Codelivery of Sorafenib and Curcumin by Directed Self-Assembled

Article pubs.acs.org/molecularpharmaceutics

Codelivery of Sorafenib and Curcumin by Directed Self-Assembled Nanoparticles Enhances Therapeutic Effect on Hepatocellular Carcinoma Haiqiang Cao,†,§ Yixin Wang,†,‡,§ Xinyu He,† Zhiwen Zhang,*,† Qi Yin,† Yi Chen,† Haijun Yu,† Yongzhuo Huang,† Lingli Chen,† Minghua Xu,† Wangwen Gu,† and Yaping Li*,† †

State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China ‡ School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China S Supporting Information *

ABSTRACT: Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related mortality worldwide. Herein, we first reported the codelivery of sorafenib and curcumin by directed selfassembled nanoparticles (SCN) to enhance the therapeutic effect on HCC. SCN was formed by employing the hydrophobic interactions among the lipophilic structure in sorafenib, curcumin, and similar hydrophobic segments of polyethylene glycol derivative of vitamin E succinate (PEG-VES), which comprised uniform spherical particles with particle size of 84.97 ± 6.03 nm. SCN presented superior effects over sorafenib, curcumin, and their physical mixture (Sora + Cur) on enhancing in vitro cytotoxicity and cell apoptosis in BEL-7402 cells and Hep G2 cells, and antiangiogenesis activities in tube formation and microvessel formation from aortic rings. Moreover, the tissue concentration of sorafenib and curcumin in gastrointestinal tract and major organs were significantly improved after their coassembly into SCN. In particular, in BEL-7402 cells induced tumor xenograft, SCN treatment displayed the obviously enhanced inhibitory effect on tumor progression over free drug monotherapy or their physical mixture, with significantly increased antiproliferation and antiangiogenesis capability. Thereby, the codelivered nanoassemblies of sorafenib and curcumin provided a promising strategy to enhance the combinational therapy of HCC. KEYWORDS: sorafenib, curcumin, nanoparticles, codelivery, hepatocellular carcinoma

1. INTRODUCTION Hepatocellular carcinoma (HCC) is the most common primary liver tumor and remains the third leading cause of cancer-related death worldwide.1−3 In clinic, HCC is characterized by its aggressiveness, poor diagnosis, and limited therapeutic opportunities. At early stages, surgery resection, liver transplantation and thermal ablation are potentially curative interventions for HCC.3 For advanced stage diseases, the systemic pharmacotherapy is usually the final and main treatment modality.2 However, advanced HCC shows intrinsic resistance to chemotherapy agents, which remains a severe barrier for successful systemic chemotherapy.2,3 As a result, the conventional chemotherapy has been used for the treatment of advanced HCC over 30 years but demonstrates a very limited survival benefit.3,4 Sorafenib, an oral multiple kinase inhibitor, is successful to prolong the survival of HCC patients at advanced stages and is an encouraging progress for treating HCC.2,3,5 Moreover, sorafenib is the only molecular targeting medicine approved for advanced HCC and is commonly used as a standard treatment. Sorafenib can significantly inhibit the activities of multiple © XXXX American Chemical Society

tyrosine kinases and serine/threonine kinases and induce inhibitory effects toward tumor angiogenesis, cell proliferation, and apoptosis to achieve the anticancer activities.5,6 However, during the clinical application, sorafenib presents low tumor response rates to a majority of HCC patients and is beneficial in only about 30% of patients.5,7 Moreover, in most patients who initially respond to sorafenib, tumor refractory and progression often develop after a few months of sorafenib therapy.5,7−9 Worse still, no alternative effective therapeutic regimens are available after sorafenib failure. Therefore, it is urgent to find a new approach to improve therapeutic efficacy of sorafenib on HCC. Curcumin, a natural polyphenolic compound, has a variety of therapeutic properties including antioxidant, anti-inflammatory, antiangiogentic, and antiproliferative activities.10−13 Curcumin has chemopreventive effects and therapeutic value in the treatment Received: November 13, 2014 Revised: January 12, 2015 Accepted: January 26, 2015

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DOI: 10.1021/mp500755j Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics and prevention of a wide range of cancers.14,15 Moreover, curcumin can act as a depressor of chemoresistance by sensitizing cancer cells to conventional chemotherapeutic agents.16−19 Accordingly, in this work, we first put forward the combinational therapy of sorafenib and curcumin on HCC and expect to reduce the drug resistance and improve the antitumor efficacy. However, both sorafenib and curcumin are extremely insoluble in water because they both possess phenyl groups and other hydrophobic segments (Scheme 1), which result in their high lipophilic

(St. Louis, USA). Primary mono-antibody against Ki-67 (ab28364) and CD-31 (ab16667) were provided by Abcam (Cambridge, U.K.). Matrigel matrix was provided by BD Bioscience (San Jose, USA). All other reagents and solvents were of analytical or high performance liquid chromatography (HPLC) grade. Water was treated with the Milli-Q Plus System (Millipore, Bedford, USA). 2.2. Cells and Animals. Human hepatocellular carcinoma cell lines of BEL-7402 and Hep G2 cells were provided by Shanghai Cell Resource Center of Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (CAS). BEL7402Cells and Hep G2 cells were respectively cultured in RPMI1640 and Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) culture media. Human microvascular endothelial cells (HMEC-1) were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA) and grown in MCDB131 medium. All culture media were supplemented with 10% fetal bovine serum (FBS, Biochrom, Germany), 100 units/mL penicillin G sodium, and100 mg/mL streptomycin sulfate. Cells were maintained at 37 °C in a humidified and 5% CO2 incubator. Balb’c nude mice were supplied by Shanghai Laboratory Animal Center, CAS. The animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Institute of Materia Medica, CAS. Animals were kept at the animal care facility, given with daily fresh diet with free access to water, and acclimatized at least 3 days for the experiments. 2.3. Preparation and Characterization of SCN. SCN was fabricated as the following: sorafenib, curcumin, and PEGVES (1:2:10.5, w/w) was dissolved in methanol and evaporated to dryness to form a thin film. Then, the film was dispersed into double-distilled water by gentle shaking. SCN could be formed spontaneously with 4 mg/mL of sorafenib and 8 mg/mL of curcumin. The morphology of SCN was measured under a transmission electronic microscope (TEM, JEOL JEM-2100F, Japan). Samples were diluted with double-distilled water and negative stained with 2% phosphotungstic acid solution (w/v) for the observation. Meanwhile, the particle size distribution of SCN was determined by dynamic light scattering (DLS) on an instrument of Nano ZS 90 (Malvern, U.K.). To evaluate the crystalline state of sorafenib and curcumin in SCN, the experiments were performed on an XRD-6000 X-ray diffractometer (XRD, Shimadzu, Tokyo, Japan) in the range of 3−30° at a scan speed of 4°/min. By contrast, the XRD profiles of free PEG-VES, sorafenib, and curcumin powder were measured as control. Meanwhile, the thermal behavior of PEG-VES, sorafenib, curcumin, and SCN were assessed on a differential scanning calorimeter (DSC 822e, Mettler Toledo, Switzerland) between 30 and 280 °C with the temperature rising at 10 °C/min. To determine the encapsulation efficiency, the drug amount of sorafenib and curcumin in SCN was determined by HPLC analysis. Briefly, free sorafenib and curcumin were separated from SCN by an ultrafiltration method (10 000 Da, Millipore) with centrifugation at 3000 × g for 5 min. The sorafenib concentration in the filtrate and SCN was determined by HPLC system (Waters Alliance, USA) with the following conditions: column, sunfireC18 column (4.6 × 150 mm, 5 μm, Waters); temperature, 37 °C; mobile phase, acetonitrile/methanol/1% acetic acid (35:38:27, v/v); flow rate, 1.0 mL/min, detection wavelength, 254 nm. Meanwhile, the curcumin concentration in the filtrate and SCN was measured on the same HPLC system

Scheme 1. Formation of SCN by Directed Self-Assembly to Enhance the Combinational Therapy of HCC

property and exhibit poor absorption upon oral administration,10,20−22 thereby restricting their potential therapeutic efficacy. Moreover, the practical application of combination therapy is usually hampered by the lack of feasible vehicles for simultaneous delivery of multiple anticancer agents.23 Directed self-assembly provides a powerful and feasible strategy for fabrication of nanoparticles from small molecules, DNA, and polymers, as a result of various intermolecular interactions.24−26 Hydrophobic interactions are one of the most important types of intermolecular forces in the formation of nanoparticles, which make it a potential platform for entrapping a wide range of lipophilic drugs.27−29 In order to codeliver multiple hydrophobic anticancer drugs with strong intermolecular interactions, a polyethylene glycol (PEG) derivative of vitamin E succinate (PEG-VES) with lipophilic phenyl groups and alkyl chains in the hydrophobic segments is employed for codelivery of sorafenib and curcumin by directed selfassembly. We suppose that these two lipophilic drugs could be coassembled with the hydrophobic segments of PEG-VES into the inner hydrophobic core of nanoparticles via intermolecular hydrophobic interactions and surrounded with a hydrophilic outer corona from the PEG segments of PEG-VES (Scheme 1). Then, the sorafenib and curcumin codelivered nanoparticles (SCN) are expected to improve the therapeutic efficacy on HCC. In particular, we focused on the effects of SCN on the antiproliferative and antiangiogenesis activities against HCC by in vitro and in vivo evaluations, to validate its feasibility in the combinational therapy of HCC.

2. MATERIALS AND METHODS 2.1. Materials. Sorafenib tosylate was obtained from Shengxin Pharmaceuticals Ltd. (Zhejiang, China). Curcumin and nile red were supplied by J and K Chemical Co. Ltd. (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and PEG-VES were purchased from Sigma−Aldrich B


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Molecular Pharmaceutics

was evaluated with HMEC-1 cells as in a previously described method.30 Briefly, 50 μL of Matrigel was added to each well of 96-well plate and maintained at 37 °C for 1.0 h. HMEC cells were collected and suspended in MCDB culture media. Then, 100 μL of culture media with 3 × 104/well were seeded to each well and respectively incubated with sorafenib, curcumin, Sora + Cur, or SCN at 1.57 μM sorafenib and 5.43 μM curcumin for 4.0 h. By contrast, cells without any treatment were performed as negative control. The tube formation was observed under microscopy (IX81, Olympus, Japan) and photographed. Then, the mouse aortic ring assay was performed to detect the formation of microvessel.31 The aortic ring models were obtained from thoracic aorta of mouse, cut into rings ∼1 mm in width, and immersed in FBS free-DMEM culture media. The rings were put into 50 μL of Matrigel-coated wells of 24-well plate, embedded in another 50 μL of Matrigel, and incubated at 37 °C for 10−15 min. Then, 1.0 mL of Opti-MEM culture media with sorafenib, curcumin, Sora + Cur, or SCN (0.078 μM sorafenib and 0.271 μM curcumin) was added to each well. The growth media was changed at day 3 and then every other day until the experiments end. The formations of microvessels were visualized under microscopy and photographed. 2.9. In Vivo Distribution after Oral Administration. The distribution of SCN in the gastrointestinal (GI) tract and major organs was investigated in tumor xenograft models and compared with the physical mixture of sorafenib and curcumin suspension at equivalent doses. SCN, the physical mixture of sorafenib and curcumin suspension were orally administered to mice at 40 mg/kg of sorafenib and 80 mg/kg of curcumin. At 4.0 h after oral administration, different segments of GI tract from stomach to colon, and major organs including heart, liver, spleen, lung, kidney, and tumor were carefully removed, rinsed with cold saline solution, weighted, and homogenized for further analysis. The sorafenib and curcumin amounts in these homogenates were respectively determined by HPLC methods as described above. The drug concentration in these tissues was normalized by the weight of selected organs. 2.10. In Vivo Therapeutic Efficacy of SCN. The in vivo therapeutic efficacy of SCN on tumor growth, inhibitory effects on cell proliferation and tumor angiogenesis were measured in BEL-7402 induced xenograft models. When tumors reached 60−80 mm3, animals were randomly divided into 5 groups. Sorafenib, curcumin, Sora + Cur, and SCN were respectively daily given to mice at 40 mg/kg of sorafenib and 80 mg/kg of curcumin by oral administration. Animals without any treatment were performed as negative control. The width and length of tumors were measured every 3 days to monitor the tumor growth. At the end point, mice were sacrificed and tumors from each group were removed, weighted, and photographed. Then, the tumor tissues were fixed in 4% paraformaldehyde solution overnight, embedded in paraffin and sectioned for further detections. The cell proliferations and tumor angiogenesis in tumor tissues were measured by in situ immunohistochemistry assay. Sections were respectively incubated with anti-Ki67 monoclonal antibody (ab28364, Abcam, UK) and anti-CD31 monoclonal antibody (ab16667, Abcam, U.K.), and then treated with secondary antibody for visualization under microscopy. The cell proliferations were evidenced as brown spots, while the neovasculature were depicted as the dark circles with lumen structure. 2.11. Statistical Analysis. One-way analysis of variance (ANOVA) was used to determine the difference in multiple comparisons, followed by Tukey’s posthoc test. Statistical difference was considered at p < 0.05.

with the following conditions: column, sunfireC18 column (4.6 × 150 mm, 5 μm, Waters); temperature, 25 °C; mobile phase, methanol/3.6% acetic acid (70:30, v/v); flow rate, 1.0 mL/min, detection wavelength, 428 nm. The encapsulation efficiency and drug loading capacity of sorafenib and curcumin in SCN were calculated. 2.4. In Vitro Antiproliferative Activity. To determine the effect of SCN on in vitro antiproliferative activities, the in vitro cytotoxicity of SCN was measured in BEL-7402 and Hep G2 cells. Briefly, cells were seeded into 96-well plate at 6000 cells/well and culture overnight. Then, sorafenib, curcumin, physical mixture of sorafenib and curcumin (Sora + Cur), and SCN were respectively added to each well at predetermined concentrations and incubated for further 24, 48, and 72 h. The concentration of sorafenib, curcumin, or Sora + Cur was equivalent to SCN. Then, the cytotoxicity was measured by MTT assay (Enspire, PerkinElmer, Singapore). The cell viability was defined as the absorbance values of samples compared to that of negative controls. Moreover, the half-maximal inhibitory concentration (IC50) of each group was calculated. 2.5. Cellular Uptake. The cellular uptake behavior of SCN in BEL-7402 and Hep G2 cells was visualized under laser confocal scanning microscopy (LCSM, Fluoview FV 1000, Olympus, Japan). SCN was fluorescently labeled with hydrophobic nile red for the measurements. Prior to the experiments, cells were seeded on the glass coverslips (Ø10 mm) in 24-well culture plate at 1 × 104 cells/well and culture overnight for the attachments. The fluorescent SCN was added to each well at 1.57 μM sorafenib and 5.43 μM curcumin and incubated for 2.0 h. Then, cells were respectively stained with LysoTracker Green DND-26 (Molecular Probe, USA) and Hoechst 33342 according to the manufacturer’s protocols. Afterward, the coverslips were placed onto the glass microscope slides, and the subcellular localization of SCN was visualized under LCSM. 2.6. Mitochondrial Membrane Potential. The effects of SCN on the mitochondrial membrane potential (MMP) were measured in BEL-7402 and Hep G2 cells. Cells were seeded into 12-well plate and cultured overnight for the attachments. Then, sorafenib, curcumin, Sora + Cur, and SCN were added to each well with 1.57 μM sorafenib and 5.43 μM curcumin and incubated for 24 h. Afterward, cells were stained with the JC-1 mitochondrial membrane potential assay kit (Beyotime, China) and measured by flow cytometry (FACSCalibur, BD, USA). The variation of MMP, a switch from red to green fluorescence, was defined as the ratio between the red and green fluorescence intensity (JC-1 Red/Green). The effect of sorafenib, curcumin, Sora + Cur, and SCN on MMP was characterized as the JC-1 Red/Green values of samples compared to that of negative control. 2.7. Cell Apoptosis. To evaluate the effects of SCN on cell apoptosis, the cell populations at early and late apoptotic stages in BEL-7402 and Hep G2 cells were measured by flow cytometry (FACSCalibur, BD, USA). Cells were respectively incubated with sorafenib, curcumin, Sora + Cur, and SCN at 1.57 μM sorafenib and 5.43 μM curcumin for 48 h. By contrast, cells without any treatment were performed as negative control. Then, cells were stained with the Annexin V-FITC/PI Apoptosis Detection kit (Invitrogen, USA) according to the manufacturer’s protocols and further analyzed using the FACSCalibur system (BD, USA). 2.8. In Vitro Antiangiogenesis Measurements. The effects of SCN on the antiangiogenesis activity were evaluated by tube formation and aortic ring assays. Tube formation assay C


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Figure 1. In vitro characterizations of SCN. (A) Typical TEM images of SCN, scale bar = 100 nm; (B) Particle size distribution of SCN; (C) XRD patterns of PEG-VES, sorafenib, curcumin, and SCN; (D) DSC thermographs of PEG-VES, sorafenib, curcumin, and SCN.

3. RESULTS 3.1. Physicochemical Characterization of SCN. SCN was self-assembled by employing the intermolecular hydrophobic interactions between the lipophilic groups of sorafenib, curcumin and similar structures in the hydrophobic parts of amphiphilic PEG-VES, which was illustrated in Scheme 1. The formation of SCN was verified by in vitro characterizations. The TEM images showed that SCN was made of uniform spherical nanometer-sized particles (Figure 1A). Meanwhile, the DLS measurements indicated that the mean particle size of SCN was 84.97 ± 6.03 nm with the polydispersivity index (PDI) of 0.176 ± 0.034 (Figure 1B), which was in accordance with the TEM observations. The encapsulation efficiency of sorafenib and curcumin in SCN was 98.16 ± 0.23% and 91.66 ± 0.53%, respectively. Meanwhile, the drug loading efficiency was 6.54 ± 0.01% for sorafenib and 13.58 ± 0.07% for curcumin in SCN, which resulted in a 20% total loading capacity of the PEG-VES. These experimental results indicated the formation of SCN and the high encapsulation efficiency of lipophilic drugs in the coassembled SCN. Moreover, the crystalline state of sorafenib and curcumin in SCN was determined by XRD and DSC analysis. The measured results showed that the characteristic diffraction peaks of sorafenib and curcumin were not detected in the XRD profiles of SCN (Figure 1C). Similarly, in the DSC thermographs, the typical melting peaks of sorafenib and curcumin disappeared in the thermal profiles of SCN (Figure 1D). These results indicated that the hydrophobic sorafenib and curcumin could be dispersed with the amphiphilic PEG-VES and existed as amorphous or molecular state in SCN. 3.2. In Vitro Antiproliferation and Cell Apoptosis. The anticancer activity of sorafenib resulted from a dual inhibitory effect toward cell proliferation and tumor angiogenesis.5,6,32 The in vitro antiproliferative activity of SCN on HCC cells was measured in comparison with free sorafenib, curcumin, and physical mixture of sorafenib and curcumin. The in vitro cytotoxicity of SCN in BEL-7402 and Hep G2 cells determined by MTT assay indicated that all the four groups exhibited

dose- and time-dependent cell proliferation inhibition behaviors. In particular, the coassembled SCN presented significantly enhanced antiproliferation effects comparing to sorafenib, curcumin, and their physical mixture (Figure S1, Supporting Information). Then, the difference of the antiproliferation effects were reflected more clearly by the IC50 values after 24, 48, and 72 h incubation (Table 1). Typically at 48 h of Table 1. IC50 Values of Sorafenib and Curcumin in BEL-7402 Cells and Hep G2 Cells Treated with Sorafenib, Curcumin, Physical Mixture of Sora + Cur, and SCN at 24, 48, and 72 h after Incubations IC50 in BEL-7402 cells (μM)

IC50 in Hep G2 cells (μM)

group

24 h

48 h

72 h

24 h

48 h

72 h

sorafeniba curcumina Sora + Cura SCNa sorafenibb curcuminb Sora + Curb SCNb

>31.40

13.19

6.59

11.62

8.63

10.68

13.19 6.59

9.42 2.35

5.02 1.10

8.16 3.14

5.49 3.30

4.71 2.83

>108.58 45.60 22.53

50.49 32.30 8.14

36.64 17.37 3.80

25.52 28.50 11.13

21.99 19.27 11.67

19.27 16.29 10.04

a Refers to the IC50 values of sorafenib. bRefers to the IC50 values of curcumin.

incubation, the IC50 value from SCN in BEL-7402 cells was 2.35 μM for sorafenib and 8.14 μM for curcumin, which was respectively 17.8% and 16.1% of that from free sorafenib and curcumin. Meanwhile, in Hep G2 cells, the IC50 values of SCN was 3.3 μM for sorafenib and 11.67 μM for curcumin, which was respectively 38.7% and 52.8% of that from free sorafenib and curcumin. In particular, the IC50 value of SCN was respectively 25.1% and 60.4% of that from their physical mixture in BEL-7402 cells and Hep G2 cells. Thereby, the antiproliferation activities were significantly improved by coassembled nanoparticles of SCN. Then, the effect of SCN on cell apoptosis in BEL-7402 cells and Hep G2 cells was evaluated. At first, the mitochondrial D


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Figure 2. Mitochondrial membrane potential (MMP) and cell apoptosis in BEL-7402 cells and Hep G2 cells treated with sorafenib, curcumin, Sora + Cur, and SCN with 1.57 μM sorafenib and 5.43 μM curcumin. (A) Relative MMP values, *p < 0.05; (B) cell percentages at early apoptotic and late apoptotic stages.

(combination of red and green fluorescence) in BEL-7402 cells and Hep G2 cells. Accordingly, SCN could be colocalized with lysosomes after internalization. 3.3. In Vitro Antiangiogenesis Measurements. HMEC-1 tube formation and aortic ring models were used for assessing the antiangiogenesis activities of SCN (Figure 4). In the tube formation assay, HMEC-1 cells aggregated in Matrigel and elongated into sparse meshes with tube-like structure in the negative control (Figure 4A). When cells were treated with free sorafenib, curcumin, or Sora + Cur, the enclosed tube-like structures were still detectable with some separate endothelial cells, which indicated their mild inhibition ability on tube formation. However, in SCN group, the typical enclosed capillary networks were disrupted, and only a few capillary tubes and plenty of endothelial cells were readily detected, which indicated the obvious inhibition efficacy on tube formation. Then, the ex vivo mouse aortic ring assays were utilized to evaluate the inhibitory effect on microvessel sprouting from the aortic rings (Figure 4B). Compared with the negative control, the treatment of free sorafenib, curcumin, and Sora + Cur exhibited limited inhibitory effects on the microvessel formation. However, SCN treatment resulted in a significant inhibition on the microvessel sprouting from the aortic rings. Thereby, the tube formation and aortic ring assays clearly confirmed the significant enhancement of antiangiogenesis activity by coassembled nanoparticles of SCN. 3.4. In Vivo Distribution in Tumor Xenograft Model. Following oral delivery, the tissue concentration of sorafenib and curcumin from SCN in the GI tract or major organs was significantly higher than that from the physical mixture of sorafenib and curcumin suspension (Figure 5). In the GI tract, SCN was mainly distributed in stomach and duodenum, and then significantly decreased in jejunum, ileum, and colon (Figure 5A,C). Compared with the free drug suspension, the concentration of sorafenib and curcumin from SCN was respectively increased by 16.87 and 54.36 times in stomach, and

membrane potential (MMP) was an important parameter of mitochondria function, and the loss of MMP was usually regarded as a hallmark of apoptosis.12,33 Compared with the negative control, the MMP values in BEL-7402 cells and Hep G2 cells was not significantly changed after the treatment of free sorafenib, curcumin, and their physical mixture of Sora + Cur (Figure 2A). However, the SCN treatment resulted in a reduction of 53% and 68% in BEL-7402 cells and Hep G2 cells, respectively. The significant reduction of MMP by SCN could be promising to induce apoptosis in HCC cells, which was further verified by cell apoptosis measurements. Cells were stained with the Annexin V-FITC/PI Apoptosis Detection kit for analysis by flow cytometry. The induced cell apoptosis was characterized by counting the cell populations at early and late apoptotic stages (Figure 2B). When cells were treated with 1.57 μM sorafenib and 5.43 μM curcumin, the induced apoptosis percentages by sorafenib, curcumin, Sora + Cur, and SCN was 25.64%, 22.05%, 22.05%, and 56.22% in BEL-7402 cells, and 18.59%, 25.7%, 24.63%, and 62.86% in Hep G2 cells, respectively. Compared with free sorafenib and curcumin monotherapy, the apoptotic cell percentages were not significantly changed by Sora + Cur, but obviously enhanced by SCN. Typically, SCN treatment respectively resulted in an enhancement of 3.38 and 2.45 times in BEL-7402 cells and 2.45- and 2.85-fold in Hep G2 cells over free sorafenib and curcumin monotherapy. Moreover, the induced cell apoptosis in BEL7402 cells and Hep G2 cells by SCN was respectively 2.55- and 2.85-fold higher than Sora + Cur, which clearly verified the significant enhancement of cell apoptosis by coassembled nanoparticles of SCN. In addition, the subcellular localization of SCN in BEL-7402 cells and Hep G2 cells was visualized under LCSM (Figure 3). SCN was fluorescently labeled with nile red for the measurements. By contrast, the lysosomes were stained with LysoTracker Green DND-26, while the nuclei were counterstained with Hoechst 33342. The captured images showed the yellow fluorescent spots E


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Figure 3. Subcellular localization of SCN with lysosomes in BEL-7402 cells (A) and Hep G2 cells (B) at 2.0 h after incubation, scale bar = 10 μm.

3.88-fold than that from free drug suspension (Figure 5B). Similarly, the tissue concentration of curcumin was higher in lung and kidney, which was respectively improved 7.53 and 7.32 times when applied as SCN in place of free drug suspension (Figure 5D). In particular, in the tumor tissues, the sorafenib and curcumin concentration was significantly enhanced by 4.23and 5.85-fold by SCN. Thereby, the distribution of sorafenib and curcumin in the GI tract and major organs was evidently increased by coassembled nanoparticles of SCN. 3.5. In Vivo Efficacy on Inhibiting Tumor Growth. The in vivo antitumor efficacy of SCN on tumor growth, cell proliferation, and tumor angiogenesis were investigated in BEL-7402 induced tumor xenograft models. Compared with the control group, all the chemotherapeutic groups showed the inhibition of the tumor progression (Figure 6), and the inhibition efficacy on tumor growth was in the following order: SCN > Sora + Cur ≈ sorafenib > curcumin (Figure 6A). The curcumin monotherapy exhibited slight but not significant inhibitory effects, whereas the sorafenib monotherapy induced an obvious reduction of tumor growth. The tumor volume of coassembled nanoparticles of SCN was significantly lower than that of sorafenib, curcumin, or Sora + Cur, indicating the obviously enhanced inhibitory effects of SCN on tumor growth. Meanwhile, at the end point, the tumor tissues were removed, weighted, and photographed (Figure 6B). The relative tumor weight compared to the saline group was used to characterize the inhibition of tumor progression. The SCN treatment resulted in a 78.8% reduction of tumor growth, which was respectively 6.0- and 1.5-fold higher than curcumin and sorafenib monotherapy (Figure 6C). Moreover, the inhibition efficacy of SCN was significantly higher than that of Sora + Cur. Therefore, the coassembled SCN provided higher therapeutic efficacy against tumor progression compared with free drug monotherapy or their free combination. Then, the in vivo antiproliferation and antiangiogenesis activities of SCN were determined by immunohistochemistry measurements. For the in vivo antiproliferation assay, the tumor tissue sections were stained with anti-Ki 67 antibody, and cell proliferation was denoted as brown spots in the captured images (Figure 6D). The Ki-67 positive cells were largely detected in saline, sorafenib, curcumin, or Sora + Cur group, but less

Figure 4. In vitro antiangiogenesis activities of sorafenib, curcumin, Sora + Cur, and SCN. (A) Tube formations of HMEC-1 cells in Matrigel assay with 1.57 μM sorafenib and 5.43 μM curcumin; (B) microvessel formations from mouse aortic ring models with 0.078 μM sorafenib and 0.271 μM curcumin. Scale bar = 100 μm.

then obviously enhanced by 4.79- and 14.54-fold in duodenum. Moreover, in the major organs of tumor xenograft models, the sorafenib concentration from SCN was higher in liver, kidney, and lung, which was significantly increased by 3.48-, 3.86-, and F


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Figure 5. Tissue concentration of sorafenib and curcumin in the GI tract and major organs in tumor xenograft models treated with free sorafenib, curcumin, and SCN. The difference was detected between SCN and free drug suspension, *p < 0.05, **p < 0.01.

Figure 6. In vivo therapeutic efficacy on inhibiting tumor growth in BEL-7402 induced xenograft models. The mice were daily treated with sorafenib, curcumin, Sora + Cur, and SCN by oral gavage at 40 mg/kg of sorafenib and 80 mg/kg of curcumin. (A) Tumor growth profiles; (B) photographs of tumor tissues from each group; (C) relative tumor weight of each group when compared to the negative control, *p < 0.05, **p < 0.01; (D) cell proliferation in tumor tissues after staining with anti-Ki 67 antibody, scale bar = 20 μm; (E) the neovasculature in tumor tissues after incubation with anti-CD 31 antibody, scale bar = 100 μm. G


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treatment of advanced HCC. In this study, sorafenib and curcumin are coassembled into nanoparticles of SCN to improve the therapeutic efficacy on HCC, which has not been reported previously. The combinational chemotherapy of sorafenib and curcumin on HCC was significantly improved by SCN, which was evidenced by in vitro and in vivo evaluations. The dual inhibitory effect on cell proliferation and tumor angiogenesis of SCN were much higher than that of free sorafenib, curcumin, and their free combination of Sora + Cur. Moreover, the SCN treatment demonstrated significantly enhanced inhibitory effects on tumor progression in BEL7402 cell induced xenograft models when compared with other chemotherapy groups. These experimental data effectively evidenced the obviously enhanced therapeutic efficacy on HCC by coassembled nanoparticles of SCN. However, in the free combination of Sora + Cur, the therapeutic efficacy was not obviously increased in comparison with that of free sorafenib group, which suggested that there was no additive effect between these two drugs. Thereby, the enhanced inhibition efficacy on HCC by SCN could be mainly ascribed to the codelivery of sorafenib and curcumin in the nanoassemblies of SCN other than their free combination.

detectable in SCN group, which effectively verified the significantly enhanced antiproliferative activity of SCN. Then, for the in vivo antiangiogenesis measurements, samples were stained with anti-CD31 antibody and the neovasculature was depicted as the dark circles with lumen structure (Figure 6E). The captured images showed that the neovasculature was detected in the tumor sections from control, sorafenib, curcumin, and Sora + Cur groups, but barely detected in SCN treated group, which implicated the higher antiangiogenesis capability of SCN over other chemotherapy groups. Taken together, these experimental data clearly verified the greatly enhanced therapeutic efficacy on in vivo antiproliferation and antiangiogenesis activities by SCN.

4. DISCUSSION Nanoparticles can be self-assembled from versatile small molecules and polymers, which demonstrate great potential to simultaneously deliver multiple therapeutic agents for combination chemotherapy.17,26,34−37 Directed self-assembly of nanoparticles can be mediated by employing various intermolecular interactions among these involved ingredients, such as hydrophobic interactions, hydrogen bonding, π−π stacking forces, etc.26,27,29 We have recently developed directed self-assembled nanoparticles of probucol due to the intermolecular hydrophobic interactions between the lipophilic drugs and hydrophobic segments of Triton X-100, which verified the potential of directed self-assembly for drug delivery.25 In this work, the hydrophobic drugs of sorafenib and curcumin comprised hydrophobic structures similar to the lipophilic part of PEG-VES. These two drugs and PEG-VES could be directly self-assembled into SCN due to the intermolecular hydrophobic interactions among them, which could combine two drugs within a single nanovehicle. The formation of SCN was verified by in vitro characterizations, which showed uniform nanometersized particles with high encapsulation efficiency and drug loading capacity. To our knowledge, this could be the first attempt for codelivery of multiple anticancer drugs by directed self-assembly due to the intermolecular hydrophobic interactions, which provided a feasible platform for drug delivery to achieve the combination therapy on HCC. Furthermore, the enhanced oral delivery could provide an essential prerequisite for the combinational therapy of HCC. Previously, we developed a nanodelivery system of sorafenib with greatly enhanced oral delivery and tumor targeting in a gastric cancer xenograft model, which dramatically improved the therapeutic efficacy on tumor growth and live metastasis.20 With respect to the coassembled nanoparticles of SCN in this work, the distribution of sorafenib and curcumin in the GI tract was evidently enhanced in comparison with the free drug suspension. The enhancement could result from the nanometer-sized particles of SCN, which could improve the affinity and accessibility to the intestinal membrane, thereby increasing their absorption in the intestine.38,39 Moreover, the increased absorption in the GI tracts could provide a drug reservoir for further enhancement of biodistribution in major organs.25 The tissue concentration in major organs of tumor xenograft model was undoubtedly increased by SCN. Particularly in tumor, the tissue concentration of sorafenib and curcumin was respectively enhanced 4.23- and 5.85-fold by SCN over free drug concentration, which was beneficial for combination therapy on HCC. Combining sorafenib with other chemotherapeutic agents has been shown to exert synergistic effects in recent preclinical studies,2,3,40,41 which could provide promising strategies for

5. CONCLUSION The codelivered nanoparticles of sorafenib and curcumin (SCN) were developed by directed self-assembly, which comprised homogeneous nanometric spherical particles with the mean diameter of 84.97 ± 6.03 nm. The in vitro cytotoxicity, cell apoptosis in HCC cells, and in vitro antiangiogenesis capability were greatly enhanced by SCN. Moreover, the tissue concentration of sorafenib and curcumin in GI tract and major organs of tumor xenograft were also significantly increased. In particular, SCN presented superior inhibitory effect on tumor growth over sorafenib, curcumin monotherapy, or their free combination. Thereby, the coassembled nanoparticles of SCN could provide a promising platform for codelivery of multiple anticancer drugs for achievement of combinational therapy, and the coassembly of sorafenib and curcumin in SCN could offer a potential and effective strategy for enhancing the therapeutic efficacy on HCC.

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ASSOCIATED CONTENT

S Supporting Information *

In vitro cytotoxicity in BEL-7402 and Hep G2 cells are listed in Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Tel/Fax: +86-21-2023-1979. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The National Basic Research Program of China (2015CB932103 and 2012CB932502) and the National Natural Science Foundation of China (81270047 and 81373359) are gratefully acknowledged for financial support. H


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Codelivery of Sorafenib and Curcumin by Directed Self-Assembled

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