The multilayer nanoparticles formed by layer by layer approach for cancer-targeting therapy

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The multilayer nanoparticles formed by layer by layer approach for cancer-targeting therapy Keun Sang Oh a, Hwanbum Lee a, Jae Yeon Kim a, Eun Jin Koo a, Eun Hee Lee a, Jae Hyung Park b, Sang Yoon Kim c, d, Kwangmeyung Kim d, Ick Chan Kwon d, Soon Hong Yuk a,⁎ a

College of Pharmacy, Korea University, 2511 Sejongro, Sejong 339-700, Republic of Korea Department of Polymer Science and Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Republic of Korea d Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Republic of Korea b c

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Article history: Received 23 August 2012 Accepted 17 October 2012 Available online 24 October 2012 Keywords: Multilayer nanoparticles Layer by layer approach Pluronic Vesicle Magnetic resonance imaging Cancer-targeting therapy

a b s t r a c t The multilayer nanoparticles (NPs) were prepared for cancer-targeting therapy using the layer by layer approach. When drug-loaded Pluronic NPs were mixed with vesicles (liposomes) in the aqueous medium, Pluronic NPs were incorporated into the vesicles to form the vesicle NPs. Then, the multilayer NPs were formed by freeze-drying the vesicle NPs in a Pluronic aqueous solution. The morphology and size distribution of the multilayer NPs were observed using a TEM and a particle size analyzer. In order to apply the multilayer NPs as a delivery system for docetaxel (DTX), which is a model anticancer drug, the release pattern of the DTX was observed and the tumor growth was monitored by injecting the multilayer NPs into the tail veins of tumor (squamous cell carcinoma)-bearing mice. The cytotoxicity of free DTX (commercial DTX formulation (Taxotere®)) and the multilayer NPs was evaluated using MTT assay. We also evaluated the tumor targeting ability of the multilayer NPs using magnetic resonance imaging. The multilayer NPs showed excellent tumor targetability and antitumor efficacy in tumor-bearing mice, caused by the enhanced permeation and retention (EPR) effect. These results suggest that the multilayer NPs could be a potential drug delivery system for cancer-targeting therapy. © 2012 Elsevier B.V. All rights reserved.

1. Introduction A simple yet highly versatile process, called the layer-by-layer (LbL) approach, has been explored to form new types of nanoengineered materials for drug delivery and has proven to be a useful technique for the assembly of nanoengineered polymeric nanoparticles (NPs) [1–4]. Usually, these types of NPs are generated by the sequential deposition of polymer layers from aqueous solutions onto a template and most type of interaction (for example, electrostatics, hydrogen bonding, covalent bonding, and specific recognition) can be used as the driving force for the assembly of the multilayer shell. In cancer therapy, the efficient delivery of anticancer drugs to tumor sites is strongly required and this has been demonstrated by accumulating NPs containing anticancer drugs at tumor site based on the enhanced permeation and retention (EPR) effect. Basic requirement for the accumulation of at tumor site is the prolonged half-life in the

⁎ Corresponding author. Tel.: +82-44-860-1612 Fax.: +82-44-860-1606. E-mail address: [email protected] (S.H. Yuk). 0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jconrel.2012.10.013

systemic circulation. Through LBL approach, appropriate size and surface modification of NPs can lead to achieve this requirement with enhanced targetability [1–4]. Liposomes (vesicles) have attracted attention for medical applications due to their ability to protect and carry hydrophilic and hydrophobic molecules [5,6]. Furthermore, liposomes have been used extensively for drug delivery systems due to the lack of cytotoxicity [7,8]. Liposomes are generally more biocompatible than polymers, however, they often lack stability in vivo due to the host enzymes. A number of attempts to integrate advantages and reduce disadvantages have been made through the combination of liposomes with polymers and this has demonstrated the enhanced stability and targeted delivery of the encapsulated bioactive compounds in the liposomes [9–11]. One example of the combination of polymers and liposomes was presented using core/shell NPs with a drug-loaded lipid core [12,13]. The lipid core was composed of lecithin, which formed a spherical supramolecular structure (multilamellar liposomes) in a concentrated state and the polymeric shell was composed of Pluronic. It was reported that the interactions between Pluronic and lipid bilayer membrane, which is the main component of liposomes, contributed to the formation of composite structures that preserve the integrity of the liposome [14,15].

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During the freeze-drying process, hydrophobic blocks (i.e., polypropylene blocks) enable Pluronics to physically anchor on the liposome surface and these interactions replace the water in the lipid bilayer membrane. This enabled to design of Pluronic/liposome composite systems with a core/ shell structure using a freeze-drying method. In this study, the multilayer NPs with therapeutics or diagnostic modalities were prepared using the LbL approach through intermolecular interactions between Pluronic and lipid bilayer membranes. For this purpose, Pluronic NPs containing therapeutic agents (docetaxel (DTX), anticancer drug) or molecular imaging probes (iron oxide NPs, magnetic resonance imaging probe) were prepared for a template. In our previous study [16], paclitaxel (PTX)-loaded Pluronic NPs were successfully prepared by a temperature-induced phase transition (120 °C for 90 min) in a mixture of Pluronic F-68 and liquid PEG (polyethylene glycol, molecular wt: 400) containing PTX. The liquid PEG is used as solubilizer of PTX and the polymer for the encapsulation of PTX is composed of Pluronic F-68. However, the oxidation of DTX was observed during the formation of DTX-loaded Pluronic NPs by a temperature-induced phase transition. To prevent DTX from the oxidation during the formation of DTX-loaded Pluronic NPs, the temperature-induced phase transition was performed at 90 °C for 10 min. The minimal oxidation of DTX was observed, however, the precipitation of DTX from the Pluronic NPs was observed in the aqueous medium within 10 min [17]. To improve the stability of DTX-loaded Pluronic NPs in the aqueous medium, the vesicle NPs were formed through vesicle fusion [17] and further improvement in the stability of NPs was made by adding Pluronic layer to form the multilayer NPs through LbL approach as shown in Fig. 1. This process resulted in the powdery form of the multilayer NPs, which may increase the stability of dosage form during the storage. We then evaluated the nanoparticle drug release, and in vivo tumor targeting using magnetic resonance (MR) imaging. Finally, the antitumor efficacy of the multilayer NPs was evaluated by measuring the changes of tumor volumes in tumorbearing mice. 2. Materials and methods 2.1. Materials Pluronic F-68, (Poly(ethylene oxide)–poly(propylene oxide)– poly(ethylene oxide) triblock copolymer) (Mw = 8350; (EO)79(PO)

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was obtained from BASF Corp., Korea, and was used as received. Polyethylene glycol (PEG, molecular wt 400) was purchased from CRODA (Yorkshire, UK). DTX (anhydrous form) was purchased from Parling PharmaTech Co., Ltd. (Shanghai, China). The iron(II) chloride tetrahydrate (FeCl2·4H2O), iron(III) chloride hexahydrate (FeCl3·6H2O), hydrogen tetrachloroaureate(III) hexahydrate (HAuCl4·6H2O), trisodium citrate dyhydrate, and L-α-phosphatidylcholine from egg yolk were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Resovist (40 mol Fe kg−1) was purchased from Schering AG (Berlin, Germany).

2.2. Preparation of the vesicles NPs containing DTX-loaded Pluronic NPs Firstly, the DTX-loaded Pluronic NPs (template) were prepared via a temperature-induced phase transition. 200 mg of PEG and the weighed amount of DTX were mixed to form a drug-loaded phase, which was subsequently mixed with 800 mg of Pluronic F-68. While increasing the temperature to 90 °C, the mixture melted into the liquid phase. After the equilibrium at 90 °C for 10 min, the liquid mixture was cooled to 0 °C for 10 min to induce a phase transition. For the formation of the vesicle NPs, the vesicle suspension was separately prepared in the aqueous medium by the sonication using a probe type ultrasonic wave homogenizer (Branson Sonifier Model 185) and it was composed of 25 wt.% aqueous solution of L-α-phosphatidylcholine with the spherical form. Then, weighed amounts of DTX-loaded Pluronic NPs were mixed with the vesicle suspension to form the vesicle NPs containing DTX-loaded Pluronic NPs [17].

2.3. Preparation of the vesicle NPs containing Pluronic-coated iron oxide NPs Gold-deposited iron oxide NPs were formed through the reduction of Au3+ on the surface of iron oxide nanoseeds. Firstly, the iron oxide nanoseeds were prepared via the alkaline coprecipitation of ferrous (Fe2+) and ferric (Fe 3+) chlorides following the procedure described elsewhere [18]. In order to deposit the iron oxide nanoseeds with the gold, 50 ml of aqueous solution containing the prepared iron oxide nanoseeds were refluxed in a three-necked round bottom flask at 80 °C for 30 min. Then, 3 ml of 1 mM tetrachloroauric acid (HAuCl4) and 3 ml of 1 wt.% trisodium citrate dyhydrate were added stepwise. Vigorous stirring was continued for another 60 min. The solution was cooled to room temperature and was filtered with a 0.2 μm filter. For the formation of Pluronic-coated iron oxide NPs (template), this colloidal solution (0.19 mg/ml) was freeze-dried in 5 ml of F-68 (5 wt.%) aqueous solution to form the lyophilized NPs. For the formation of the vesicle NPs, the vesicle suspension was separately prepared in the aqueous medium by the sonication using a probe type ultrasonic wave homogenizer (Branson Sonifier Model 185) and it was composed of 25 wt.% aqueous solution of L-α-phosphatidylcholine with the spherical form. Then, weighed amounts of Pluronic-coated iron oxide NPs were mixed with the vesicle suspension to form the vesicle NPs containing Pluronic-coated iron oxide NPs.

2.4. Preparation of the multilayer NPs

Fig. 1. The multilayer NPs and the application as a nanocarrier for cancer-targeting therapy.

The F-68 aqueous solution (20 wt.%) was mixed with the vesicle NPs containing Pluronic NPs with DTX or iron oxide NPs and the ratio was 50/50 (w/w). Then, solution mixture was freeze-dried in order to induce the formation of a polymeric shell on the surface of the vesicle NPs. The F-68 aqueous solutions were prepared with trehalose (5 wt.% of total NPs), which was used as a cryoprotectant, to preserve of core/shell structure integrity of the NPs during the freeze-drying [12,13]. The loading amounts of DTX and iron oxide NPs were 3.5 wt.% and 0.47 wt.%, respectively.

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Fig. 2. TEM pictures of (a) the vesicle NPs containing DTX-loaded Pluronic NPs, (b) the multilayer NPs containing DTX-loaded Pluronic NPs, (c) Pluronic-coated iron oxide NPs, and (d) the multilayer NPs containing Pluronic-coated iron oxide NPs.

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The average diameter and size distribution of the NPs (1 mg/ml of NPs dispersed in phosphate buffered saline (PBS, pH 7.4)) were measured via dynamic light scattering (Zeta Sizer Nano Series, Malvern, UK) at 632.8 nm and 25 °C. Transmittance electron microscopy (TEM) measurement images were also taken in order to observe the morphology of NPs. For the TEM measurements, the freeze-dried NPs were dispersed in distilled-deionized water to obtain the solution of 0.1 wt.%. To prepare a sample, each solution (5 μl of the aqueous solution containing NPs (1 mg/1 ml in distilled water)) was dropped on a carbon-coated grid and was stained with 2% uranyl acetate. The grids were dried at 25 °C in a vacuum oven for 24 h. The samples were examined with a FEI Tecnai G2 Spirit BioTWIN TEM operating at 120 kV. The digital images were obtained using a SIS Megaview III camera. For the cryo-TEM measurement, samples were prepared as a thin aqueous film supported on a cryo-grid. Thin aqueous films can be produced by applying approximately 7 μl of the aqueous solution containing NPs (1 mg/30 ml in distilled water) to holey-carbon film-supported grids, followed by the removal of excess fluid by blotting onto a filter paper for 7 s. The frozen grids were stored in liquid nitrogen and transferred to a GATAN model 630 cryotransfer (Gatan, Inc., Warrendale, PA) under liquid nitrogen at approximately − 190 °C. Direct imaging was performed at a temperature of approximately −170 °C with a 120 kV acceleration voltage, using the images acquired with a Multiscan 600 W CCD camera (Gatan, Inc., Warrendale, USA). 2.6. In vitro drug release characteristics of the NPs In order to measure the release pattern of the DTX from the NPs, 10 mg of NPs was dispersed in 10 ml of PBS (phosphate buffered saline, pH 7.4) and put into a dialysis bag (MWCO: 500,000, Spectrum®, Rancho Dominquez, USA), which was immersed in 20 ml of PBS (pH 7.4) containing 0.1% (w/v) Tween 80. The experimental setup was placed in a shaking water bath maintained at

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37 °C and shaken horizontally at 100 rpm. At predetermined time intervals, 2 ml of aliquots of the release medium (PBS) were withdrawn and the total release medium was replaced with 20 ml of fresh PBS in order to maintain the sink conditions. The quantification of the released DTX was determined by reverse-phase high performance liquid chromatography (RP-HPLC) using a Capcell-pack C18 column and an acetonitrile/water (50/50, v/v) mobile phase over 15 min at a flow rate of 1.0 ml/min. The eluent was monitored by UV absorption at 227 nm.

2.7. MTT assay of the multilayer NPs Murine SCC-7 (squamous cell carcinoma) cells were cultured in RPMI 1640 (Gibco, Grand Island, USA) containing 10% (v/v) FBS (Gibco) and 1% (w/v) penicillin–streptomycin at 37 °C in a humidified 5% CO2–95% air atmosphere. The cytotoxicity of free DTX (commercial DTX formulation (Taxotere®)) and various NPs was evaluated using MTT assay. The cells were seeded at a density of 5 × 103 cells/well in 96-well flat bottomed plates, and were allowed to adhere overnight. The cells were washed twice with PBS and incubated for 24, 48, and 72 h with various concentrations of free DTX, and NPs. The cells were then washed twice with PBS to eliminate the remaining drugs. Twenty five microliters of MTT solution (5 mg/ml in PBS) was added to each well and the cells were incubated further for 2 h at 37 °C. The cells were added and dissolved in 200 ml of DMSO. The absorbance at 570 nm was measured with a microplate reader (VERSAmax™, Molecular Devices Corp., Sunnyvale, USA).

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3. Results and discussion

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3.1. Preparation and characterization of the multilayer NPs

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2.8. In vivo MR characteristics of the multilayer NPs in tumor-bearing mice For the in vivo experiments, SCC-7 (squamous cell carcinoma) cells were induced in male C3H/HeN mice (7 weeks old, 20–25 g, ORIENT BIO Inc. Korea) by subcutaneous injection of 1.0 × 106 cells suspended in cell culture media (RPMI medium 1640 at 37 °C in a humidified 5% CO2 incubator, 10% fetal bovine serum, 1% antibiotic agent). When the tumor volume reached approximately 250–300 mm3, 0.75 mg of the multilayer NPs containing iron oxide NPs mixed with 0.1 ml of saline was administered through the tail veins of the tumor-bearing mice. In vivo MR images were collected in order to observe the accumulation of multilayer NPs at the tumor sites. The accumulation of the multilayer NPs containing iron oxide at the tumor site shortened the spin–spin relaxation time (T2⁎) by dephasing the spin of neighboring water protons and resulted in the darkening of T2⁎-weighted images. The MR images were acquired at 1 h, 3 h, and 6 h post-injection. The MR images were acquired using a 4.7 T Bruker BioSpin imager (Bruker Medical Systems, Karlsruhe, Germany) with a conventional T2⁎-weighted gradient-(GRE) sequence (repetition time ms/echo time ms, 400/10.4; flip angle, 30°). The specific parameters of the images were as follows: the spatial resolution was 256 × 256 matrix; the field of view, 30× 30 mm; the section thickness, 0.7 mm; the section gap, 0.7 mm; and the number of sections, 12. All animal experiments were carried out in accordance with the guidelines for animal experiments at the Korea Institute of Science and Technology, Republic of Korea. 2.9. Antitumor efficacy of the multilayer NPs in tumor-bearing mice The tumors were produced in the C3H/HeN mice as described above. When tumors grew to approximately 50–100 mm 3, the mice were divided into five groups: 1) normal saline (the control group, n = 5); 2) free DTX (commercial DTX formulation (Taxotere®)) at 10 mg DTX/kg (n = 5); 3) empty multilayer NPs (n = 5); 4) the vesicle NPs at 10 mg DTX/kg (n = 5); and 5) the multilayer NPs at 10 mg DTX/kg (n = 5). Each sample was injected once every 3 days for 12 days. The tumor size was measured on the first day after the first injection. After that, it was measured every second day using Vernier calipers in two dimensions. The individual tumor volumes (V) were calculated using the formula V = [length × (width) 2] / 2, where the length is the longest diameter and the width is the shortest diameter perpendicular to length. 2.10. Statistical analysis Data are expressed as the mean ± S.E.M. of at least three experiments. All data processing was performed using the ORIGIN® 7.0 statistical software program (OriginLab Corp., USA).

Because the multilayer NPs were designed as nanocarrier systems with therapeutic or diagnostic modalities for chemotherapy, the components for the therapeutic or diagnostic modalities were prepared individually. For the therapeutic component, the vesicle NPs containing DTXloaded Pluronic NPs were prepared according to the previous method reported by us [16,17]. After preparing the DTX-loaded Pluronic NPs by a temperature-induced phase transition, the incorporation of the DTX-loaded Pluronic NPs was accomplished by vesicle fusion. When spherical vesicles encounter one another in suspension, they are prone to adhere and fuse to form a larger one. Because spherical vesicles open during the fusion process, the leakage of the entrapped molecules was usually observed [19–21]. However, the incorporation of NPs into the vesicles was also observed during the vesicle fusion as reported previously [17]. By varying the DTX-loaded Pluronic NPs/ vesicles (w/w) ratio in the aqueous media, the optimum composition for the formation of the vesicle NPs was determined by observing the precipitation of DTX from the vesicle NPs. When the loading amount was more than 12.5 wt.%, the DTX precipitated from the vesicle NPs indicating that a limited amount of DTX-loaded Pluronic NPs could be incorporated efficiently into the vesicles. Therefore, the vesicle NPs with the loading amount of 7.5 wt.% were used as the therapeutic components. The spherical shape and size of the vesicle NPs (the loading amount: 7.5 wt.%) were confirmed using a cryo-TEM (Fig. 2(a)). The vesicle NPs containing iron oxides NPs were prepared as the component for the diagnostic modality. To avoid the agglomeration between the anionic vesicles and cationic iron oxide NPs in the aqueous media during the fusion process, gold was deposited on the surface of the iron oxide NPs and the gold-deposited iron oxide NPs were stabilized with Pluronic (Fig. 2(c)). For the formation of diagnostic component, the incorporation of the Pluronic-coated iron oxide NPs into vesicles was induced. After preparing the components for therapeutic or diagnostic modalities, the multilayer NPs were prepared by freeze-drying the vesicle NPs containing DTX-loaded NPs or Pluronic-coated iron oxide NPs in the Pluronic aqueous media. It has been reported that hydrophobic blocks (i.e., polypropylene blocks) enabled Pluronic to physically anchor on the liposome surface during the freeze-drying [14,15]. This allowed the formation of the multilayer NPs as shown in Fig. 2. Fig. 3 shows the size distribution of the vesicle NPs and multilayer NPs. With the formation of the multilayer NPs, the diameter was increased to approximately 300 nm as expected from the TEM images. It was impossible to obtain the freeze-dried form of the vesicle NPs, because the vesicle NPs were agglomerated into microparticles during the freeze-drying. However, the multilayer NPs were obtained in the nano-sized powder form due to the presence of Pluronic layer on the surface. 3.2. Physicochemical properties of the multilayer NPs The in vitro release of DTX was examined with the variation of nanoparticle structure (Fig. 4). The Pluronic NPs showed a rapid release pattern and much (77%) of the loaded DTX was released within 30 min with the precipitation of DTX in the release medium. This was due to the unstable and inefficient capability of the Pluronic NPs. With the formation of the vesicle NPs, the release rate was decreased and this was due to the control of the lipid bilayer on the release of DTX from the Pluronic NPs. A further decrease in the release rate of DTX was observed with the multilayer NPs due to the presence of additional Pluronic layer on the surface. The cytotoxicity of free DTX (commercial DTX formulation (Taxotere®)) and NPs was evaluated by determining the viability of the

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SCC7 murine cancer cells (Fig. 5(a)). All samples exhibited cytotoxicity, which was enhanced as the concentration increased. However, the cell viability of the empty multilayer NPs was found to be significantly higher than that of other NPs (Fig. 5(b)). This result indicated that the empty multilayer NPs exhibited minimal cytotoxicity compared with the other NPs. As expected, the empty multilayer NPs exhibited almost 100% cell viability at a concentration of 250 μg/ml, indicating excellent cytocompatibility in the cell culture system (Fig. 5(b)). Also, free DTX and the multilayer NPs demonstrated the drug concentrationdependent cytotoxicity in the cell culture system. When the DTX concentration was increased, the cytotoxicity of the vesicle NPs and the multilayer NPs was lower than that of the free DTX (Fig. 5(a)). The lowest cell viability, i.e. the highest cell mortality, was observed at the highest concentration of DTX after treatment for the longest time, which proved the controlled release of DTX from the NPs. This new DTX formulation of the multilayer NPs in the aqueous medium may minimize the adverse effects of the current free DTX formulation containing ethanol, such as anaphylaxis and severe hypersensitivity [22–24]. If the multilayer NPs is accumulated at the tumor site, enhanced antitumor efficacy is expected with reduced adverse effects [25]. The effect of iron oxide NPs on the cytotoxicity was also expected [18]. However, the concentration-dependent cytotoxicity due to the presence of the iron oxide NPs in the multilayer NPs was minimal due to the efficient incorporation of the iron oxide NPs in the multilayer NPs (Fig. 5(b)).

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Fig. 5. Cytotoxicity of (a) the vesicle NPs containing DTX-loaded Pluronic NPs, the multilayer NPs containing DTX-loaded Pluronic NPs, and free DTX (commercial DTX formulation (Taxotere®)) and (b) Pluronic-coated iron oxide NPs, the multilayer NPs containing Pluronic-coated iron oxide NPs, and empty multilayer NPs. The cell viability of each sample was measured using MTT assays. The data represents mean ± S.E.M. (n = 7).

When drugs and imaging agents are delivered using the same NPs, both are assumed to have similar behaviors, such as biodistribution and targeted accumulation, in living systems [26,27]. By observing the targeted accumulation of the multilayer NPs containing iron oxide NPs using MR, it may be possible to expect the targeted accumulation of the multilayer NPs containing DTX. The significant enhancement of MR intensity was observed at the tumor site which was described by the dotted line in Fig. 6. After 1 h post-injection, the MR intensity at the tumor site was increased. At up to 6 h after injection, a significant enhancement of MR intensity at the tumor site was observed, indicating the excellent tumor targetability of the multilayer NPs (Fig. 6(a)). The tumors were distinguishable from the surrounding background tissue 1 h post-injection. In order to target tumors, NPs need a long half-life and the polyethylene oxide (PEO) in the Pluronic F-68 can facilitate this requirement [28,29]. The multilayer NPs showed the enhanced MR intensity at the tumor site compared with Resovist (Fig. 6(b)). This was because the multilayer NPs predominantly accumulated in the tumor tissue and led to the excellent tumor targetability of the multilayer NPs. Note that the dark images in Fig. 6(b) was due to the bone in the tumor-bearing mouse. To obtain the clear image, the tumor site was magnified in the axial images in Fig. 6(b). 3.4. In vivo antitumor efficacy of the multilayer NPs in the tumorbearing mice We subsequently determined whether the multilayer NPs had therapeutic activity in vivo in the SCC-7 tumor-bearing mice (Fig. 7). As controls, the mice were injected with free DTX (commercial DTX formulation (Taxotere®)), the vesicle NPs containing DTX-loaded Pluronic NPs, empty multilayer NPs (the multilayer NPs without DTX), or saline (200 μl). The tumor size was similar for all treatments on day 3. By day 5, the tumor size had increased less after treatment with the multilayer NPs compared with free DTX, indicating that the multilayer NPs were more effective at reducing the tumor volume due to their tumor targeting. In addition, the empty multilayer NPs also blocked the tumor growth, potentially via the accumulation in the tumor tissue by changing the local environment. Previous study speculated that large amounts of glycol chitosan NPs accumulated in the tumor tissue and affected tumor growth by changing the local environment in the

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4. Conclusions We verified the formation of the multilayer NPs using the layer by layer approach for cancer-targeting therapy. The TEM and size distribution analyses demonstrated the formation of the multilayer NPs. Based on MR images with the multilayer NPs and Resovist, we could expect the extended retention of the multilayer NPs in the blood stream at the tumor tissue with the enhanced targeting efficiency. This led to the increase of the therapeutic efficacy above that of the surfactant-based DTX. These results indicate that the multilayer NPs may be very useful as drug carriers for cancer-targeting therapy.

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This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (MEST) (20110027932 and 2012028831).

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tumor tissue, for example by increasing the viscosity in the neovasculature [30] and the similar results were also reported using Pluronic based NPs [16]. By considering the minimal cytotoxicity of empty multilayer NPs, a slight reduction in the tumor volume by the empty multilayer NPs may be due to the accumulation of the NPs in the tumor tissue.

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14 * : P value > 0.05 ** : P value < 0.05

Fig. 7. Therapeutic efficacy of (a) normal saline, (b) empty multilayer NPs, (c) free DTX (commercial DTX formulation (Taxotere®)), (d) the vesicle NPs, and (e) the multilayer NPs in squamous cell carcinoma cells allograft.

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