uptake on both cells.

Fig. 3. A, B) Cellular uptake of FITC-RnH3 (n ¼ 5, 6, 7, 8, 9) at the concentration of 10 mM on HepG2 cells at pH 7.4 and pH 6.4 investigated by CLSM (A) and flow cytometry (B). C, D) Cellular uptake of FITC-R6Hm (m ¼ 3, 4, 5) at the concentration of 10 mM on HepG2 cells at pH 7.4 and pH 6.4 investigated by CLSM (C) and flow cytometry (D). Scar bars are 50 mm. **P < 0.01.

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3.1.3. Effect of histidine on pH-response of RH CPPs to tumor microenvironment Based on the results above, to further estimate the role of histidine in tumor pH-response of RH CPPs, the cellular uptake of FITC-R6Hm (m ¼ 3, 4, 5) in HepG2 cells at pH 7.4 and pH 6.4 were also assessed by CLSM and flow cytometry, respectively. As shown in Fig. 3C, the fluorescence intensities of FITC-R6H4 and FITC-R6H5 remarkably enhanced in comparison with that of R6H3 at both pH values as the ratio of histidines in R6Hm increased. FITC-R6H3, FITC-R6H4 and FITC-R6H5 presented the pH-responsive ratios of 2.99, 3.90 and 2.06 in HepG2 cells, respectively (Fig. 3D), indicating that R6H4 has the optimal pH-response to tumor microenvironment. Additionally, the similar phenomenon was also found in A549 cells by using CLSM (Fig. S2C) and flow cytometry (Fig. S2D), respectively. Arginine has the isoelectric point (pI) of about 10.76, and thus constantly carries positive charge at both neutral and acidic conditions. By comparison, histidine, a special one of twenty amino acids with the pI of around 7.4, has the capability of pH-response to the acidity. Histidine-containing peptides have a net positive charge at the acidic condition, whereas are predominantly uncharged at the physiological environment. This characteristic of histidine has been used to design many pH-dependent peptides [13e15]. As a consequence, histidine plays an important role in providing a difference in the protonation of RH CPPs at pH 7.4 and at pH 6.4 for pH-response to tumor extracellular matrix. Compared with R6H3 and R6H5, R6H4 had the strongest pH-dependent cellular uptake regarded as the highest pH-response. It is speculated that there is a key balance between arginine and histidine in R6H4, in which they not only develop individual features, but also produce synergetic effect. On the one hand, arginine with positive charge clothes R6H4 with the strong and tunable capability of cell penetration. On the other hand, histidine endues R6H4 with pH-responsive cellular uptake, and potentially has the proton sponge effect on endosomal escape for improved intracellular delivery. 3.1.4. Cellular uptake kinetics of R6H4 To further demonstrate acid-activated cellular uptake of R6H4, the cellular uptake kinetics of R6H4 was estimated at the different pH values. Fig. 4 and Fig. S3 showed the cellular uptake of FITCR6H4 at pH 7.4 and pH 6.4 on HepG2 and A549 cells with the increase of the cell incubation time, respectively. R6H4 had the higher cellular uptake at pH 6.4 compared with at pH 7.4 at both

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cells regardless of the cell incubation time, reconfirming the pH-response of R6H4 to the mildly acidic tumor milieu. 3.2. Preparation and characterization of liposomes CL was comprised of SPC and cholesterol, and R6H4-C18 (2.5 mol% of lipid concentration) was anchored into the membrane of CL to construct R6H4-L by hydrophobic interaction. HA-R6H4-L was obtained by means of coating cationic R6H4-L by anionic HA via electrostatic effect. The particles size, zeta potential and EE of various liposomes were listed in Table 1 and Table S2. The bare CL had a particle size of 84.3  2.3 nm and zeta potential of 22.54  1.45 mV. By comparison, CPP-modified liposomes showed significantly a larger size and a converse surface charge. The zeta potential of R6H4-L and R8-L were about þ27.45  1.80 and þ32.82  1.60 mV, respectively. Interestingly, the coating effect of HA provided HA-R6H4-L or HA-R8-L a notably increased size and a highly negative charge. Both HA-R6H4-L and HA-R8-L had the particle size larger than 120 nm and the zeta potential of about 25 mV. These changes in the particle size and zeta potential of the liposomes present a substantial evidence of the liposomal surface modification by cationic CPPs and anionic HA via hydrophobic and electrostatic interactions, respectively. PTX, a water-insoluble anticancer drug, is encapsulated in the lipid bilayer of the liposomes. Compared with the bare liposomes, PTX-loaded liposomes had a larger particle size and a similar variation in zeta potential from CL to HA-coated CPP-modified liposomes. EE of PTX in the liposomes was all higher than 95%. The concentration of the total lipids and PTX in the liposomes were 30 mg/mL and 1 mg/mL, respectively. The AFM image confirmed the spherical shape of PTX-loaded HA-R6H4-L (PTX/HA-R6H4-L) with a good dispersity (Fig. S4). 3.3. Hemolysis and plasma stability The application of the liposomal formulations in vivo in the pharmaceutical field counts on several aspects including drugloading efficiency, safety and stability. Hemolysis of the liposomes was investigated for their hemocompatibility, a safety guide for the intravenous injection in clinical studies. As shown in Fig. 5A, all of the different kinds of the liposomes displayed little and no hemolysis, compared with Cremophor EL, the component of TaxolÒ (the commercial PTX formulation), with much higher hemolysis, suggesting the relatively better safety of various liposomes than TaxolÒ despite highly positive charged CPP-modified liposomes. On the other hand, it is also important for the liposomes to keep stable away from the attack of plasma proteins in blood until reaching the final target. The change in the particle size of the liposomes is generally monitored in the presence of plasma as a simple but essential index to estimate the stability of the liposomes in vitro. As shown in Fig. 5B, for 15 min incubation with the plasma, the particle sizes of negatively charged HA-R6H4-L and CL slightly changed with approximately a fluctuation of 8%, while that of cationic R6H4-L had a increase of 23%. It is indicated that cationic

Table 1 Particle size, zeta potential and entrapment efficiency (EE) of various PTX-loaded liposomes.

Fig. 4. Cellular uptake kinetics of FITC-R6H4 at the concentration of 10 mM on HepG2 cells at pH 7.4 and pH 6.4 by flow cytometry.

Liposomes

Particle size (nm)

PTX/CL PTX/R8-L PTX/R6H4-L PTX/HA-R8-L PTX/HA-R6H4-L

87.8 105.6 116.2 135.5 147.8

    

2.3 2.4 4.1 1.9 3.3

Zeta potential (mV)

EE (%)

28.64 35.15 28.63 16.78 20.80

98.42 99.05 97.30 97.28 96.55

    

3.23 2.13 1.83 1.15 1.70

    

3.57 0.32 2.50 2.23 1.70

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20

R6H4-L

HA-R6H4-L

R8-L

HA-R8-L

Cremophor EL

10 5 0

0. 75 0 0. 30 0 0. 15 0 0. 07 5 0. 03 0 0. 01 5

50 0 1.

3.

00 0

-5

Concentration (mg/mL)

B

150

CL

R6H4-L

HA-R6H4-L

Particle size (nm)

140 130

as cancers, osteoarthritis and mucopolysaccharidosis [41,42,49], is able to specifically hydrolyze HA, which allows the R6H4 exposure of HA-R6H4-L at HAase-rich tumor microenvironment for maximizing the targeting and internalization. HA as a macromolecule dissolved in aqueous solution has a certain degree of viscosity compared with purified water, and is degraded by HAase together with the decrease of its viscosity. In viscosity assay, a remarkably sharp reduction of viscosity of HA at the concentration range from 0.3 mg/mL to 3 mg/mL by incubation with 2 mg/mL HAase, suggesting the hypersensitive cleavage reaction of HA response to specific HAase. In particular, the presence of HAase resulted in the rapid and complete degradation of HA at the concentration of 0.3 mg/mL and 0.67 mg/mL for 30 min incubation (Fig. 6A). As shown above, HA-R6H4-L has a highly negative charge, while R6H4-L without HA coating presents a positive charge. To confirm the exposure of R6H4 in the surface of HA-R6H4-L, the variation in the zeta potential of HA-R6H4-L at HA concentration of 0.67 mg/mL was measured in the presence of HAase. Along with the hydrolysis by HAase, long-chain HA breaks down, yielding fragments and shedding from HA-R6H4-L as a result of impaired electrostatic interaction, followed by the exposure of R6H4 and charge conversion from negative of HA-R6H4-L to positive of R6H4-L. As shown in Fig. 6B, the zeta potential of HA-R6H4-L changed sharply from negative to approximately neutral for a short exposure time of

120

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50 40

100 0

15

30

60

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240

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Time (min) Fig. 5. A) Hemolysis of the different liposomes and Cremophor EL at various concentrations. B) Variation in the particle size of CL, R6H4-L and HA-R6H4-L incubated with the rat plasma for different time.

liposomes tend to aggregate with anionic plasma components, leading to the instability of the liposomes, the rapid clearance from reticuloendothelial system (RES) and the risk of perturbing the plasma membrane structure to induce high cytotoxicity and excessive immune response [20e22]. By comparison, anionic liposomes, particularly polymer-coated liposomes such as HA-R6H4-L, have much less interaction with plasma proteins under the protection of the anionic HA hydrophilic shell, thereby latently improving the stability, bioavailability and blood persistence. Hence, the instability of R6H4-L in the presence of plasma implies the inferior behavior in vivo and the necessary introduction of HA to shield the positive charge of R6H4-L for enhanced stability. 3.4. Exposure of R6H4 in HA-R6H4-L by degradation of HA HA is involved in numerous biological and physiological functions including cell motility, cell matrix adhesion, and cell proliferation, and has superior properties, such as non-immunogenicity, non-toxicity, and total biodegradability [49]. Besides, HA-binding receptors, such as CD44 and RHAMM, are overexpressed on the cell surface of several malignant tumors [35e37]. According to these features, HA has been widely used in tumor-targeted drug delivery. More importantly, HAase, an endo-glucosidase extensively existing in pathophysiology of many disorders, such

Viscosity (mPa/s)

Hemolysis (%)

15

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T. Jiang et al. / Biomaterials 33 (2012) 9246e9258

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pH 6.4

pH 7.4

15

0

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-30 0

2

4

6

8

Time (h) Fig. 6. A) Change in the viscosity of the different concentrations of HA solutions incubated with 2 mg/mL HAase for different time. B) Change in the zeta potential of HA-R6H4-L incubated with 2 mg/mL HAase for different time.

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12 10

R8-C18

CL

R6H4-C18

R8-L

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on” the penetrating activity of R6H4 for improved tumor cellular uptake.

R6H4-L

3.5. Buffering capacity

pH

9 7.4 6 4 3 0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

HCl volume (mL) Fig. 7. Acid titration profiles of aqueous solutions of the different liposomes. Solutions of each liposome were adjusted to pH 10 using 0.3 M NaOH and then titrated with 0.02 M HCl.

30 min to HAase regardless of pH values. Moreover, the increasing acidity had a concomitant increase in HAase hydrolysis capability. At 8 h, the zeta potential of HA-R6H4-L continuously rose to þ10 mV at pH 6.4 and þ20 mV at pH 5.4, while HA-R6H4-L still presented slightly negative charge at pH 7.4. The results certify the exposure of R6H4 and the transition from HA-R6H4-L to R6H4-L by the hydrolytic effect of HAase, which can be promoted and enhanced under the tumor microenvironmental acidity. Collectively, HA as an outer corona is introduced via electrostatic interaction for increased longevity of R6H4-L in blood circulation, which can be detached at weak acidic HAase-rich tumor microenvironment to “turn off” the protective function of HA and “turn

A myriad of nanoparticles, including CPPs-modified liposomes, enter the tumor cells mainly through energy-dependent endocytosis, followed by transporting into the endosomes/lysosomes [50e54], which are regarded as a primary barrier for intracellular drug delivery owing to their bio-function on degrading inclusions by the acid and hydrolase. Although oligoarginine has been demonstrated as efficient CPPs for membrane penetration, it has a great difficulty in transporting its cargo with a large size, such as liposomes, to penetrate the endosomal/lysosomal membranes before degradation [50,55e57]. R6H4 different from R8 contains 40% histidines, which have an established proton sponge effect for endosomal/lysosomal escape similar to polyethylenimine (PEI), and this effect is generally evaluated as a pH-buffering capacity by a acid titration method [17,58]. As shown in Fig. 7, the acid titration profiles of R6H4-C18 and R6H4-L were obtained from the change in pH value titrated by 0.02 M HCl, respectively. The slope of the plot of pH value versus the amount of HCl indicates the buffering capacity. R6H4-C18 displayed a slower downtrend and gentler slope of the titration curve than R8-C18, and additionally provided R6H4-L a stronger buffer capacity compared with R8-L and CL from neutral (pH 7.4) to acidic condition (pH 4.0). Other histidine lipids [17,59] for endosomal escape showed similar titration profiles. Accordingly, R6H4-L containing histidines with imidazole rings has the proton sponge effect accompanied by cell-penetrating capability for endosomal/lysosomal escape, able to absorb protons for increased osmotic pressure inside the endosomes/lysosomes, followed by membrane disruption and liberation into the cytoplasm.

Fig. 8. Intracellular delivery of the different C6-loaded liposomes on HepG2 cells at various time observed by CLSM. C6/CL (A), C6/HA-R8-L (B) and C6/HA-R6H4-L (C) were pretreated with 2 mg/mL HAase for 30 min before their addition to the cells. The late endosomes and lysosomes were stained by LysoTracker Red. 1: green fluorescent C6; 2: red fluorescent endosomes/lysosomes; 3: overlay of 1 and 2. White arrows indicate the occasions of coincidence between the liposomes and endosome/lysosomes. Green arrows signify the endosomal/lysosomal escape of the liposomes into the cytoplasm. Red arrows imply the destruction of endosomes/lysosomes. Scale bars are 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

T. Jiang et al. / Biomaterials 33 (2012) 9246e9258

3.7. In vitro cytotoxicity of PTX-loaded liposomes HA-R6H4-L accumulates at the tumor site by means of the combination of passive targeting as EPR effect and active targeting of HA, and then encounters the hydrolysis by HAase and converses to cationic R6H4-L. The tumor extracellular pH-response of R6H4 has been demonstrated that the cellular uptake of R6H4 at pH 6.4 was extremely higher than that at pH 7.4. To further identify that R6H4 endues R6H4-L with pH-response to the tumor microenvironment for enhanced internalization and antiproliferation, in vitro cytotoxicity of HA-R6H4-L against HepG2 cells at pH 7.4 and pH 6.4 was performed after HAase treatment for 30 min by using MTT assay. As shown in Fig. 9, PTX/HA-R6H4-L killed cancer cells in a dose-dependent manner at pH 6.4, whereas it had no cytotoxicity at pH 7.4 until a high PTX concentration of 20 mg/mL, indicating that the killing activity of PTX/HA-R6H4-L is pH-dependent. On the contrary, the antiproliferative effects of PTX/CL and PTX/HA-R8-L had no noticeable improvement from pH 7.4 to pH 6.4. Moreover, PTX/HA-R6H4-L presented even higher cytotoxicity than PTX/HA-R8-L and PTX/ CL at pH 6.4 at all the PTX concentrations investigated. Accordingly, PTX/HA-R6H4-L based on pH-response of R6H4 showed remarkably high cytotoxicity and selectivity to cancer cells in a pH-dependent manner.

120

pH 7.4

pH 6.4

100 Cell viability (%)

On the basis of these findings that R6H4-L has the stronger proton sponge effect and membrane penetrating capability in comparison with R8-L, we are highly interested in whether HA-R6H4-L after HAase treatment exhibits an effective intracellular trafficking. Double-labeling experiment for subcellular localization was carried out by using CLSM. Lysosomes as well as the late endosomes were selectively stained as a red fluorescence by LysoTracker Red, a specific marker for these acidic organelles, while the liposomes encapsulating C6 were observed as a green fluorescence. Co-localization of the liposomes with the specific organelle dyes was viewed as a yellow fluorescence. As shown in Fig. 8, a majority of endocytosed green fluorescent HA-R6H4-L was located in the red fluorescent endosomes/lysosomes, judging by the yellow fluorescence, after cell incubation with HA-R6H4-L for 1 h. Interestingly, as time extended to 3 h, a portion of green fluorescence had a significant dissociation from the red fluorescence resulted from the initiation of the efficient endosomal/lysosomal escape of HA-R6H4-L, although there were a range of yellow fluorescence in the cells. At 6 h, HA-R6H4-L showed broader cytoplasmic release and distribution, judging from the great separation between the green and red fluorescence. Furthermore, the staining of endosomes/lysosomes by the tracker depended upon the acidity of the endosomal/lysosomal compartments, and thus, the red fluorescence of endosomes/lysosomes became attenuated, suggesting the destruction of the acid endosomal/lysosomal environment as a result of the proton sponge effect of HA-R6H4-L. By comparison, HA-R8-L displayed relatively weaker capability of endosomal/lysosomal escape. Only a part of green fluorescent HA-R8-L was released into the cytoplasm at 6 h. In contrast, almost all the green fluorescence was highly overlaid with the red fluorescence when the cells were incubated with C6/ CL even after 6 h, indicating that it is difficult for the CL to penetrate through the endosomes/lysosomes. Consequently, R6H4 provides HA-R6H4-L endosomal/lysosomal escape and cytoplasmic liberation for efficient intracellular delivery dependent on the proton sponge effect and membrane penetrating capability, followed by internalization of HA-R6H4-L and subsequent transportation into endosomes/lysosomes.

A

80 60 40 20 0

0.2

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3.6. Intracellular delivery

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C

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*

pH 6.4

**

**

80 60

**

40 20 0

0.2

0.5

2

5

20

PTX concentration (µg/mL) Fig. 9. Cytotoxicity of the different PTX-loaded liposomes on HepG2 cells at pH 7.4 and pH 6.4 for 24 h. PTX/CL (A), PTX/HA-R8-L (B) and PTX/HA-R6H4-L (C) were pre-treated with 2 mg/mL HAase for 30 min before their addition to the cells. *P < 0.05, **P < 0.01.

3.8. In vivo imaging analysis Most of the currently developed nanosystems, including active targeting ones, have a discrepancy between targetability in vitro and in vivo that they have superior targeting ability in the tumor cells in vitro but inferior accumulation at the tumor site in vivo. This is mainly attributed to the clearance from the circulation by the RES and the fleeing from the tumor sites owing to lack of cellular

T. Jiang et al. / Biomaterials 33 (2012) 9246e9258

uptake. To address this dilemma, HA-R6H4-L was functionalized with a tumor extracellular pH-triggered CPP and a detachable HA corona. HA-R6H4-L presents improved blood persistence by the protective corona and negatively charged surface, and enhanced tumor accumulation by the affinity between HA and its receptors. On the other hand, it can turn the protection off by HA degradation and turn on the targeting/internalizing functions at the tumor site. Accordingly, HA-R6H4-L may have a promising efficient tumor targeting in vivo. For the purpose of verifying this potential, the biodistribution of NIRD15/R6H4-L and NIRD15/HA-R6H4-L administrated intravenously into Heps tumor-bearing nude mice was visualized by a non-invasive near infrared optical imaging technique, respectively. In addition, a high dose of HA polymer was intravenously injected before administration of NIRD15/HA-R6H4-L to further

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estimate the competitive inhibition of free HA to active tumor targeting of HA-R6H4-L. Fig. 10 showed the real-time images of the liposomes in the tumor-bearing nude mice, in which the whole bodies of the live mice were monitored at 4 h and 24 h after administration, respectively. NIRD15 signal of R6H4-L was visualized mainly in liver and little in tumor at 4 h, revealing a high hepatic uptake and a rapid clearance of cationic liposomes. Excitingly, HA-R6H4-L exhibited much stronger fluorescence intensity in tumor regions in a quite short time compared with the whole body and R6H4-L without HA coating. As time increased, a preferential accumulation of fluorescence was obvious in the tumor site rather than liver or other normal tissues within 24 h post-injection. By comparison, the NIRD15 signal of HA-R6H4-L was remarkably attenuated at both 4 h and 24 h, when free HA was pre-injected into the mice. It is suggested that free HA can competitively bind to HA

Fig. 10. In vivo imaging of Heps tumor-bearing nude mice after intravenous injection of NIRD15/R6H4-L (A), NIRD15/HA-R6H4-L (B) and NIRD15/HA-R6H4-L with pre-injection of free HA (C).

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Fig. 11. In vivo therapeutic efficacy of the different PTX formulations in Heps tumor-bearing mice. A, B) Changes of tumor volumes (A) and survival rates (B) of tumor-bearing mice after intravenous injection of the different PTX formulations at a dose of 10 mg/kg and saline as a negative control. The arrows signify the time of intravenous administration. *P < 0.05, **P < 0.01. C) Representative images of tumor sections separated from mice stained by HE at 16 days post-implantation.

receptors against HA-R6H4-L, resulting in the decreased fluorescent intensity, which provided a considerable evidence that the coating of HA onto the surface of cationic liposomes not only provide stability in the blood circulation, but also strengthen active tumor targeting. Such a powerful tumor targeting of HA-R6H4-L is ascribed to the protection of R6H4-L by HA for improved stability and prolonged circulation to attain a preferable EPR effect, the affinity of HA with its receptors overexpressed in tumor for active tumor targeting, and more notably, the exposure of R6H4 with pH-induced enhanced cell-penetrating ability at the tumor site for increased cellular uptake and decreased escape from tumor into blood. 3.9. In vivo antitumor efficacy In order to confirm the feasibility of HA-R6H4-L for cancer therapy in vivo, the antitumor efficacy of PTX/HA-R6H4-L was estimated in Heps tumor xenograft models. As shown in Fig. 11A, the tumor volume of mice receiving saline as control rapidly increased at 3 weeks, while the growth of tumor was significantly suppressed by the treatment of various PTX formulations. Noting that TaxolÒ inhibited tumor growth to some extent but with two mice death during the administration period resulted from the side effect and wide distribution of TaxolÒ. PTX/R6H4-L had an evidently higher tumor inhibitive effect than TaxolÒ, whereas PTX/ CL showed a comparable effect to TaxolÒ on tumor inhibition, indicating that in addition to EPR effect for liposomal accumulation

at the tumor site, a prompt penetration in the tumor cells avoiding the drainage into blood, is a prerequisite for enhanced antitumor activity of liposomal drugs. More significantly, PTX/HA-R6H4-L displayed a prominent effect on tumor size inhibition in comparison with other formulations. Furthermore, it also possessed the most distinguished effect on extending survival period of tumorbearing mice (Fig. 11B). The images of stained tumor tissue section by HE showed the greatest massive cancer cell remission after applying PTX/HA-R6H4-L (Fig. 11C), which presents a substantial evidence of the efficient antitumor activity of PTX/HAR6H4-L in vivo. Overall, dual-functional HA-R6H4-L with degradable HA and pH-responsive R6H4 attains the goal of overcoming sequential physiological and biological barriers and improving antitumor efficacy in vivo. 4. Conclusion In summary, we developed a dual-decorated liposome (HA-R6H4-L) for tumor-targeted drug delivery. HA-R6H4-L, with the protection by HA turning on, accumulates at the tumor site through passive and active targeting, and detaches the HA outer corona by HAase followed by disassembly to cationic R6H4-L, as the HA protection switches off. The exposure of R6H4 with pH-response to tumor microenvironment enhances the cellular uptake of the liposomes as the cell-penetrating effect of R6H4 turns on. The proton sponge effect accompanied by membrane penetrating ability of R6H4 produces endosomal/lysosomal escape for

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efficient intracellular delivery. These properties endue HA-R6H4-L with precise and safe anticancer drug delivery for tumor cells, with largely improved therapeutic efficacy and decreased drug toxicity. Acknowledgment We thank Prof. Hongping Ying for providing cell laboratory, and also acknowledged Dr. Ran Mo for technical assistance and writing improvement. The National Natural Science Foundation of China (No. 30973649 and No. 30901867) is gratefully acknowledged for financial support. This work was also supported by the Research Fund for the Doctoral Program of Higher Education of China (No. 20090096110002) and the National Basic Research Program of China (CX10B-373Z). Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2012.09.027. References [1] Cheng CJ, Saltzman WM. Enhanced siRNA delivery into cells by exploiting the synergy between targeting ligands and cell-penetrating peptides. Biomaterials 2011;32:6194e203. [2] Lee JY, Bae KH, Kim JS, Nam YS, Park TG. Intracellular delivery of paclitaxel using oil-free, shell cross-linked HSA-multi-armed PEG nanocapsules. Biomaterials 2011;32:8635e44. [3] Liu J, Zhao Y, Guo Q, Wang Z, Wang H, Yang Y, et al. TAT-modified nanosilver for combating multidrug-resistant cancer. Biomaterials 2012;33:6155e61. [4] Jiang QY, Lai LH, Shen J, Wang QQ, Xu FJ, Tang GP. Gene delivery to tumor cells by cationic polymeric nanovectors coupled to folic acid and the cellpenetrating peptide octaarginine. Biomaterials 2011;32:7253e62. [5] Chen JX, Wang HY, Li C, Han K, Zhang XZ, Zhuo RX. Construction of surfactantlike tetra-tail amphiphilic peptide with RGD ligand for encapsulation of porphyrin for photodynamic therapy. Biomaterials 2011;32:1678e84. [6] Rijt SH, Kostrhunova H, Brabec V, Sadler PJ. Functionalization of osmium arene anticancer complexes with (poly) arginine: effect on cellular uptake, internalization, and cytotoxicity. Bioconjug Chem 2011;22:218e26. [7] Wender PA, Galliher WC, Goun EA, Jones LR, Pillow TH. The design of guanidinium-rich transporters and their internalization mechanisms. Adv Drug Deliv Rev 2008;60:452e72. [8] Katayama S, Hirose H, Takayama K, Nakase I, Futaki S. Acylation of octaarginine: implication to the use of intracellular delivery vectors. J Control Release 2011;149:29e35. [9] Takayama K, Nakase I, Michiue H, Takeuchi T, Tomizawa K, Matsui H, et al. Enhanced intracellular delivery using arginine-rich peptides by the addition of penetration accelerating sequences (Pas). J Control Release 2009;138:128e33. [10] Liu BR, Huang YW, Winiarz JG, Chiang HJ, Lee HJ. Intracellular delivery of quantum dots mediated by a histidine- and arginine-rich HR9 cellpenetrating peptide through the direct membrane translocation mechanism. Biomaterials 2011;32:3520e37. [11] Gerweck LE, Seetharaman K. Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer. Cancer Res 1996;56: 1194e8. [12] Thurlkill RL, Grimsley GR, Scholtz JM, Pace CN. pK values of the ionizable groups of proteins. Protein Sci 2006;15:1214e8. [13] Zhang W, Song J, Zhang B, Liu L, Wang K, Wang R. Design of acid-activated cell penetrating peptide for delivery of active molecules into cancer cells. Bioconjug Chem 2011;22:1410e5. [14] Tu Z, Volk M, Shah K, Clerkin K, Liang JF. Constructing bioactive peptides with pH-dependent activities. Peptides 2009;30:1523e8. [15] Makovitzki A, Fink A, Shai Y. Suppression of human solid tumor growth in mice by intratumor and systemic inoculation of histidine-rich and pH-dependent host defense-like lytic peptides. Cancer Res 2009;69:3458e63. [16] Tanaka K, Kanazawa T, Ogawa T, Takashima Y, Fukuda T, Okada H. Disulfide crosslinked stearoyl carrier peptides containing arginine and histidine enhance siRNA uptake and gene silencing. Int J Pharm 2010;398:219e24. [17] Mo R, Sun Q, Xue J, Li N, Li W, Zhang C, et al. Multistage pH-responsive liposomes for mitochondrial-targeted anticancer drug delivery. Adv Mater 2012;24:3659e65. [18] Lee ES, Na K, Bae YH. Super pH-sensitive multifunctional polymeric micelle. Nano Lett 2005;5:325e9. [19] Koren E, Apte A, Sawant RR, Grunwald J, Torchilin VP. Cell-penetrating TAT peptide in drug delivery systems: proteolytic stability requirements. Drug Deliv 2011;18:377e84.

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