Synergistic combination therapy of lung cancer: Cetuximab functionalized nanostructured lipid carriers for the co-delivery of paclitaxel and 5-Demethylnobiletin

Biomedicine & Pharmacotherapy 118 (2019) 109225

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Synergistic combination therapy of lung cancer: Cetuximab functionalized nanostructured lipid carriers for the co-delivery of paclitaxel and 5Demethylnobiletin

T

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Shenghu Guo, Yuehua Zhang, Zheng Wu, Lei Zhang, Dongwei He, Xing Li, Zhiyu Wang Department of Immuno-oncology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, 050011, Hebei Province, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Combination therapy Cetuximab Nanostructured lipid carriers Paclitaxel 5-Demethylnobiletin

Lung cancer remains the leading cause of cancer associated deaths worldwide. Recent efforts have been focused on combinational and nanoparticulate therapies that can efficiently deliver multiple therapeutics. Herein, we reported cetuximab (CET) functionalized, paclitaxel (PTX) and 5-Demethylnobiletin (DMN) co-loaded nanostructured lipid carriers (NLCs) (CET-PTX/DMN-NLCs). The morphology, particle size, zeta potential, stability and drug release were tested. Cellular uptake, cell viability, synergistic effects and in vivo anti-tumor effects were evaluated on human lung adenocarcinoma cells (A549 cells), human embryonic lung cells (MRC-5 cells) and A549 paclitaxel-resistant cells bearing mice models. NLCs had sizes of around 130 nm and zeta potentials of +20-30 mV. The release of drugs from NLCs was relatively fast at the first 12 h and then became slow until completion of sustained release behavior. Cells uptake of CET-PTX/DMN-NLCs (65.8%) was remarkably higher than that of PTX/DMN-NLCs (35.5%) in A549 cells. The combination treatment with PTX and DMN synergistically decreases the viability of cells than the single PTX-NLCs and DMN-NLCs. CET-PTX/DMN-NLCs exhibited the most remarkable in vivo tumor inhibition efficiency, which suspended the tumor growth from 1010.23 to 211.18 mm3 at the end of the study. The highest tumor accumulation amount and low toxicity made CET-PTX/ DMN-NLCs a promising system for the synergistic combination therapy of lung cancer.

1. Introduction Lung cancer remains the leading cause of cancer associated deaths worldwide, of which non-small cell lung cancer (NSCLC) accounts for more than 80 percent [1,2]. Late diagnosis is a major obstacle to treating NSCLC patients with surgery and chemotherapy. Targeted therapy is recommended for patients with metastatic or last stage (stage IIIb/IV) NSCLC [3,4]. According to the NCCN guidelines in 2019, recommended chemotherapy includes platinum agents, taxanes such as paclitaxel (PTX), albumin-bound paclitaxel, and so on. Because of serious side effects and multi-drug resistance of monotherapies, recent efforts have been focused on combinational and nanoparticulate therapies that can efficiently deliver multiple drugs or therapeutics to solve these challenges [5]. Combination therapies of chemotherapeutic drugs and traditional Chinese medicines have become attractive fields in recent cancer treatments [6–8]. Paclitaxel (PTX), a representative microtubule-stabilizing chemotherapeutic drug, could block the cell cycle at the G2/M

phase [9]. 5-Demethylnobiletin (DMN), a hydroxylated polymethoxyflavone from citrus plants, shows much higher cytotoxicity against NSCLC cell lines such as A549, H460 and H1299 cells [10,11]. Tan et al have reported the synergistic effects of PTX and DMN in lung cancer cells in vitro cytotoxicity and in vivo antitumor effect [12]. The combination of PTX and DMN makes the dosage of PTX lower, thus reducing its adverse effects [13]. However, the poor water solubility of PTX and DMN is still an obstacle for their application. In clinics, some drug products including paclitaxel bound albumin nanoparticles, CRLX101 (cyclodextran-poly-ethylene glycol polymer comprising camptothecin), Nanoplatin (micelle form of cisplatin) and Genexol-PM (micellar formulation of paclitaxel containing block of mPEG and D, L-PLA) have been approved by the FDA or in their clinical periods for treatment of NSCLC patients [13]. Even more, the progresses of nanoparticulate drug delivery in the field of combination therapy provides an effective solution to load two or more different mechanisms agents for controlled release, synergistic effects, lower side effects and drug resistance [14–16]. Herein, we designed and reported PTX and DMN

⁎ Corresponding author at: No.12 Jiankang Road, Department of Immuno-oncology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, 050011, Hebei Province, PR China. E-mail address: [email protected] (Z. Wang).

https://doi.org/10.1016/j.biopha.2019.109225 Received 14 May 2019; Received in revised form 9 July 2019; Accepted 10 July 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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DDAB (w/v) were dissolved in Milli-Q water (8 mL) to form aqueous phase. Aqueous phase was stirred (800 rpm) and the lipid phase was rapidly injected into the aqueous phase, resulting in a suspension of PTX/DMN-NLCs. The suspension was then dialyzed against Milli-Q water for 24 h, washed twice and filtered through a membrane (0.45 μm) to wash out the unloaded drugs and materials, filtered the larger particles to ensure a uniform nano-system. Then PTX/DMN-NLCs were lyophilized and stored at 2–8 °C. Single PTX or DMN loaded NLCs were prepared by the same method using PTX or DMN only and named PTX-NLCs and DMN-NLCs. Blank NLCs prepared by the same method using no drugs.

nanoparticles for NSCLC treatment. Combination therapy including chemotherapy and targeted therapy has become one of the important treatment options for advanced NSCLC patients. Epidermal growth factor receptor (EGFR) of NSCLC cells is a crucial target [17–19]. Cetuximab (CET), a monoclonal antibody that targets EGFR, has been approved as the first line of treatment for advanced colorectal cancer, NSCLC and head and neck cancers [20,21]. Nanosized drug delivery systems were widely used for systemic applications [22,23]. Nanostructured lipid carriers (NLCs) have a solid matrix blended with a liquid lipid to form an unstructured matrix that improved the drug loading capacity of nanoparticles and reduced drug expulsion from the matrix during storage [24]. In order to conquer the dose-related adverse effects, CET functionalized nanostructured lipid carriers (NLCs) were engineered to co-deliver PTX and DMN. Herein, we reported PTX and DMN co-loaded NLCs (PTX/DMNNLCs) and CET functionalized PTX/DMN-NLCs (CET-PTX/DMN-NLCs). The morphology, particle size, zeta potential, stability and drug release were tested. Cellular uptake, cell viability, synergistic effect and in vivo anti-tumor effect were evaluated on human lung adenocarcinoma cells, human embryonic lung cells and A549 paclitaxel-resistant cells bearing mice models.

2.3. Preparation of CET-PTX/DMN-NLCs CET-PTX/DMN-NLCs (Fig. 1) were developed by a reaction between thiolated CET and DSPE-PEG-Mal containing PTX/DMN-NLCs [26]. CET (1 equivalent) and Traut’s reagent (20 equivalents) were added in PBS and reacted for 1 h at room temperature under nitrogen. CET-PTX/ DMN-NLCs were prepared by incubating the thiolated CET with PTX/ DMN-NLCs for 20 h at 18 °C under nitrogen. CET-PTX/DMN-NLCs were purified through Sepharose CL-4B gel filtration. Absorbance of the eluates of CET-PTX/DMN-NLCs and free CET was measured using an enhanced BCA protein assay kit at 562 nm to confirm that the CET had been linked to the NLCs. Blank CET conjugated NLCs were prepared by the reaction between the CET and blank NLCs, named CET-NLCs.

2. Materials and methods 2.1. Materials CET was purchased from Sunrise Technology Development (Wuhan, China). PTX, DMN, Oleic acid (OL), soybean phosphatidylcholine (SPC), dimethyl formamide (DMF), coumarin-6 (C6), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Aldrich (St Louis, MO, USA). Traut’s reagent was provided by Thermo Scientific (Waltham, MA). COMPRITOL® 888 ATO (ATO) was provided by Gattefossé (Paramus, NJ, USA). DSPE-PEG2000Mal (DSPE-PEG-Mal) was obtained from Shanghai Ponsure Biotechnology Co., Ltd (Shanghai, China). Enhanced bicinchoninic acid (BCA) protein assay kit was obtained from Beyotime Biotechnology (Shanghai, China).

2.4. Characterization of morphology, particle size and zeta potential The prepared CET-PTX/DMN-NLCs or PTX/DMN-NLCs were dispersed air dried, stained with sodium phosphotungstate, and observed on a JEM transmission electronic microscopy (TEM) microscope (JEOL Ltd., Tokyo, Japan) [27]. The mean particle size, polydispersity index, and zeta potential were measured by NanoZS Zeta sizer (Malvern Instruments, Malvern, UK) [28]. 2.5. Entrapment efficiency and drug loading The entrapment efficiency (EE) and drug loading (DL) of drugs loaded NLCs was determined by measuring the concentration of free drugs in the aqueous phase of the NLCs dispersion and the total drug amount present in the system [29]. The NLCs were diluted (20 times) in mobile phase of acetonitrile and water (50/50, v/v) and samples (20 μL) were injected into the HPLC with a set of flow rate (1 mL/min) and injection volume (10 μL). The detection wavelength of the UV detector was 227 m (for PTX) or 330 nm (for DMN) [14,27]. The EE and DL were calculated using the following equations: EE (%) = (The total

2.2. Preparation of PTX/DMN-NLCs PTX/DMN-NLCs (Fig. 1) were prepared by solvent diffusion method [25]. Injectable soya lecithin (100 mg), PTX (50 mg), and DMN (50 mg) were dissolved in DMF (1 mL) to get lipid phase 1. SPC (100 mg) and ATO (100 mg) were dispersed in OL (1 mL) to form lipid phase 2. Lipid phase 1 was added to lipid phase 2 with heating at the temperature of 85 °C to prepare the lipid phase. DSPE-PEG-Mal (100 mg), and 0.5%

Fig. 1. Scheme graphs and TEM images of CET-PTX/DMN-NLCs and PTX/DMN-NLCs. PTX/DMN-NLCs were prepared by solvent diffusion method. CET-PTX/DMN-NLCs were developed by a reaction between thiolated CET and DSPE-PEG-Mal containing PTX/DMNNLCs The prepared CET-PTX/DMN-NLCs or PTX/DMN-NLCs were dispersed air dried, stained with sodium phosphotungstate, and observed on a JEM transmission electronic microscope.

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amount of drugs – the amount of free drugs)/The total amount of drugs × 100; DL (%) = (The total amount of drugs – the amount of free drugs)/The weight of the lipid phase × 100.

2.10. Animals and tumor xenografts Female BALB/c nude mice (16–19 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All animal experiments should comply with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978) and approved by the Animal and Ethics Review Committee of Hebei Medical University. The lung tumor xenografts were generated by subcutaneous injection of A549/PTX cells (5.0 × 106 cells in PBS) into the right flank of mice and tumors were allowed to reach approximately a volume of 100 mm3 before administration. During the whole in vivo experiments, tumor diameters were not allowed to exceed 12 mm and tumor weight must beneath 10% of the body weight of the mice.

2.6. Stability and drug release The stability of NLCs was examined for up to 90 days when stored at 2–8 °C [30]. The particle size and EE of NLCs were measured at predetermined time points. A dialysis method was applied to evaluate the drug release from NLCs using a membrane (3500 Da cutoff) in two kinds of release mediums (PBS of pH 7.4 and acetate buffer of pH 5.0) containing Tween 80 (1%, w/v) [31]. The concentrations of drugs released and EE during storage were determined by HPLC described in section 2.5.

2.11. In vivo anti-tumor effect 2.7. Cells and culture Lung tumor xenografts mice were randomly divided into the ten groups (six mice each group) and administrated intravenously through the tail vein with the following samples every three days: (1) CET-PTX/ DMN-NLCs (5 mg/kg of PTX and 5 mg/kg of DMN), (2) PTX/DMN-NLCs (5 mg/kg of PTX and 5 mg/kg of DMN), (3) PTX-NLCs (10 mg/kg of PTX), (4) DMN-NLCs (10 mg/kg of DMN), (5) CET-NLCs, (6) free PTX/ DMN (5 mg/kg of PTX and 5 mg/kg of DMN), (7) free PTX (10 mg/kg of PTX), (8) free DMN (10 mg/kg of DMN), (9) blank NLCs, (10) 0.9% saline control [38,39]. The tumor volumes and body weights of mice were recorded every three days [40]. Tumor volume (TV) was calculated according to the following formula: TV = (the length of the tumor) × (the width of the tumor)2 / 2. At the end of the experiment, all mice were scarified to remove the heart, lung, kidney, liver, spleen and the tumor to quantify drug distribution. They were washed twice with 0.9% saline solution, weighed, and homogenized with saline (containing 1 mM EDTA) to prepare 20% (w/v) homogenate solution. Then acetonitrile and water (50/50, v/v) was added to extract drugs. After centrifugation (5000 rpm, 10 min), the supernatants (20 μL) were injected onto the HPLC system and concentrations of drugs were determined by HPLC described in section 2.5.

A549 cells (human lung adenocarcinoma cells) and MRC-5 cells (human embryonic lung cells) were obtained from American Type Culture Collections (ATCC, Manassas, VA). A549/PTX cells (A549 paclitaxel-resistant cells) were purchased from Shanghai Meixuan Biological Science and Technology Co, Ltd (Shanghai, China). Cells were cultured in RPMI-1640 (5% CO2, 37 °C) and supplemented with 10% fetal bovine serum (FBS) [32].

2.8. Cellular uptake C6 loaded CET-PTX/DMN-NLCs or PTX/DMN-NLCs were prepared by the same method in section 2.2 by adding C6 (1 mg/mL of the lipid phase) into the lipid phase 1. A549 cells and MRC-5 cells were seeded at 24-well black plates (5 × 104 cells per well) separately and incubated until the cells reached 80% confluence. The culture medium was changed with C6 loaded NLCs (100 μL, 500 μg/mL) and incubated with the cells (37 °C, 4 h). Then, the cells were washed using PBS (pH 7.4) and photographed by fluorescence microscopy [33]. Cells were washed three times with D-Hank's solution, collected and centrifugated (1500 rpm, 5 min). The fluorescence of cells was analyzed using a flow cytometer [34].

2.12. Statistical analysis Statistical analysis was performed using SPSS 20.0 software (SPSS, Chicago, IL). All data are presented as mean ± the standard error of the mean (SEM). All experiments were repeated at least three times and the statistical significance of differences among groups was evaluated with Dunnett’s test subsequent to ANOVA. P < 0.05 was considered statistically significant.

2.9. Cell viability and synergistic effect Cell viability was estimated on A549/PTX cells using the MTT assay [35]. A549/PTX cells (4 × 103 cells per well) were seeded in 96-well plates and let them attach for 24 h at 37 °C. Then fresh medium with various concentrations of drugs loaded NLCs and free drugs (0.5 1, 5, 10, and 50 μg/mL) was introduced and incubated for 72 h. For single drug formulations, concentrations of PTX or DMN were presented as they are in the figure. For dual drugs containing formulations, concentrations of PTX or DMN were divided equally (for example 10 μg/mL free PTX/DMN means 5 μg/mL PTX and 5 μg/mL DMN). At the indicated time points, 20 μL MTT (5 mg/mL) reagent was added to each well and incubated at 37 °C for 4 h. Then, 150 μL of DMSO was added after the medium was discarded. The absorbance was measured at a wavelength of 570 nm. The drug concentration causing 50% inhibition (IC50) was calculated using the CalcuSyn software (Biosoft, Ferguson, MO). Combination index (CI) was measured to study the synergy in PTX and DMN in the drugs combination system on A549/PTX cells [36,37]. CI when the drug concentration causing 50% inhibition (CI50) was calculated as follows: CI50 = (D)PTX/(D50)PTX + (D)DMN/(D50)DMN. CI50 < 1 and > 1 represent synergism and antagonism, respectively. The CI50 values curves were drawn according to the fraction of affected cells, which are considered validate with the values between 0.2 and 0.8.

3. Results 3.1. Preparation of CET-PTX/DMN-NLCs The absorbance curves following elution of CET-PTX/DMN-NLCs and free CET were presented in Fig. 2. There is one peak during 10–18 min for free CET formulation, which represented free CET. There are two peaks separated using a BCA kit at 562 nm for CET-PTX/DMNNLCs formulation, one of which is overlapped with the peak of free CET, thereby demonstrating successful linking of CET to NLCs. 3.2. Characterization of NLCs Table 1 summarized the characteristics of NLCs. NLCs had sizes of around 130 nm and zeta potentials of +20-30 mV. The presence of CET did not enlarge the sizes, but reduced the surface charge. The polydispersity index of the NLCs was below 0.200. PTX and DMN EE of NLCs were about 90%. 3

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combination treatment with PTX and DMN synergistically decreases the viability of cells than the single PTX-NLCs and DMN-NLCs. Whether DMN could enhance the efficacy of PTX was further evaluated by CI50 according to the IC50 of the formulations (Fig. 6B). The effect of each drug combination treatment was compared with the effect of treatment with either agent alone. When the fraction of affected cells was between 0.2 and 0.8, all CI values of DMN plus PTX in the tested combination groups were < 1, which indicated that all of the tested combination groups showed a synergistic effect. The smaller the CI value was, the stronger the synergistic efficacy. It was evident that the best combination effect was achieved by CET-PTX/DMN-NLCs. 3.6. In vivo anti-tumor effect and tissue distribution To evaluate the in vivo anti-tumor effect of NLCs, lung cancer bearing mice were used and the tumor growth was individually monitored during the study (Fig. 7). Tumor growth in the 0.9% saline control group increased statistically in comparison with drugs treated groups (P < 0.05). In addition, antitumor effect for drugs loaded or CET contained NLCs was found statistically higher than for the free drugs formulations (P < 0.05). It was also observed that CET-NLCs alone could significantly inhibit tumor growth without loading drugs. On the other side, blank NLCs did not show any influence on tumor growth. Body weight of the NLCs groups increased slightly with time, while the free drugs groups showed decreases in body weight. In vivo tissue distribution results showed that CET-PTX/DMN-NLCs with CET led to a higher tumor accumulation of drugs compared to other NLCs and the free drugs (P < 0.05) (Fig. 8). In addition, the drug concentrations in heart and kidney of NLCs were lower than that of free drugs formulations (P < 0.05). These results likely explained the higher delay in tumor growth found in mice treated with CET-PTX/ DMN-NLCs.

Fig. 2. The absorbance curves following elution of CET-PTX/DMN-NLCs and free CET. There is one peak during 10–18 min for free CET formulation, which represented free CET. There are two peaks separated using a BCA kit at 562 nm for CET-PTX/DMN-NLCs formulation, one of which is overlapped with the peak of free CET, thereby demonstrating successful linking of CET to NLCs.

3.3. Stability and drug release NLCs were lyophilized and stored at 2–8 °C. The mean particle size and EE were evaluated for 90 days to evaluate the storage stability. Fig. 3 showed that slight variations were found during and after the storage period with no significant change. The release of drugs from NLCs was relatively fast at the first 12 h and then became slow until completion of sustained release behavior (Fig. 4). The release profiles of PTX and DMN showed no obvious difference at pH 7.4. Also the release at pH 5.0 showed no difference with the profiles at pH 7.4 (data not shown). The release of PTX and DMN followed Higuchi models.

4. Discussion Antibodies have been used extensively for the targeting of nanocarriers [41–43]. For an example of CET, Zalba et al introduced CEToxaliplatin-liposomes for epidermal growth factor receptor targeted chemotherapy of colorectal cancer [44]. However, CET contained NLCs for the co-delivery of PTX and DMN has not been reported. Particle sizes of NLCs revealed that they are nano-sized particle with homogeneous diameter at around 130 nm. TEM images further confirmed that NLCs was spheroid nanoparticles with uniform sizes. It was noted that a change in zeta potential was decreased from 28.1 to 19.5 mV when conjugated with CET. These results were in accordance with the results reported by Zhang et al with similar tendency [45]. In their research, the zeta potential of nanoparticles decreased from positive to neutral with the successful conjugation of CET. The physical stability of NLCs was evaluated after storage at 2–8 °C. Tan et al observed that the nanoparticles were stable for up to 30 days [46]. In this study, no significant change was noted in size and EE for 90 days, indicating the storage stability of the systems. In vitro drug release of the drug loaded NPs may be controlled by the erosion, corrosion, and

3.4. Cellular uptake The cellular uptake efficiency results are displayed in Fig. 5. The result clearly showed that A549 cells uptake of CET-PTX/DMN-NLCs (65.8%) was remarkably higher than that of PTX/DMN-NLCs (35.5%) (P < 0.05), which indicated that CET has the potential to improve the uptake of the NLCs. On the contrary, no obvious difference was found on the uptake of MRC-5 cells, which may prove the targeting of CET to the cancer cells but not the normal cells. 3.5. Cell viability and synergistic effect Single PTX or DMN loaded NLCs decreases the viability of A549/ PTX cancer cells in a concentration-dependent manner (Fig. 6A). The

Table 1 The mean particle size, size distribution, surface charge, EE, and DL of NLCs (Mean ± SD, n = 6). Formulations

CET-PTX/DMN-NLCs PTX/DMN-NLCs PTX-NLCs DMN-NLCs CET-NLCs Blank NLCs

Particle sizes (nm)

131.6 129.7 127.9 130.1 131.2 128.9

± ± ± ± ± ±

3.5 3.9 3.2 3.5 2.9 3.2

Polydispersity index

0.131 0.127 0.121 0.125 0.118 0.129

± ± ± ± ± ±

0.026 0.029 0.019 0.018 0.0185 0.021

Zeta potentials (mV)

+19.5 +28.1 +28.9 +29.4 +20.7 +30.3

4

± ± ± ± ± ±

2.7 2.9 2.3 2.1 2.6 1.9

EE (%)

DL (%)

PTX

DMN

PTX

DMN

90.5 ± 4.2 91.3 ± 3.2 89.7 ± 3.6 / / /

91.1 ± 3.7 90.9 ± 2.9 / 91.5 ± 3.3 / /

9.6 ± 0.9 8.8 ± 0.7 10.2 ± 1.0 / / /

8.9 ± 0.7 9.2 ± 0.8 / 9.6 ± 0.9 / /

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Fig. 3. The mean particle size and EE evaluated for 90 days to evaluate the storage stability. Mean ± SEM, n = 8. The mean particle size and EE showed slight variations during and after the storage period with no significant change.

Fig. 4. The release profiles of PTX (A) and DMN (B) from NLCs. Mean ± SEM, n = 8. The release of drugs from NLCs was relatively fast at the first 12 h and then became slow until completion of sustained release behavior. Fig. 5. The cellular uptake efficiency of NLCs. Mean ± SEM, n = 8. * P < 0.05. C6 loaded CET-PTX/DMN-NLCs or PTX/DMN-NLCs were prepared and incubated with A549 cells and MRC-5 cells. Then, the cells were photographed by fluorescence microscopy (A) and the fluorescence of cells was analyzed using a flow cytometer (B). In Fig. 5 B, a: CET-PTX/ DMN-NLCs incubated with A549 cells, b: PTX/ DMN-NLCs incubated with A549 cells, c: CETPTX/DMN-NLCs incubated with MRC-5 cells, d: PTX/DMN-NLCs incubated with MRC-5 cells.

Internalization of nanoparticles in cancer cells more or less reflects the therapeutic effect [49]. In this study, the fluorescent marker C6 was applied to visualize cellular uptake of NLCs. The green fluorescence of C6 loaded NLCs was internalized by cancer cells and directly visualized and quantified. Cellular uptake efficiency of CET-PTX/DMN-NLCs was remarkably higher than that of PTX/DMN-NLCs, which indicated that CET has the potential to improve the uptake of the NLCs. These results indicated that nanocarrier-based formulations effectively avoided p-

diffusion process [47]. Nanocarriers could achieve drug depot effects, which would lead to the sustained release of hydrophobic drugs. To evaluate the release behavior of drugs loaded in the NLCs and study whether conjugation of CET has effects on the release of drugs, the drugs released from NLCs were quantitatively analyzed and expressed as a function of time. The release profiles of CET-PTX/DMN-NLCs and non CET NLCs showed no obvious difference in drug release profiles, which illustrated that CET did not affect the drug release [48]. 5

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Fig. 6. Cell viability of NLCs estimated using the MTT assay (A) and synergistic effect evaluated by CI50 (B). Mean ± SEM, n = 8. * P < 0.05. Single PTX or DMN loaded NLCs decreases the viability of A549/PTX cancer cells in a concentration-dependent manner (Fig. 6A). For single drug formulations, concentrations of PTX or DMN were presented as they are in the figure. For dual drugs containing formulations, concentrations of PTX or DMN were divided equally (for example 10 μg/mL free PTX/DMN means 5 μg/mL PTX and 5 μg/mL DMN). The combination treatment with PTX and DMN synergistically decreases the viability of cells than the single PTX-NLCs and DMN-NLCs. When the fraction of affected cells was between 0.2 and 0.8, all CI values of DMN plus PTX in the tested combination groups were < 1, which indicated that all of the tested combination groups showed a synergistic effect.

Fig. 7. In vivo anti-tumor effect of NLCs evaluated on lung cancer bearing mice in terms of tumor volume changes (A), tumor images (B), and body weight changes (C). Mean ± SEM, n = 8. * P < 0.05. The antitumor effect for drugs loaded or CET contained NLCs was found statistically higher than for the free drugs formulations. Body weight of the NLCs groups increased slightly with time, while the free drugs groups showed decrease in body weight.

Fig. 8. In vivo PTX (A) and DMN (B) tissue distribution results of NLCs evaluated on lung cancer bearing mice. Mean ± SEM, n = 8. * P < 0.05. In vivo tissue distribution results showed that CET-PTX/DMN-NLCs with CET led to a higher tumor accumulation of drugs compared to other NLCs and the free drugs. In addition, the drug concentration in heart and kidney of NLCs were lower than that of free drugs formulations.

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Acknowledgement

gpmediated efflux, thus offering an approach for reversing multidrug resistance [50]. On the contrary, no obvious difference was found on the uptake of MRC-5 cells, which may prove that CET was targeted to the cancer cells but not the normal cells. Anticancer therapies work by the induction of apoptosis in cancer cells without damaging the surrounding normal cells [37]. The results showed that DMN by itself did not have a strong effect on apoptosis; however, when combined with PTX, the fraction of cells in early and late apoptosis increased, along with a significant increase in the necrotic cells, corroborating the synergistic anticancer effect. The use of multiple drugs in combination has possible favorable outcomes, such as synergism, additive and antagonism [51]. Evaluation of drug-drug interaction is important in the areas of cancer chemotherapy. In order to determine the possible effect of the drug combination mathematical model based method has been introduced. The combination index (CI) values of PTX plus DMN were evaluated by the median-effect method proposed by Chou and Talalay [52]. All CI values of DMN plus PTX in the tested combination groups were < 1, which indicated that all of the tested combination groups showed a synergistic effect. The smaller the CI value was, the stronger the synergistic efficacy. CET-PTX/DMN-NLCs had better ability and showed obvious synergism effect than PTX/DMNNLCs and free PTX/DMN. In vivo antitumor efficiency study revealed that the antitumor effect for drugs loaded or CET contained NLCs was found statistically higher than for the free drugs formulations. These could be explained by the SLNs system constructed could exhibit high structural integrity, stability during use, and sustained release capability [53]. Also, the structure of NLCs has a higher affinity to the lipid structured cell surface, which can promote the fusion of the system to the cell membrane and deliver drugs more efficiently into the tumor site. It was also observed that CET-NLCs alone could significantly inhibit tumor growth without loading drugs. Similar result has also been reported by Li et al [54], which found that nanoparticles in combination with CET showed stronger inhibition of tumor growth under the same conditions than non CET contained systems. PTX/DMN-NLCs demonstrated stronger antitumor effect than PTX-NLCs and DMN-NLCs. This may be explained by the co-delivery of two drugs could get the best anti-tumor effect due to the synergetic effect of the two drugs, which is in accordance with the observation of Li et al [55]. In the study carried out by Zhu et al [56], double drugs loaded nanoparticles improved the anticancer activity than the free drugs. In the meanwhile, a lower toxicity was obtained which could be observed by the body weight loss of the animals. This point is the same with our study and such increased anticancer activity and less toxicity may be explained by the biodistribution data. In vivo tissue distribution results showed that CET-PTX/DMN-NLCs with CET led to a higher tumor accumulation of drugs compared to other NLCs and the free drugs. In addition, the drug concentrations of NLCs in the heart and kidney were lower than that of free drugs formulations. Lu et al pointed out that the administration of modified NLCs could lead to a dramatic increase of drug accumulation in the tumor tissue, in comparison with the free drugs solutions [57]. Distribution mainly in tumor tissue compared with the other tissues could decrease the side effects and lead to better anti-tumor therapeutic efficiency. The same conclusion was got in this section and CET-PTX/DMN-NLCs could be used as a promising system for the synergistic combination therapy of lung cancer.

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