Co-delivery of paclitaxel and gemcitabine via CD44-targeting nanocarriers as a prodrug with synergistic antitumor activity against human biliary cancer

Biomaterials 53 (2015) 763e774

Contents lists available at ScienceDirect

Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Co-delivery of paclitaxel and gemcitabine via CD44-targeting nanocarriers as a prodrug with synergistic antitumor activity against human biliary cancer Ilkoo Noh a, d, Hyun-Ouk Kim a, Jihye Choi a, e, Yuna Choi b, Dong Ki Lee c, Yong-Min Huh b, **, Seungjoo Haam a, * a

Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, South Korea Department of Radiology, Severance Hospital, Yonsei University College of Medicine, Seoul 120-752, South Korea Department of Internal Medicine, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul 135-720, South Korea d Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, South Korea e Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 March 2015 Accepted 4 March 2015 Available online

Multi-drug delivery focuses on different signaling pathways in cancer cells that have synergistic antiproliferative effects. In this study, we developed multi-prodrug nanocarriers (MPDNCs) consisting of poly (L-lysine)ecarboxylate PTX (PLL-PTX) and hyaluronic acid-conjugated GEM (HA-GEM) for CD44targeted synergistic biliary cancer therapy. An in vitro study of cell viability and mRNA expression levels and an in vivo study showed that MPDNCs more effectively inhibit proliferation in CD44overexpressing cancer cells (HuCCT1) than in cells with lower CD44 expression (SCK) by synergistically inducing apoptosis. Consequently, these results demonstrate that MPDNCs are prodrugs with synergistic cancer therapeutic efficacy and effective cellular uptake at target cells compared to free drugs, indicating their strong potential as efficient multi-drug-carrying nano-platforms for cancer treatment. © 2015 Elsevier Ltd. All rights reserved.

Keywords: CD44 targeting Polymeric prodrug Gemcitabine Paclitaxel Biliary cancer Combined cancer therapy

1. Introduction Simultaneous administration of a hydrophilic drug such as gemcitabine (GEM) or 5-fluorouracil and hydrophobic paclitaxel (PTX) to cancer patients, especially those with biliary and pancreatic cancer, has become a standard clinical treatment and has proven to be an effective way to suppress the proliferation of cancer cells [1e3]. In particular, combinational chemotherapy using GEM with PTX through multiple specific treatment pathways has synergistically superior therapeutic efficacy, thus reducing intolerable cytotoxicity and maximizing the therapeutic effect even at a lower dose [4e6]. Furthermore, this combination is capable of modulating the genetic barriers of the cancer cell caused by mutation, thereby delaying adjustment to cancer [6,7]. However, co-delivery of GEM and PTX without carriers is often limited by several problems. For instance, free GEM has poor stability in the bloodstream

* Corresponding author. Tel.: þ82 2 2123 2751; fax: þ82 2 312 6401. ** Corresponding author. Tel.: þ82 2 2228 2375; fax: þ82 2 362 8647. E-mail addresses: [email protected] (Y.-M. Huh), [email protected] (S. Haam). http://dx.doi.org/10.1016/j.biomaterials.2015.03.006 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

and low permeability across the cellular membrane, and free PTX has limited efficacy due to its low water solubility [8,9]. Moreover, co-administration of GEM and PTX leads to pharmacokinetic discrepancy in the bloodstream and non-specific uptake, which consequently engenders relapse or metastasis of tumors due to low accumulation in malignancy [10,11]. Accordingly, in order to determine an effective method for the concurrent drug delivery of GEM and PTX into target cells, various approaches incorporating nanoparticles as the delivery carriers have been investigated [7,12,13]. The drug containing nanocarriers have the primary advantages of nanomedicine as follow; protecting enzymatic degradation and immune recognition, effective deliver to targeted cancer cell owing to corresponding biomarkers on cancer cell membrane compared to non-encapsulated drugs, and escaping reticuloendothelial system which induce sustained drug delivery [14]. However, drug-delivering nanoparticles, such as polymeric micelles simply loaded with anticancer agents, face the hurdle of the solubility issue: drug loaded polymeric micelles are suitable for loading of hydrophobic drugs such as PTX, but not for hydrophilic ones such as GEM [15,16]. Furthermore, liposomes are potential delivery vehicles of hydrophilic and hydrophobic drugs,

764

I. Noh et al. / Biomaterials 53 (2015) 763e774

but there are several drawbacks to their use, such as a lower localized hydrophobic drug concentration in the lipid bilayer and a higher initial burst, as well as thermodynamic instability, which limit the duration of their circulation in co-delivery systems [17]. In contrast, nanocarriers based on the conjugation of a polymer with a drug, a polymeric prodrug, have several advantages such as enhancing drug bioavailability, balancing the pharmacokinetics of hydrophilic and hydrophobic drugs, allowing the tumor-specific activation of drugs, and condensing complex formulations for advanced drug delivery [18e20]. Thus, nanocarriers containing polymeric prodrugs offer new strategies to create ideal cancer therapy systems by synchronizing the advantages of polymeric prodrug and co-delivery systems, and thus are able to have synergistic therapeutic effects via simultaneous multi-drug accumulation [21e23]. Furthermore, the limitations that have resulted from low delivery efficiency to target cells can be overcome by exploiting selective tumor targeting [24]. Biliary cancer, which originates in the bile duct, is a devastating malignant cancer, and its incidence rate is increasing worldwide [25]. In addition, biliary cancer is mostly diagnosed at the unresectable stage due to nonspecific initial symptoms. However, carcinoembryonic antigen (CEA), cell surface receptors of cluster determinant 44 (CD44), and carbohydrate antigen (CA) 19-9 expression levels have been recognized among the criteria for malignancy [26]. Among these receptors, CD44, which plays an important role in cell invasion, tumor progression, and cell survival, is regarded as a significant tumor indicator in that it is only overexpressed in malignancy and not in the normal bile epithelium [27,28]. Thus, hyaluronic acid (HA), which has a strong affinity for CD44, enhances the therapeutic efficacy of biliary cancer treatments, as well as their bioavailability, owing to its nonimmunogenic shielding of cationic polymers and biocompatibility [29]. In this report, we designed a CD44-targeted combinatorial drug delivery strategy to synergistically induce apoptosis in human biliary cancer. Fig. 1 illustrates the formation and synergistic apoptosis induction of multi-prodrug nanocarriers (MPDNCs) after their cellular uptake. We formulated a polymeric prodrug complex using electrostatic attractions between synthesized poly (L-lysine)e carboxylate PTX (PLL-PTX) and hyaluronic acid-conjugated GEM (HA-GEM) with hydrolysable linkers. PLL-PTX, a cationic polymer, is able to overcome chemotherapeutic hindrances by enhancing PTX water solubility [8]. In addition, HA-GEM, an anionic polymer, is able to specifically bind to the CD44 receptor and deliver the hydrophilic GEM anticancer agent. MPDNCs are internalized into target cancer cells via CD44-mediated endocytosis. In detail, GEM and PTX are hydrolyzed in acidified endosomes, and GEM suppresses DNA repair by being incorporated into DNA in competition with normal deoxycytidine and inhibiting ribonucleotide reductase [30]. Meanwhile, PTX acts as a tubulin stabilizer and influences cell replication by causing mitotic arrest [31]. The actions of these components of the multi-drug on different pathways lead to synergistic therapeutic effects at desirable cancer sites when conveyed in MPDNCs [32]. In vitro and in vivo studies of MPDNCs were carried out to assess the efficacy of their anti-tumor activity in target cells via synergistic induction of programmed apoptosis. 2. Materials and methods 2.1. Materials Sodium hyaluronic acid (30 kDa) was obtained from Lifecore Biomedical, LLC (Chaska, MN, USA). Paclitaxel (PTX) was purchased from Samyang Genex, Inc (Seoul, South Korea). Succinic anhydride (SA), Nε-carbobenzyloxy-L-lysine (Lys(Z)), hexylamine, hydrogen bromide in acetic acid (HBr), N,N0 -dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), dichloromethane (DCM), dimethyl sulfoxide anhydrous (DMSO), pyridine anhydrous, tetrahydrofuran (THF), N,N-dimethyformamide anhydrous (DMF), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid

sodium salt (HEPES), phosphate buffered saline tablets (PBS), chloroform-d (CDCl3), dimethyl sulfoxide-d6 (DMSO-d6) and deuterium oxide (D2O) were obtained from SigmaeAldrich (St. Louis, MO, USA). Gemcitabine hydrochloride (GEM), trifluoroacetic acid (TFA), triphosgen, and N-hydroxysuccinimide (NHS) were obtained from Tokyo Chemical Industry (Tokyo, Japan). Diethyl ether, n-hexane, acetonitrile, and methanol were purchased from Duksan Pure Chemicals Co., Ltd (Ansan, South Korea). Dulbecco's phosphate buffered saline (DPBS) (10 mM, pH 7.4), Dulbecco's modified Eagle's medium (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI1640) were obtained from Invitrogen, Co (Carlsbad, CA, USA). All of the other chemicals and reagents were of analytical grade. 2.2. Synthesis of poly (L-lysine)ecarboxylate paclitaxel (PLL-PTX) 2.2.1. Synthesis of Nε-carbobenzyloxy-L-lysine N-carboxyanhydrides (Lys(Z)-NCA) Lys(Z) (5.00 g, 17.85 mmol) was reacted with triphosgen (2.12 g, 7.15 mmol) suspended in THF (65 mL) at 50  C for 3 h under nitrogen purging [33]. Lys(Z)-NCA was precipitated with the excess cold n-hexane and the product was purified by repeated filtration. The purified Lys(Z)-NCA was then dried at room temperature. 1H NMR (400 MHz, DMSO-d6, ppm) (Avance II, Bruker Co., Billerica, MA, USA): d ¼ 9.08 (aeNH), 7.41e7.24 (ArH in Z groups), 5.03 (eCH2e in Z groups), 4.44 (aeCH), 2.99 (εeCH2), 1.81e1.58 (beCH2), and 1.43e1.22 (geCH2 and deCH2) (Supplementary Fig. 1). FT-IR (cm1) (Spectrum two, Perkin Elmer Inc., Akron, OH, USA): 1720e1840 (C]O in carboxyl anhydride of NCA) (Supplementary Fig. 4A). 2.2.2. Synthesis of poly (Nε-carbobenzyloxy-L-lysine) (PLL(Z)) Lys(Z)-NCA (5.60 g, 18.28 mmol) was dissolved in DMF (25 mL) under nitrogen gas. Hexylamine (80.89 mL, 0.61 mmol) dissolved in DMF (1 mL) was injected into the Lys(Z)-NCA solution and the reaction was performed at 35  C for 48 h under nitrogen purging. Then, the excess of cold diethyl ether was added to quench the reaction and the product was purified by repeated filtration. The purified PLL(Z) was dried at room temperature. The average molecular weight and the degree of polymerization were determined by gel permeation chromatography (GPC; Acme 9200, Young Lin Instrument Co., Ltd., Anyang, South Korea). The dissolution solvent and mobile phase were composed of DMF containing 0.01 M LiBr. GPC was performed on a GPC KD805 column (Showa Denko K.K. Co., Tokyo, Japan) at a 1 mL/min flow rate with a refractive index detector (Supplementary Fig. 3). The average molecular weight (4525 Da) and polydispersity index (1.06) were calculated from the GPC retention time (9.07 min, y ¼ 0.39965x þ 7.26276, R2 ¼ 0.99). 1H NMR (400 MHz, DMSO-d6, ppm): d ¼ 7.41e7.18 (ArH in the Z group), 4.99 (eCH2e in the Z group), and 0.83 (eCH3 in hexylamine) (Supplementary Fig. 1). FT-IR (cm1): 1690 (C]O in the Z group), 1640 and 1500 (eNHCOe, amide bond of the PLL(Z) repeating unit) (Supplementary Fig. 4A). 2.2.3. Synthesis of poly (L-lysine) (PLL) In order to deprotect the Z groups of PLL(Z), PLL(Z) (4.33 g, 0.43 mmol) dissolved in trifluoroacetic acid (30 mL) was added to HBr (5.28 g, 65.37 mmol, 4 eq. with respect to the Z groups). The reaction was carried out at room temperature for 2 h. The reaction was quenched using the excess cold diethyl ether, and the product was purified by repeated filtration. PLL was dialyzed against distilled water (DIW) using a dialysis tube (1K MWCO, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA), followed by lyophilization. 1H NMR (400 MHz, D2O, ppm): d ¼ 4.42 (aeCH), 3.82 (εeCH2e), 1.10e1.8 (eCH2e in PLL, and eCH2e in hexylamine), and 0.79 (eCH3 in hexylamine) (Supplementary Fig. 1). FT-IR (cm1): 3331 (eNH2 in the ε-amine of PLL) (Supplementary Fig. 4A). 2.2.4. Synthesis of paclitaxel-succinic anhydride (PTX-SA) PTX (50.00 mg, 0.06 mmol) and SA (23.44 mg, 0.23 mmol) were dissolved in pyridine (5 mL). Then, DMAP (0.72 mg, 0.01 mmol) dissolved in pyridine (1 mL) was injected into the PTX and SA mixture, which then was stirred at room temperature for 3 h. After 3 h, the reaction was quenched with DCM (20 mL), followed by DMAP and pyridine extraction with DIW. The separated DCM solution was concentrated by evaporation and precipitated with the excess cold n-hexane. The purified PTX-SA was dried at room temperature [34]. 1H NMR (400 MHz, CDCl3, ppm): d ¼ 5.98 (CH-30 ), 5.51 (CH-20 ), and 4.45 (CH-70 ) (Supplementary Fig. 2). FT-IR (cm1): 1730 (eCOO in the PTX-SA conjugated site), and 1700 (eC]O in Paclitaxel) (Supplementary Fig. 5A). 2.2.5. Synthesis of poly (L-lysine)ecarboxylate paclitaxel (PLL-PTX) PTX-SA (50.00 mg, 0.05 mmol) dissolved in DMSO (1 mL) was activated using DCC (20.07 mg, 0.11 mmol) and NHS (12.05 mg, 0.11 mmol). The activated PTX-SA was injected into PLL (13.93 mg, 0.11 mmol of eNH2 group) dissolved in DMSO/ DIW (4 mL; 70:30, v/v), and the reaction was carried out at room temperature for 24 h. Subsequently, the reaction mixture was combined with methanol/DIW (120 mL; 70:30, v/v), and by-products were removed using Amicon Ultra (MWCO 3000, Millipore Co., Billerica, MA, USA) (5500 g, 1 h). The washed product was lyophilized, and stored at 70  C. The degree of substitution of PLL-PTX was calculated from 5.98 ppm (CH-30 of PLL-PTX) using chemical shift from 0.79 ppm (eCH3 of hexylamine) as a reference. 1H NMR (400 MHz, DMSO-d6, ppm): d ¼ 0.79 (eCH3 in hexylamine), 5.98 (CH-30 ), and 5.51 (CH-20 ) (Fig. 3A). FT-IR (cm1): 1735

I. Noh et al. / Biomaterials 53 (2015) 763e774

765

Fig. 1. Schematic illustration of the preparation of multi-prodrug nanocarriers (MPDNCs) and the activated release behavior of PTX and GEM in target tumor cells. MPDNCs were formed though electrostatic interactions between PLL-PTX (a cationic charged species) and HA-GEM (an anionic charged species). MPDNCs were taken up by CD44-mediated endocytosis, followed by accelerated cleavage of the drug conjugates in acidified endosomes, thereby leading to synergistic apoptosis.

(C]O), 1100 (CeO), 1640 and 1500 (eNHCOe in the amide bond of repeating units), and 3331 (eNH2 in the ε-amine of the PLL fraction) (Supplementary Fig. 5A). 2.3. Synthesis of hyaluronic acid-gemcitabine (HA-GEM) HA (30 kDa, 100.00 mg) was dissolved in DIW (20 mL), and dialyzed against the dilute HCl solution using a dialysis tube (8K MWCO, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) for 6 h, followed by lyophilization. The desalted HA (90 mg, 0.22 mmol of carboxyl group) and GEM (26.35 mg, 0.88 mmol) were dissolved in DMSO (10 mL) in the dried 3-neck flask under nitrogen gas. Subsequently, DCC (11.34 mg, 0.55 mmol) and DMAP (8.06 mg, 0.66 mmol) dissolved in DMSO (1 mL) were added to the flask, respectively, and the reaction was performed at 40  C for 24 h. Then, the reaction was quenched using the excess cold methanol, and further repeated filtration was used to purify the product. Purified HA-GEM was dried under the vacuum, and stored at 70  C. The degree of substitution of HAGEM was calculated from 7.90 ppm (eCH]CHe of the cytosine group) using chemical shift from 2.00 ppm (eCH3 of the acetamido moiety of N-acetyl-Dglucosamine) as a reference. 1H NMR (400 MHz, D2O, ppm): d ¼ 1.98 (eCH3-a), 7.90 ppm (CH-50 ), 6.75 (CH-1), 6.18 (CH-10 ) (Fig. 3B). FT-IR (cm1): 1720 (C]O in ester bond), and 1110 and 1120 (CeO in ester bond) (Supplementary Fig. 6A). 2.4. Preparation and characterization of multi-prodrug nanocarriers (MPDNCs) composed of HA-GEM and PLL-PTX Multi-prodrug nanocarriers (MPDNCs) were prepared by electrostatic interaction with polymeric prodrugs. Briefly, HA-GEM (3.20 mg) was dissolved in HEPES buffer (10 mM, pH 7.4), and mixed with various amounts of the PLL-PTX stock solution (0.50 mg/100 ml) resulting in various HA-GEM/PLL-PTX mass ratios. The mixture of HA-GEM and PLL-PTX was incubated at 4  C for 3 h. Size distributions and zeta potentials of MPDNCs at various mass ratios were measured using dynamic light scattering (DLS) and zeta potential analyzer (ELS-Z, Otsuka Electronics Co., Ltd., Osaka, Japan) (n ¼ 3, error bars represent the standard deviation) (Fig. 4AeB). The morphologies of the MPDNCs at different mass ratios were investigated using atomic force microscopy (AFM; Dimension 3100, Veeco Instruments Inc., NY, USA) (Fig. 4C).

2.5. Release profile of GEM and PTX The release profiles of PTX and GEM from MPDNCs were investigated using an ultra-performance liquid chromatograph (UPLC) (Acquity UPLC™, Waters Co., MA, USA). The MPDNCs-dispersed solution was placed in a dialysis tube (Tube-O-DIALYZER™ Medi 4K MWCO, Geno Technology Inc., St Louis, MO, USA), and then immersed in either acetate buffer (10 mM, pH 5.5) or DPBS buffer (10 mM, pH 7.4) at 37  C [35]. At predetermined time intervals, the solution was lyophilized, and then an equivalent amount of fresh buffer was added. The mixture of ACN/DIW (1 mL; 75:25, v/v) was added to the lyophilized solution, and the precipitate formed was filtered using a hydrophilic syringe filter (ADVANTEX Co., Markham, ON, Canada). The mobile phase was composed of DPBS/ACN (50:50, v/v) with a flow rate of 1 mL/ min. UPLC was performed on a C18 column (Symmetry C18 5 mm 3.9  150 mm column, Waters Co, MA, USA) with detection at 230 nm (n ¼ 3, error bars represent the standard deviation) (PTX: 0.93 min, y ¼ 23960747x, R2 ¼ 0.99, GEM: 5.94 min, y ¼ 23182716x, R2 ¼ 0.95). 2.6. In vitro CD44 target efficacy of MPDNCs In order to investigate CD44 expression levels in biliary cancer cell lines (HuCCT1 and SCK cells), flow cytometry (FACSCalibur™; Becton Dickinson and Co., Franklin Lakes, NJ, USA) was performed. The cells (1  106 cells) were washed in blocking buffer composed of 0.2% (v/v) fetal bovine serum and 0.02% (v/v) sodium azide, and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD44 (20 mL, Becton Dickinson and Co.) and FITC-conjugated mouse IgG (20 mL, Becton Dickinson and Co.) at 4  C for 30 min each. Then the cells were re-suspended in 400 mL of 4% (v/v) paraformaldehyde (PFA), and monitored by flow cytometry [36]. Thereafter, confocal laser scanning microscopy (CLSM; LSM710, Carl Zeiss AG, Oberkochen, Germany) was performed to verify CD44-mediated cellular uptake of MPDNCs. Additionally, rhodamine B (RhoB)-conjugated HA-GEM (RhoB-HA-GEM) were synthesized following the same preparation process previously described for HA-GEM [37]. Then, RhoB-labeled MPDNCs (RhoB-MPDNCs) were formulated from RhoB-HA-GEM and PLL-PTX through the same synthetic route as MPDNCs (RhoBHA-GEM/PLL-PTX mass ratios 32). HuCCT1 and SCK cells (2  105 cells) were seeded in coverglass-bottom dishes (SPL Life Sciences Co, Ltd., Pocheon, South Korea), and

766

I. Noh et al. / Biomaterials 53 (2015) 763e774

Fig. 2. Synthetic routes of polymeric prodrugs. (A) PLL, synthesized by ring opening polymerization, was conjugated with PTX-SA and (B) HA was conjugated with GEM by the carbodiimide reaction. i) Triphosgen, Tetrahydrofuran, 50  C, 3 h ii) Hexylamine, N,N-dimethyformamide, 35  C, 48 h iii) Hydrogen bromide in acetic acid, 33 wt%, Trifluoroacetic acid, room temperature, 3 h iv) Succinic anhydride, 4-dimethylaminopyridine, pyridine, room temperature, 3 h v) N,N0 -Dicyclohexylcarbodiimide, N-Hydroxysuccinimide, dimethyl sulfoxide/distillated water (70:30, v/v), room temperature, 24 h vi) Dialysis against a dilute HCl solution. vii) Gemcitabine hydrochloride, N,N0 -Dicyclohexylcarbodiimide, 4dimethylaminopyridine, dimethyl sulfoxide, 40  C, 24 h.

then incubated for 24 h at 37  C in 5% CO2. After 24 h, the corresponding medium was replaced with fresh medium supplemented with 4% (v/v) each of fetal bovine serum (FBS) and RhoB-MPDNCs. For CD44 blocking experiments, the cells were preincubated with excess HA (10 mg/mL) solution at 37  C for 2 h in 5% CO2, and washed with DPBS before treatment with RhoB-MPDNCs [38]. After 1.5 h, the RhoB-MPDNCs treated cells were fixed using 4% PFA at 37  C for 30 min. Nuclei were counterstained with the DNA-binding dye Hoechst 33258 at 37  C for 1 h. Subsequently, the each medium was replaced with fresh DPBS, and RhoB-MPDNCs internalization was monitored by CLSM. (Fig. 6A, B, and D). Finally, the relative fluorescence intensity was calculated using the following equation: Relative fluorescence intensity ¼

RhoB intensityðA:U:Þ Dash line inðAÞðmmÞ 

1 Intensity of HuCCT1ðRhoB  MPDNCsÞðA:U:Þ

2.7. In vitro therapeutic efficacy The in vitro therapeutic efficacy assay (MTT assay) of MPDNCs was carried out in human biliary cancer cell lines (SCK and HuCCT1 cells) [39]. The cells were cultured in DMEM and RPMI1640, respectively. Each medium was supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics. Briefly, SCK and HuCCT1 cells were seeded (1  104 cells) in 96-well plates and incubated at 37  C for 24 h in 5% CO2. Since the cellular uptake of MPDNCs via receptor-mediated endocytosis occurred in a short time period, MPDNCs were treated for 4 h, and the medium was replaced with fresh medium supplemented with 4% (v/v) FBS. Equivalent concentrations of GEM and PTX were maintained in all cases (GEM, PTX, GEM þ PTX, HA-GEM, PLL-PTX, and MPDNCs) [40]. After 4 h, the medium was replaced with fresh medium supplemented with 4% (v/v) FBS and cells were further incubated at 37  C for 24 h in 5% CO2. The cells were washed with DPBS and treated with the MTT solution at 37  C for 3 h in 5% CO2. After 3 h, the resultant formazan crystals were solubilized in 0.01 M HCl with 10% sodium dodecyl sulfate. The absorbance of the resulting solution was measured at 575 nm and at 650 nm as the reference using a spectrophotometer (Epoch™, BioTek Instruments Inc.,

Winooski, VT, USA). Finally, the cell viability was determined from the absorbance ratio of treated to non-treated cells (n ¼ 6, error bars represent the standard deviation). 2.8. In vitro mRNA expression level In order to determine Bcl-2, Bcl-xl and Bax mRNA expression levels in HuCCT1, real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed with internal standards as described previously [41]. Briefly, the cultured HuCCT1 and SCK cells (1  106 cells) were treated with GEM, PTX, GEM þ PTX, HA-GEM, PLL-PTX, HA-GEM and MPDNCs, while equivalent GEM and PTX concentrations were maintained. After 4 h, the medium was replaced with fresh medium supplemented with 4% (v/v) FBS and cells were further incubated at 37  C in 5% CO2. After 24 h, the treated cells were collected. The total RNA from treated HuCCT1 and SCK cell lines was isolated using the RNeasy plus Mini Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer's instructions. The total RNA concentration was determined using the biophotometer (BioPhotometer plus, Eppendorf AG., Seoul, South Korea). Complementary DNA (cDNA) was synthesized from total RNA (2 mg) using the High Capacity RNA-to-cDNA kit (Applied Biosystems Inc., Foster City, CA, USA). qRT-PCR analysis and quantification were performed using the QuantiMix SYBR Kit (PhileKorea Technology Co., Ltd., Daejeon, Korea) on a Realtime PCR System (LightCycler 480 System, HNS Bio Co., Ltd., Seoul, South Korea). The reactions were carried out in a reaction mixture (20 mL) containing cDNA (1 mL), the SYBR Green mixture (10 mL), each forward (1 mL) and reverse primer (1 mL), and DEPC water (7 mL). The primer sequences (Bioneer Inc., Daejeon, South Korea) were as follows: Bcl-2 (forward: 30 -CTG GGG GAG GAT TGT GGC CTT CTT TG-50 ), (reverse: 50 TCC AGG TGT GCA GGT GCC GGT TC-30 ); Bcl-xL (forward: 30 - CAC CCT TTC GCA TTT GTT CCT-50 ), (reverse: 50 - GGT CGC ATT GTG GCC TTT-30 ); Bax (forward: 30 -ACT CCC CCC GAG AGG TCT T-50 ), (reverse: 50 -CAA AAG TAG AAA AGG GCC GAC AA-30 ); GAPDH (forward: 30 -GCT CTC TGC TCC TCC TGT TC-50 ), (reverse: 50 -TGA CTC CGA CCT TCA CCT TC-30 ). The PCR was initiated at 95  C for 5 min, followed by 45 cycles of amplification (95  C for 10 s, 60  C for 10 s, and 72  C for 10 s). The melting curve for each PCR reaction was generated to confirm the purity of the amplification products. PCR was performed in triplicate for each sample. Data were analyzed according to

I. Noh et al. / Biomaterials 53 (2015) 763e774

767

Fig. 3. 1H NMR spectra of (A) PLL-PTX with DMSO-d6, and (B) HA-GEM with D2O. The degree of substitution of PLL-PTX was calculated from 5.98 ppm (CH-30 of PLL-PTX) using chemical shift from 0.79 ppm (eCH3 of Hexylamine) as a reference. The degree of substitution of HA-GEM was calculated from 7.90 ppm (eCH]CHe of the cytosine group) using chemical shift from 2.00 ppm (eCH3 of the acetamido moiety of N-acetyl-D-glucosamine) as a reference.

the comparative Ct method in which the fold change for each mRNA was calculated by 2DDCt, and GAPDH was used as an internal standard for each sample (n ¼ 3, error bars represent the standard deviation). 2.9. In vivo therapeutic efficacy All animal experiments were carried out with the approval of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The xenograft model was prepared through the implantation of HuCCT1 cells (5  106 cells with 200 mL of RPMI1640) by subcutaneous injection into male BALB/c nude mice, which were 4 weeks of age. Beginning 12 days postimplantation, 4 groups of tumor-bearing mice (n ¼ 3) were injected through the tail vein twice per week (a total of six times) with 100 mL of saline, HA-GEM, GEM þ PTX, or MPDNCs. The animals were treated with equivalent doses of the drugs (GEM: 108.80 mg, PTX: 54.00 mg). The tumor sizes and body weights of the mice were measured at 3-day intervals. The length of the minor axis (2a) and the major axis (2b) of each tumor was measured using a caliper. The volume of each tumor was then calculated using the formula for a prolate spheroid [(4/3) p  a2b].The relative tumor growth was calculated from the tumor volume at the end of the experimental period. Tumors from mice treated with MPDNCs were frozen-sectioned and stained using hematoxylin and eosin (H&E) according to the manufacturers' instructions. Stained tissue sections were analyzed using a virtual microscope (BX51, Olympus Optical Co., Ltd., Tokyo, Japan).

3. Results and discussion 3.1. Synthesis and characterization of MPDNCs 3.1.1. Synthesis of paclitaxel-conjugated poly L-lysine (PLL-PTX) PLL-PTX, a cationic moiety, was synthesized as illustrated in Fig. 2A. First, Lys (Z)-NCA was prepared using Lys(Z) with triphosgen. Then, PLL (Z) was polymerized by ring opening polymerization

from Lys(Z)-NCA using hexylamine as a nucleophilic initiator. Successful elimination of the Z group was verified by comparison of the 1H NMR and FT-IR spectra from PLL (Z) and PLL. In the 1H NMR spectrum of PLL, the chemical shift of the benzyl group (eCH2e: 4.99 ppm) and the aromatic group (ArH: 7.41e7.18 ppm) disappeared after the specific deprotection process (Supplementary Fig. 1). Additionally, amide bond species in the repeating unit (1640e1500 cm1), the ε-amine of the PLL fraction (3331 cm1) and a difference in the fingerprint region (1200e400 cm1) (Supplementary Fig. 4AeB) indicated complete elimination of the Z group from PLL. The degree of polymerization (20) of PLL was quantified from the peak integration ratio of hexylamine (eCH3, 0.79 ppm) to the a-carbon proton of PLL (eCHe, 4.42 ppm) (Supplementary Fig. 1). In order to conjugate PTX to the side chains of the amine groups in PLL, PTX was first carboxylated using SA in the presence of catalytic amounts of DMAP at room temperature for 3 h [42]. Thereafter, PLL and PTX-SA were conjugated under carbodiimide reaction conditions, formulating an amide linker between the amine group of PLL and the carboxyl group of PTX-SA. PLL-PTX was partially positive and slightly water soluble due to the free ε-amine of PLL. The ester linkers between PTX and PLL enabled higher cleavability from the prodrug form of PTX to PTX under acidic conditions. In 1H NMR and FT-IR spectra of PLL-PTX, proton signals of hexylamine (eCH3: 0.79 ppm), paclitaxel signal coexistence (CH-30 : 5.98 ppm, and CH-20 : 5.51 ppm) (Fig. 3A) and the transmittance difference in the fingerprint region demonstrated the change in its chemical structure relative to its precursor (Supplementary Fig. 5AeB).

768

I. Noh et al. / Biomaterials 53 (2015) 763e774

Fig. 4. MPDNCs were characterized using dynamic light scattering, zeta potential analyzer, and atomic force microscopy. (A) Hydrodynamic diameters and (B) zeta potentials of MPDNCs with different HA-GEM/PLL-PTX mass ratios (H/P) in HEPES buffer (10 mM, pH 7.4) (*p < 0.001, **p < 0.0001) (n ¼ 3, error bars represent the standard deviation). (C) AFM images of MPDNCs with different H/P. The distances of various H/P are indicated by dashed lines.

Consequently, PTX molecules were successfully conjugated to the ε-amine groups of PLL, corresponding to 54 wt% of PLL-PTX determined from 5.98 ppm (CH-30 of PLL-PTX) using chemical shift from 0.79 ppm (eCH3 of hexylamine) as a reference. 3.1.2. Synthesis of gemcitabine-conjugated hyaluronic acid (HAGEM) The conjugation of HA-GEM was accomplished by a two-step reaction, as summarized in Fig. 2B. First, HA was desalted by dialysis against dilute HCl, and then the carboxyl groups of HA were conjugated with the C-50 hydroxyl group of GEM via esterification in mild experimental conditions [37,43]. HA-GEM exhibit strongly negative charges due to the hydroxyl and carboxyl groups in HA. Ester linkers between GEM and HA allow higher cleavage efficacy in low pH conditions from the GEM prodrug to GEM and HA. FT-IR spectra illustrated the peak of a carboxyl bond (1720, 1110 and 1120 cm1), which was attributed to the formation of an s-bond between GEM and HA (Supplementary Fig. 6). In 1H NMR spectra of HA-GEM, the peaks of GEM or HA appeared at 2.00 ppm (eCH3-a), 7.90 ppm (CH-50 ), 6.75 ppm (CHe1) and 6.18 ppm (CH-10 ).The degree of modification for HA-GEM was estimated from the peak integration ratio of 7.90 ppm (eCH]CHe of the cytosine group) and 2.00 ppm (eCH3 of the acetamido moiety of N-acetyl-D-

glucosamine) (Fig. 3A). Consequently, GEM was successfully conjugated with the carboxylic acid of HA, corresponding to 3.4 wt% of HA-GEM. 3.1.3. Formulation and characterization of multi-prodrug nanocarriers (MPDNCs) To investigate the suitable colloidal size for cellular internalization of MPDNCs, MPDNCs of various sizes were prepared by electrostatic interaction in HEPES buffer (10 mM, pH 7.4) with varying HA-GEM/PLL-PTX mass ratios (H/P). As shown in Fig. 4A, MPDNCs exhibited diverse hydrodynamic diameters according to H/P: H/P-1 (38118.99 ± 7968.23 nm), H/P-2 (1482.68 ± 736.82 nm), H/P-4 (1052.23 ± 187.77 nm), H/P-8 (498.89 ± 31.10 nm), H/P-16 (336.01 ± 31.10 nm), H/P-32 (217.14 ± 21.02 nm), and H/P-64 (878.10 ± 77.45 nm). With the increase in the proportion of PTX from H/P-1 to H/P-64, the average hydrodynamic diameter gradually decreased until H/P-32, but increased sharply at H/P-64. These H/P ratios can be changed into charge ratio (carboxylic acid/primary amine ratio). GEM conjugated to the carboxylic acid of HA corresponded to 3.4 wt% of HA-GEM. One chain of hyaluronic acid (3 kDa) has approximately 73 groups of free carboxylic acid and approximately 69 those of free carboxylic acid were left after HA-GEM conjugation. Subsequently, PLL (4525 Da) has

I. Noh et al. / Biomaterials 53 (2015) 763e774

769

Table 1 Hydrodynamic diameter and zeta potential of MPDNCs with varied ratios of carboxylic acid to primary amine ratio (Charge ratios). Charge ratios

0.53

1.07

2.14

4.28

8.55

17.10

34.21

Hydrodynamic diameter (nm) Zeta potential (mv)

38118.99 ± 7968.23 0.36 ± 0.54

1482.68 ± 736.82 14.99 ± 3.79

1052.23 ± 187.77 17.72 ± 1.50

498.89 ± 31.10 21.29 ± 2.16

336.01 ± 31.10 19.67 ± 1.19

217.14 ± 21.02 19.59 ± 1.36

878.10 ± 77.45 20.59 ± 1.43

approximately 35 groups of primary amine conjugated with PTXSA and the unreacted amine units are approximately 6. Thus, given that the mass ratios were converted to the molar ones indicating the charge ratios (carboxylic acid/primary amine ratio), it was shown the Table 1.These results were consistent with AFM analysis indicating that MPDNCs retained the form of an intact spherical particle at H/P-32 (ca. 200 nm), but not at other ratios (H/P-1, H/P-16 and H/P-64) (Fig. 4C). Additionally, MPDNCs exhibited nearly neutral charge (0.36 ± 0.54 mV) at H/P-1, whereas the zeta potential of MPDNCs indicated that they were negatively charged from H/P-2 to H/P-64 (H/P-2: 14.99 ± 3.79 mV, H/P-4: 17.72 ± 1.50 mV, H/P-8: 21.29 ± 2.16 mV, H/P-16: 19.67 ± 1.19 mV, H/P-32: 19.59 ± 1.36 mV, and H/P64: 20.59 ± 1.43 mV) (Fig. 4B). Based on these correlations of

hydrodynamic diameter and zeta potential, we found that an adequate charge ratio between the ε-amine group of PLL-PTX and the carboxyl group of HA-GEM played a significant role in the formulation of compact MPDNCs, because the charge imbalance promoted the aggregation of the nanocarrier and paclitaxel residues in PLL-PTX drive the condensed nanoparticle to a more thermodynamically favorable conformation [44e47]. Furthermore, as a consequence of the HA-GEM mass fraction increment in MPDNCs, the zeta potential of MPDNCs exhibited negative charge due to the exposure of HA-GEM on the surface of the MPDNCs, thereby augmenting the ability of HA to bind to the CD44 receptor on cancer cells. Thus, MPDNCs synthesized at H/P-32 were the most suitable for cellular internalization because of their appropriate particle size and targeting ability.

Fig. 5. Release profiles of PTX (A) and GEM (B) from MPDNCs in DPBS (pH 7.4) and acetate buffer (10 mM, pH 5.5) at 37  C. Images in the boxed section represent the first order exponential function in the initial burst period. Release rate constants of PTX and GEM were calculated from the first-order regression slope at the initial burst and after the initial burst, respectively (n ¼ 3, error bars represent the standard deviation).

770

I. Noh et al. / Biomaterials 53 (2015) 763e774

3.2. In vitro paclitaxel and gemcitabine release of MPDNCs In vitro drug release profiles from MPDNCs (H/P-32) were obtained in different proton environments: 1) normal blood (DPBS, 10 mM, pH 7.4) and 2) cancer endosomes (acetate buffer, 10 mM, pH 5.5). As shown in Fig. 5AeB, the cumulative proportions of GEM released within 12 h at pH 5.5 and pH 7.4 were 56.01 and 43.95%, respectively, indicating that the more acidic condition promoted faster release due to accelerated hydrolysis of the polymer matrix. On the other hand, the cumulative release of PTX from MPDNCs was slower than GEM release at both pH conditions within 12 h (pH 5.5: 43.46%, and pH 7.4: 33.98%). This implies that the hydrophobic properties of PTX and the steric bulk of the C-20 position in PTX induced degradation kinetics to a lesser extent. In contrast, HA was increasingly degraded under considerably acidified conditions, resulting in the exposure of its acidic cleavable ester bonds [48,49]. The release rate constant (k) calculated from the first-order regression slope indicated that, at pH 5.5, PTX and GEM were released 1.29- and 1.20-fold faster, respectively, than at pH 7.4 during the initial burst period (0e3 h). Subsequently, after the initial burst period (3e12 h), the release of PTX and GEM occurred 1.13- and 2.48-fold faster at pH 5.5 than at pH 7.4. The amount of GEM released from the degradation of HA was proportional to the

length of the acid treatment, which had caused the deformation of the particle by breaking the charge balance. Consequently, the cleavage of PTX- and GEM-conjugated linkers from MPDNCs was accelerated at low pH conditions in tumor endosomes and the linkers exhibited pharmacokinetic behaviors similar to PTX and GEM. 3.3. In vitro CD44 targeting efficacy of MPDNCs To verify the affinity of the MPDNCs for CD44 receptor on cancer cells, the CD44 expression levels of HuCCT1 and SCK human biliary cancer cell lines were investigated using flow cytometry. Subsequently, intracellular uptake of MPDNCs via the CD44 route into biliary cancer cell lines was quantified by CLSM with the Hoechst 33258 nuclear counterstaining dye. Flow cytometry analysis confirmed that HuCCT1 cells highly overexpressed CD44, compared to SCK cells (as a negative control) which expressed CD44 at a low level (Fig. 6C). When HuCCT1 and SCK cells were treated with RhoB-MPDNCs for 1.5 h, a noticeable RhoB signal was observed in HuCCT1 cells, whereas a small and insignificant signal was detected in SCK cell lines. Additionally, to confirm the cellular uptake of RhoB-MPDNCs via CD44-mediated endocytosis, HuCCT1 and SCK cells were pre-incubated with excess HA to block the CD44 receptor

Fig. 6. (A) CD44-specific uptake in HuCCT1 and SCK cells, and (B) Z-line fluorescence intensity of Rhodamine B were determined by confocal laser scanning microscopy (Scale bar: 10 mm). The distances are indicated by the dashed lines in (A). HuCCT1 and SCK cells were incubated with Rhodamine B (Red)-labeled MPDNCs (RhoB-MPDNCs) for 1.5 h, followed by fixation using 4% PFA. Nuclei were counterstained with the DNA-binding dye Hoechst 33258 (Dk. blue) for 1 h. Pre-incubated free HA (10 mg/mL) was used as a negative control and untreated cells were used as a positive control (Scale bar: 10 mm) (C) CD44 expression levels of HuCCT1 and SCK cells were determined by flow cytometry. (D) Relative fluorescence intensity based on (B); I: HuCCT1 (RhoB-MPDNCs), II: HuCCT1 (blocked), III: SCK (RhoB-MPDNCs), and IV: SCK (blocked). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

I. Noh et al. / Biomaterials 53 (2015) 763e774

prior to RhoB-MPDNC treatment. The RhoB signal was hardly detected in blocked HuCCT1 and SCK cells compared to those without excess HA treatment (Fig. 6A). The difference in uptake capability was obvious from the difference in relative fluorescence intensity according to the CD44 expression level, in agreement with a previous CLSM study (Fig. 6B and D). This remarkable uptake capability suggests that MPDNCs enhanced specific drug uptake into cancer cells via HA receptor-mediated endocytosis (the dominant path for the intercellular uptake of MPDNCs). 3.4. In vitro therapeutic efficacy of MPDNCs In order to demonstrate the therapeutic efficacy of MPDNCs compared to free drugs and non-particulated polymers, in vitro cytotoxicity experiments were performed using the MTT assay (Supplementary Fig. 7). Fig. 7AeB shows the in vitro cytotoxicities and the half-maximal inhibitory concentrations (IC50) of GEM, PTX, GEM þ PTX, HA-GEM, PLL-PTX and MPDNCs (H/P-32) in HuCCT1 and SCK cells according to GEM and PTX concentration. Fig. 7A illustrates that co-delivery of free drugs without nanocarriers had a lesser and insignificant therapeutic effect compared to delivery of a single drug with short-term uptake in both HuCCT1 (GEM: 73.75 ± 7.07%, PTX: 88.43 ± 2.17%, and GEM þ PTX: 82.13 ± 2.80%)

771

and SCK cells (GEM: 60.48 ± 4.25%, PTX: 80.91 ± 2.00%, and GEM þ PTX: 56.32 ± 2.60%). This was attributed to the low permeability of GEM across the cellular membrane, and therapeutic hindrance of PTX. On the other hand, the prodrugs and nanoparticles were more cytotoxic than the free drugs. In particular, MPDNCs and HA-GEM, which have CD44-targeting moieties, exhibited remarkably higher efficacy in CD44-overexpressing HuCCT1 cells (MPDNCs: 48.57 ± 1.73% and HA-GEM: 53.73 ± 2.38%) than in SCK cells with low CD44 expression (MPDNCs: 53.13 ± 3.46% and HA-GEM: 63.25 ± 1.18%), whereas this difference in efficacy was not exhibited by PLL-PTX (HuCCT1: 74.45 ± 1.05% and SCK: 72.34 ± 0.21%), which does not have a CD44targeting moiety. In addition, the cytotoxicities of HA-GEM and MPDNCs were 20% and 34% higher than those of GEM and GEM þ PTX at identical dosing conditions in HuCCT1 cells, whereas an insignificant difference in cytotoxicity was observed in SCK cells. As shown in Fig. 7B, the order of the IC50 values was: GEM þ PTX (1.26  104 mg/mL) > HA-GEM (6.31  106 mg/mL) > MPDNCs (1.58  106 mg/mL) in HuCCT1 cells, and GEM þ PTX (2.00  104 mg/mL) > MPDNCs (1.00  105 mg/mL) in SCK cells. As evidenced by the IC50 values in HuCCT1 and SCK cell lines, MPDNCs had much higher therapeutic efficacy in HuCCT1 cells than in SCK cells at low concentrations. These results demonstrate that

Fig. 7. In vitro cell viability (A) and IC50 values (B) for HuCCT1 and SCK cells were confirmed by the MTT assay. Bax/Bcl-xL ratios (C) and Bax/Bcl-2 ratios (D) for HuCCT1 and SCK cells were analyzed by qRT-PCR. Cell viability, Bax/Bcl-xL ratios, and Bax/Bcl-2 ratios were determined in HuCCT1 and SCK cell lines incubated with GEM, PTX, GEM þ PTX, HA-GEM, PLLPTX, or MPDNCs for 4 h, followed by further incubation for 24 h in fresh media containing 3% FBS (n ¼ 3, error bars represent the standard deviation). (IC50 of HA-GEM was not obtained because the viabilities of cells treated with HA-GEM were not below 50% for any of the tested concentrations). GAPDH was used as a house-keeping gene to normalize each expression level.

772

I. Noh et al. / Biomaterials 53 (2015) 763e774

MPDNCs enhanced cellular drug uptake via HA-mediated endocytosis considerably, indicating their potential for better therapeutic efficacy than existing treatments against target cells, as previously described. PTX and GEM exhibit different mechanisms of apoptotic action. Nonetheless, these two representative drugs for biliary cancer both promote the down-regulation of the antiapoptotic Bcl-2 family and the up-regulation of the proapoptotic Bax signaling pathway, leading to programmed cell death [50]. Therefore, in order to study this synergistic induction of programmed cell death in human biliary cancer cell lines, the mRNA expression levels of Bax, Bcl-2 and Bcl-xL in HuCCT1 and SCK cells were investigated using realtime qRT-PCR with an internal standard (GAPDH) under the same conditions. As shown in Supplementary Fig. 8AeB, Bcl-2 and Bcl-xL mRNA levels were down-regulated and the Bax mRNA level was up-regulated more by MPDNCs than by any other treatment. The change in Bax mRNA expression levels in response to MPDNCs (1.98-fold) in HuCCT1 cells was greater than that in SCK cells (1.52fold). Moreover, the changes in Bcl-xL and Bcl-2 mRNA levels in MPDNC-treated HuCCT1 cells (0.41- and 0.43-fold) were less than those in SCK cells (0.60- and 0.59-fold). To evaluate the apoptosis factor, the ratios of Bax/Bcl-xL and Bax/Bcl-2 (ratio > 1: inducing apoptosis, ratio < 1: anti-apoptotic) were determined based on the results in Supplementary Fig. 8AeB [51]. As shown in Fig. 7CeD, the

ratios of Bax/Bcl-xL and Bax/Bcl-2 in both HuCCT1 and SCK cells treated with MPDNCs were higher than in cells from any other treatment. Furthermore, MPDNC-treated HuCCT1 cells (Bax/Bcl-xL: 4.79, and Bax/Bcl-2: 4.63) had significantly greater apoptosis factors than SCK cells (Bax/Bcl-xL: 2.54, and Bax/Bcl-2: 2.59). These gene expression results coincided with the cell death propensity data based on CD44 expression levels shown in Fig. 7AeB, indicating that the therapeutic efficacy of MPDNCs was strongly connected to the apoptosis factor. Consequently, MPDNCs showed considerable synergistic efficacy in the treatment of CD44overexpressing human biliary cancer cells. 3.5. In vivo therapeutic efficacy of MPDNCs To confirm the in vivo applicability of MPDNCs for targeted tumor suppression, HuCCT1 cells were implanted into nude male mice to form xenograft models of human biliary cancer. Beginning on the 12th day after their implantation with HuCCT1 cells, tumorbearing xenograft mice were treated with saline, HA-GEM, GEM þ PTX or MPDNCs by tail vein injection. The tumor growth curves of the xenografts clearly showed the therapeutic efficacy in the animal model. In comparison with the anticancer agent treatment groups, the saline treatment group exhibited significant tumor growth. The relative tumor volume of the saline group

Fig. 8. Antiproliferative effects of saline, HA-GEM, GEM þ PTX, and MPDNCs injected into male BALB/c nude mice inoculated with HuCCT1 cells. (A) Relative tumor volume changes and (B) Body weights of HuCCT1 xenograft-bearing mice were monitored at predetermined times for 30 days (n ¼ 3, error bars represent the standard error). Tumor tissue sections were excised from different treatment groups of mice after the 30th day. (C) Excised tumor weights from different treatment groups and (D) excised HuCCT1 tumor images. (E) Histology analysis by H&E staining was performed for the different treatment groups using a virtual microscope. (Scale bar: 100 mm).

I. Noh et al. / Biomaterials 53 (2015) 763e774

expanded to 131.54 mm3, while those of the HA-GEM and GEM þ PTX groups reached 57.40 and 39.40 mm3, respectively. The relative tumor volume of the MPDNCs group was even reduced to approximately 9.80 mm3 within 30 days (Fig. 8A). It is noteworthy that HA-GEM had a therapeutic effect even though it had a lower amount of anticancer agent than PTX þ GEM, implying that the targeting moiety in HA-GEM is able to effectively deliver GEM. Furthermore, MPDNCs caused remarkable tumor suppression compared to PTX þ GEM at the same dosage. These results illustrate that, due to CD44-targeted delivery, tailored MPDNCs were able to accumulate locally at the tumor without drug loss, and thus could synergistically induce apoptosis, while PTX þ GEM could not. The body weights of the mice did not change in any of the sampletreated groups compared to their initial weights, meaning that all the treatments were suitable for systemic chemotherapy (Fig. 8B). After the 30th day, tumors were excised to confirm the therapeutic efficacy of the treatments. Fig. 8CeD shows that the weights of MPDNCs-treated tumors were significantly reduced (230.56 mm3) compared to saline-treated tumors. In addition, H&E staining of excised tumors from each treatment group revealed that tumors from the MPDNC treatment group were more damaged than those from the other treatment groups (Fig. 8E) [52]. Consequently, these results have successfully demonstrated the therapeutic efficacy of these polymeric prodrug nanocarriers at the tumor site. MPDNCs thus have significant potential as promising nano-platforms for targeted delivery of dual chemotherapy agents and efficient tumor treatment. 4. Conclusions In summary, we successfully fabricated CD44-targeting nanocarriers composed of paclitaxel and gemcitabine prodrugs (MPDNCs) to overcome well-recognized problems with drugloaded nanosystems, such as limited circulation times and difficulty delivering drugs with different solubilities. The synthesis of PLL-PTX enhanced the water solubility and cationic properties of PTX to form nanocarriers, which assembled with the CD44targeting HA-GEM (a conjugated hydrophilic drug of GEM). MPDNCs showed remarkable characteristics including bioavailability, reduced side effects, and conveyance of drugs with different solubilities with similar pharmacokinetics. These polymeric prodrug nanocarriers enhanced the intracellular delivery of PTX and GEM into CD44-expressing human biliary cancer cells, thereby suppressing tumor growth considerably through synergistic induction of apoptosis via the different cellular activities of the dual anticancer agent. Consequently, MPDNCs allow outstanding synergism between GEM and PTX and establish a promising platform for highly effective biliary anticancer activity based on polymeric prodrug therapy. Acknowledgments This work was supported by the Industrial Strategic Technology Development Program (10044021, Development of nonvascular drug eluting stent for treatment of gastrointestinal disease) funded by the Ministry of Knowledge Economy (MKE, Korea). This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2010-0019923). Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2015.03.006.

773

References [1] Frei 3rd E. Combination cancer therapy: presidential address. Cancer Res 1972;32:2593e607. [2] Albain KS, Nag SM, Calderillo-Ruiz G, Jordaan JP, Llombart AC, Pluzanska A, et al. Gemcitabine plus paclitaxel versus paclitaxel monotherapy in patients with metastatic breast cancer and prior anthracycline treatment. J Clin Oncol 2008;26:3950e7. [3] Ma Y, Liu D, Wang D, Wang Y, Fu Q, Fallon JK, et al. Combinational delivery of hydrophobic and hydrophilic anticancer drugs in single nanoemulsions to treat MDR in cancer. Mol Pharm 2014;11:2623e30. [4] Sun TM, Du JZ, Yao YD, Mao CQ, Dou S, Huang SY, et al. Simultaneous delivery of siRNA and paclitaxel via a “two-in-one” micelleplex promotes synergistic tumor suppression. ACS Nano 2011;5:1483e94. [5] Yan Y, Such GK, Johnston APR, Best JP, Caruso F. Engineering particles for therapeutic delivery: prospects and challenges. ACS Nano 2012;6:3663e9. [6] Liu S, Guo Y, Huang R, Li J, Huang S, Kuang Y, et al. Gene and doxorubicin codelivery system for targeting therapy of glioma. Biomaterials 2012;33:4907e16. [7] Duan X, Xiao J, Yin Q, Zhang Z, Yu H, Mao S, et al. Smart pH-sensitive and temporal-controlled polymeric micelles for effective combination therapy of doxorubicin and disulfiram. ACS Nano 2013;7:5858e69. [8] Lee J, Lee SC, Acharya G, Chang CJ, Park K. Hydrotropic solubilization of paclitaxel: analysis of chemical structures for hydrotropic property. Pharm Res 2003;20:1022e30. [9] Vrignaud S, Benoit JP, Saulnier P. Strategies for the nanoencapsulation of hydrophilic molecules in polymer-based nanoparticles. Biomaterials 2011;32: 8593e604. [10] Persidis A. Cancer multidrug resistance. Nat Biotechnol 1999;17:94e5. [11] Smith MH, Lyon LA. Multifunctional nanogels for siRNA delivery. Acc Chem Res 2012;45:985e93. [12] Mi Y, Zhao J, Feng S-S. Vitamin E TPGS prodrug micelles for hydrophilic drug delivery with neuroprotective effects. Int J Pharm 2012;438:98e106. [13] Mi Y, Zhao J, Feng SS. Targeted co-delivery of docetaxel, cisplatin and herceptin by vitamin E TPGS-cisplatin prodrug nanoparticles for multimodality treatment of cancer. J Control Release 2013;169:185e92. [14] Zhao J, Mi Y, Feng SS. siRNA-based nanomedicine. Nanomedicine (Lond) 2013;8:859e62. [15] Hu CMJ, Zhang L. Nanoparticle-based combination therapy toward overcoming drug resistance in cancer. Biochem Pharmacol 2012;83:1104e11. [16] Shin HC, Alani AWG, Rao DA, Rockich NC, Kwon GS. Multi-drug loaded polymeric micelles for simultaneous delivery of poorly soluble anticancer drugs. J Control Release 2009;140:294e300. [17] Toh M-R, Chiu GN. Liposomes as sterile preparations and limitations of sterilisation techniques in liposomal manufacturing. Asian J Pharm Sci 2013;8:88e95. [18] Anal AK, Tobiassen A, Flanagan J, Singh H. Preparation and characterization of nanoparticles formed by chitosan-caseinate interactions. Colloids Surfaces B Biointerfaces 2008;64:104e10. [19] Khandare J, Minko T. Polymer-drug conjugates: progress in polymeric prodrugs. Prog Polym Sci (Oxford) 2006;31:359e97. [20] Namgung R, Mi Lee Y, Kim J, Jang Y, Lee BH, Kim IS, et al. Poly-cyclodextrin and poly-paclitaxel nano-assembly for anticancer therapy. Nat Commun 2014;5:3702. [21] Cho K, Wang X, Nie S, Chen Z, Shin DM. Therapeutic nanoparticles for drug delivery in cancer. Clin Cancer Res 2008;14:1310e6. [22] Ma L, Kohli M, Smith A. Nanoparticles for combination drug therapy. ACS Nano 2013;7:9518e25. [23] Pang Z, Feng L, Hua R, Chen J, Gao H, Pan S, et al. Lactoferrin-conjugated biodegradable polymersome holding doxorubicin and tetrandrine for chemotherapy of glioma rats. Mol Pharm 2010;7:1995e2005. [24] Wu H-C, Chang D-K, Huang C-T. Targeted therapy for cancer. J Cancer Mol 2006;2:57e66. [25] Khan SA, Thomas HC, Davidson BR, Taylor-Robinson SD. Cholangiocarcinoma. Lancet 2005;366:1303e14. [26] Lee HM, Jeong YI, Kim do H, Kwak TW, Chung CW, Kim CH, et al. Ursodeoxycholic acid-conjugated chitosan for photodynamic treatment of HuCC-T1 human cholangiocarcinoma cells. Int J Pharm 2013;454:74e81. [27] Kunlabut K, Vaeteewoottacharn K, Wongkham C, Khuntikeo N, Waraasawapati S, Pairojkul C, et al. Aberrant expression of CD44 in bile duct cancer correlates with poor prognosis. Asian Pac J Cancer Prev 2012;13:95e9. [28] Pongcharoen P, Jinawath A, Tohtong R. Silencing of CD44 by siRNA suppressed invasion, migration and adhesion to matrix, but not secretion of MMPs, of cholangiocarcinoma cells. Clin Exp Metastasis 2011;28:827e39. [29] Meyer K. The biological significance of hyaluronic acid and hyaluronidase. Physiol Rev 1947;27:335e59. [30] Plunkett W, Huang P, Gandhi V. Preclinical characteristics of gemcitabine. Anti-Cancer Drugs 1995;6:7e13. [31] Ringel I, Horwitiz SB. Studies with RP 56976 (taxotere): a semisynthetic analogue of taxol. J Natl Cancer Inst 1991;83:288e91. [32] Hensley ML, Maki R, Venkatraman E, Geller G, Lovegren M, Aghajanian C, et al. Gemcitabine and docetaxel in patients with unresectable leiomyosarcoma: results of a phase II trial. J Clin Oncol 2002;20:2824e31. [33] Lee D, Choe K, Jeong Y, Yoo J, Lee SM, Park J-H, et al. Establishment of a controlled insulin delivery system using a glucose-responsive double-layered nanogel. RSC Adv 2015;5:14482e91.

774

I. Noh et al. / Biomaterials 53 (2015) 763e774

[34] Aryal S, Hu CMJ, Zhang L. Combinatorial drug conjugation enables nanoparticle dual-drug delivery. Small 2010;6:1442e8. [35] Kim HO, Kim E, An Y, Choi J, Jang E, Choi EB, et al. A biodegradable polymersome containing Bcl-xL siRNA and doxorubicin as a dual delivery vehicle for a synergistic anticancer effect. Macromol Biosci 2013;13:745e54. [36] Lee E, Hong Y, Choi J, Haam S, Suh JS, Huh YM, et al. Highly selective CD44specific gold nanorods for photothermal ablation of tumorigenic subpopulations generated in MCF7 mammospheres. Nanotechnology 2012;23. [37] Liu J, Li J, Huang P, Chang L, Long X, Dong A, et al. Tumor targeting and pHresponsive polyelectrolyte complex nanoparticles based on hyaluronic acidpaclitaxel conjugates and chitosan for oral delivery of paclitaxel. Macromol Res 2013;21:1331e7. [38] Jang E, Lim EK, Choi Y, Kim E, Kim HO, Kim DJ, et al. p-Hyaluronan nanocarriers for CD44-targeted and pH-boosted aromatic drug delivery. J Mater Chem B 2013;1:5686e93. [39] Choi J, Park Y, Choi EB, Kim HO, Kim DJ, Hong Y, et al. Aptamer-conjugated gold nanorod for photothermal ablation of epidermal growth factor receptoroverexpressed epithelial cancer. J Biomed Opt 2014;19:051203. [40] Harding C, Heuser J, Stahl P. Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J Cell Biol 1983;97: 329e39. [41] Jeong IK, Gao GH, Li Y, Kang SW, Lee DS. A biodegradable polymersome with pH-tuning on-off membrane based on poly(beta-amino ester) for drug delivery. Macromol Biosci 2013;13:946e53. [42] Leonelli F, La Bella A, Migneco LM, Bettolo RM. Design, synthesis and applications of hyaluronic acid-paclitaxel bioconjugates. Molecules 2008;13:360e78. [43] Cavallaro G, Mariano L, Salmaso S, Caliceti P, Gaetano G. Folate-mediated targeting of polymeric conjugates of gemcitabine. Int J Pharm 2006;307:258e69.

[44] Layek B, Singh J. Amino acid grafted chitosan for high performance gene delivery: comparison of amino acid hydrophobicity on vector and polyplex characteristics. Biomacromolecules 2013;14:485e94. [45] Lim EK, Kim HO, Jang E, Park J, Lee K, Suh JS, et al. Hyaluronan-modified magnetic nanoclusters for detection of CD44-overexpressing breast cancer by MR imaging. Biomaterials 2011;32:7941e50. [46] Wong SY, Sood N, Putnam D. Combinatorial evaluation of cations, pHsensitive and hydrophobic moieties for polymeric vector design. Mol Ther 2009;17:480e90. [47] Rata-Aguilar A, Segovia-Ramos N, Jodar-Reyes AB, Ramos-Perez V, Borros S, Ortega-Vinuesa JL, et al. The role of hydrophobic alkyl chains in the physicochemical properties of poly(beta-amino ester)/DNA complexes. Colloids Surf B Biointerfaces 2015;126:374e80. [48] Mastropaolo D, Camerman A, Luo Y, Brayer GD, Camerman N. Crystal and molecular structure of paclitaxel (taxol). Proc Natl Acad Sci U S A 1995;92: 6920e4. €m B. Effect of pH on the behavior of hyaluronic [49] Maleki A, Kjøniksen AL, Nystro acid in dilute and semidilute aqueous solutions. Macromol Symp 2008;274: 131e40. [50] Sui M, Xiong X, Kraft AS, Fan W. Combination of gemcitabine antagonizes antitumor activity of paclitaxel through prevention of mitotic arrest and apoptosis. Cancer Biol Ther 2006;5:1015e21. [51] Vaskivuo TE, Stenb€ ack F, Tapanainen JS. Apoptosis and apoptosis-related factors Bcl-2, Bax, tumor necrosis factor-a, and NF-kB in human endometrial hyperplasia and carcinoma. Cancer 2002;95:1463e71. [52] Choi J, Yang J, Bang D, Park J, Suh JS, Huh YM, et al. Targetable gold nanorods for epithelial cancer therapy guided by near-IR absorption imaging. Small 2012;8:746e53.