Etoposide-loaded nanostructured lipid carriers for gastric cancer therapy

Abstract

Purpose: Gastric carcinoma is one of the most common cancers and the second most frequent cause of cancer-related deaths. The aim of this study was to prepare and characterize etoposide-loaded nanostructured lipid carriers (ETP-NLCs) and evaluate their antitumor activity in vitro and in vivo.

Methods: Novel ETP-NLCs were constructed. The physicochemical properties of the ETP-NLCs were investigated by particle-size analysis, zeta potential measurement, drug loading, drug entrapment efficiency, stability and in vitro drug release behavior. In vitro cytotoxicity against human gastric cancer cells (SGC7901 cells) was investigated, and in vivo antitumor of NLCs was evaluated on mice bearing SGC7901 cells xenografts.

Results: ETP-NLCs have a narrow size distribution at 91 nm, a zeta potential value of +23.1 mV, high drug entrapment efficiency of 78%. The drug release of ETP-NLCs exhibited a sustained behavior, which made it an ideal vehicle for drug delivery. Furthermore, ETP-NLCs could significantly enhance in vitro cytotoxicity and in vivo antitumor effect against SGC7901 cells and gastric cancer animal model compared to the free drug.

Conclusion: The results demonstrated that the NLCs might be a promising nanomedicine for the treatment of gastric carcinoma.

• Gastric cancer therapy
• in vivo drug delivery
• in vitro cytotoxicity
• nanostructured lipid carriers

Introduction

Gastric carcinoma (GC) is one of the fourth most common cancers worldwide and the second leading cause of cancer-related deaths in eastern Asia (Jemal et al., 2011; Guggenheim & Shah, 2013; Zhang et al., 2014). Although surgery is a main method for GC therapy, chemotherapy is the supplemented treatment for patients after surgery and the standard treatment for advanced GC since it provides a survival benefit and improved quality of life compared with best supportive care alone (Cervantes et al., 2013; Lordick et al., 2014). Depending on NCCN guidelines for GC in 2012, five classes of cytotoxic agents are currently used in GC (fluoropyrimidines, platinums, taxanes, topoisomerase inhibitors and anthracyclines).

Etoposide (ETP), a semisynthetic derivative of podophyllotoxin, is the inhibitor of deoxyribonucleic acid (DNA) topoisomerase 2 (Qin et al., 2013). Its main effect appears to be at the G₂ portion of the cell cycle in mammalian cells. Thus, ETP has been widely used for various cancers such as GC, small cell lung carcinoma and germ cell tumors (Johnson et al., 1998). Because of its low solubility, poor bioavailability and drug resistance, it is urgent to engineer a drug delivery system to overcome these drawbacks and improve the clinical therapy effect (Joel et al., 1995; Lorico et al., 1997; Wang et al., 2014).

Lipid-based nanoparticles can be considered as a versatile tool with a high potential of applications, including a high capacity to incorporate lipophilic and hydrophilic drugs, a biocompatible and biodegradable matrix and well-established safety profiles (Esposito et al., 2015; Liu et al., 2015). They combine advantages of colloidal drug carrier systems such as emulsions, liposomes and polymeric nanoparticles. Nanostructured lipid carriers (NLCs), the latest generation of lipid-based nanoparticles, were introduced in the late 1990s. NLCs were composed of a binary mixture of a solid lipid and a liquid lipid (Madane & Mahajan, 2014). Given their structure and characteristics, NLCs can be considered as preferable nanovectors to load poor soluble drugs; increase drug loading and longer assure molecule stability (Shidhaye et al., 2008; Han et al., 2015; Shao et al., 2015).

In this article, novel ETP-NLCs were designed. In vitro cytotoxicity against SGC7901 cells was investigated, and in vivo antitumor of NLCs was evaluated on mice bearing SGC7901 cells xenografts. This system was anticipated to achieve stable, and controlled release ETP-NLCs, to improve anticancer effects, and to reduce toxicity.

Materials and methods

Materials

ETP was provided by Qilu Pharmaceutical Co. Ltd. (Ji'nan, China). Glycerol monostearate (GM), soybean phosphatidylcholine (SPC), oleic acid and (3-[4,5-dimehyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Labrafac PG (propylene glycol dicaprylocaprate) was a kind offer from Gattefossé (Gennevilliers, France). 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) were obtained from Avanti Polar Lipids (Alabaster, AL). All other chemicals were of analytical grade or high performance liquid chromatography (HPLC) grade.

Cells and animals

SGC7901 cells were obtained from the American type culture collection (ATCC, Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Fisher Chemicals, Fairlawn, NJ) in a 5% CO₂ fully humidified atmosphere.

BALB/c nude mice (4–6 weeks old, 18–22 g weight) were purchased from Medical Animal Test Center of Shandong Province (Ji'nan, China), and were maintained under specific pathogen-free conditions.

Construction of ETP-NLCs

ETP-NLCs were constructed by using the solvent injection technique (Tiwari & Pathak, 2011). Briefy, ETP, GM, SPC and oleic acid were dissolved in isopropyl alcohol with heating at 80 °C. The resulting solution was rapidly injected into the aqueous phase containing DOTAP as that was continuously stirred at 600 rpm for 30 min. Thereafter, the dispersion was centrifuged at 10 000 rpm for 10 min and aggregates were resuspended in double distilled water. Blank NLCs not containing ETP were prepared using the same method without adding the drug.

Particle size and zeta potential of ETP-NLCs

The particle size distribution and the zeta potential of the prepared NLCs were measured by using Malvern ZetaSizer NanoSeries (Malvern Instruments Ltd., Worcestershire, UK) according to the manufacturer's instructions (Taratula et al., 2013).

Determination of drug loading of ETP-NLCs

The amount of entrapped ETP was determined by using UV–vis method (Wang et al., 2014). A sample containing a standard weight of ETP-NLCs was dissolved by adding a specific amount of ethanol. After the sample was completely dissolved, the concentration of the ETP was determined with a UV–vis spectrophotometer. The selected wavelength for ETP measurement was 285 nm. The concentration of the ETP was calculated according to an already-obtained calibrating curve.

The drug loading (DL) and drug entrapment efficiency (EE) were calculated as follows:

In vitro ETP release from ETP-NLCs

In vitro release of ETP from ETP-NLCs and ETP solution were assessed by using the dialysis method (Shao et al., 2015). Briefly, The ETP-NLCs or ETP solution was placed in the dialysis bag separately. Then, the bag was incubated with 50 ml of release medium at 37 °C (0.1% Tween 80 in PBS, pH 7.4). One milliliter of medium was collected at predetermined time points and replaced with 50 ml of fresh medium. The concentrations of released ETP were determined by using the UV–vis method mentioned above.

Cell viability study

The SGC7901 cells were treated with ETP-NLCs, free ETP solution (ETP) and blank NLCs (Liu et al., 2011). The cells were seeded in a 96-well plate a density of 4000 cells/well and allowed to adhere for 24 h prior to the assay. The cells were then treated with 0.2 ml serum free medium containing various concentrations of NLCs. After 72 h of incubation, 20 μl of MTT solution (5 mg/ml) was added to each well of the plate. After incubation for 4 h, 200 μl/well of DMSO was added to dissolve the contents in the plate. Relative viability was obtained from the absorbance at 590 nm of the treated SGC7901 cells divided by the absorbance at 590 nm of the untreated cells.

In vivo antitumor effects

The antitumor effects of NLCs were investigated in gastric tumor-bearing BALB/c nude mice models (Jin et al., 2013). BALB/c mice were inoculating (s.c.) subcutaneously in the left armpit with SGC7901 cells suspended in PBS. When tumor volume reached about 100 mm³, transplanted mice were randomly divided into four groups separately. ETP-NLCs, ETP, NLCs and 0.9% saline were injected through the tail vein of the mice of each group, once every 4 days. Twenty-four days later, all the mice were sacrificed by cervical dislocation and the tumor tissue samples were taken out.

Tumor volume of each mouse was measured with a digital caliper every 4 days, and was calculated according to the below equation:

Tumor inhibition rates (TIR) of ETP-NLCs and ETP were calculated using the below equation: where W_t and W_c represent the mean tumor weight of the treated and control groups, respectively.

Statistical analysis

All studies were repeated three times and all measurements were carried out in triplicate. Results were reported as means ± SD (SD, standard deviation). Statistical significance was tested by two-tailed Student's t-test. Differences between experimental groups were considered significant when the p value was less than 0.05 (p < 0.05).

Results and discussion

Physicochemical properties of NLCs

Physicochemical properties of NLCs are summarized in Table 1. The size of both ETP-NLCs and blank NLCs was about 90 nm. This could be the evidence that the adding of ETP do not affect the size of the NLCs formulations. Particle size is a key influence factor of the carriers for cancer therapy, the in vitro and in vivo efficiency of the carriers could be controlled by the small size of the NLCs (Sun et al., 2014). The zeta potential of ETP-NLCs was + 23 mV. Positive surface would let the NLCs easily absorbed onto the passive charged cell surface. The DL and EE of ETP-NLCs were around 32% and 78%, respectively. The high EE achieved could offer advantages in the drug delivery and therapeutic effect for cancer.

Table 1. Physicochemical properties of NLCs.

Samples	Blank NLCs	ETP-NLCs
Particle size (nm)	90.1 ± 2.5	91.3 ± 2.8
Size distribution (PDI)	0.6 ± 0.01	0.12 ± 0.03
Zeta potential (mV)	+21.5 ± 2.7	+23.1 ± 3.3
DL (%)	–	31.6 ± 1.4
EE (%)	–	78.4 ± 3.6

In vitro ETP release from ETP-NLCs

The in vitro release profiles of ETP from ETP-NLCs and ETP solution are depicted in Figure 1. For ETP solution, ETP release was so fast and completed at about 4 h. ETP showed the sustained-release behavior from ETP-NLCs at the studied 48 h, nearly complete accumulated releases were achieved at 36 h. The drug-release mechanism could belong to drug diffusion, lipid matrix swelling and the carrier erosion or degradation. The initial fast drug release (first 8 h) could be ascribed to those drugs located on or near the surface of NLCs, while the slow and sustained release could be attributed to the diffusion of drug molecules through the lipid matrix. Burst release can be useful for improving the penetration of a drug, while sustained release can be beneficial for drugs with irritation effects at high concentrations.

Figure 1. In vitro release profiles of ETP from ETP-NLCs or ETP solutions.

Cell viability study

The ability of ETP-NLCs, ETP and NLCs was evaluated on SGC7901 cells using MTT assay. The IC₅₀ values of ETP-NLCs and ETP were 6.3 and 56.5 μg/ml, respectively. The IC₅₀ value of ETP-NLCs was ninefold than ETP solution. ETP-NLCs showed significantly higher inhibition rates and obviously higher suppression efficiency than free ETP solution. It could be explained that good dispersity and stability of NLCs formula could facilitate greater cellular uptake. These results could be the evidence of the better delivery effect of the drug-loaded NLCs than their free drug solution counterparts.

In vivo antitumor efficacy

Gastric cancer bearing BALB/c nude mice models was used for the evaluation of the antitumor efficacy of ETP-NLCs in vivo. The tumor growth inhibitions over different groups were monitored for 24 days. Figure 2 illustrates that the increase of tumor volume was significantly inhibited by ETP-NLCs than ETP solution groups (p < 0.05). The blank NLCs did not have any impact on tumor inhibition (p > 0.05). The tumor volume of ETP-NLCs group was 368 mm³ after 24 days of treatment, smaller than control (0.9% saline) group which reached 1710 mm³. The tumor volume of ETP solution group and blank NLCs group reached 1030 and 1651 mm³, respectively. The TIR of ETP-NLCs and ETP solution were 78% and 40%, respectively, compared with control. ETP-NLCs inhibited tumor growth two times higher than that treated with ETP solution.

Figure 2. In vivo anticancer efficiency of ETP-NLCs.

Moreover, noticeable body weight losses were observed in ETP solution group and reduction in food intake, energy sag and inactive in moving were observed during the test. In contrast, no significant body weight loss was observed in ETP-NLCs group, demonstrating the reduced systemic toxicity by NLCs formulation. The ETP-NLCs showed far better antitumor activity than free ETP on the treatment of gastric cancer in vivo.

Conclusions

Novel ETP-NLCs were constructed. ETP-NLCs have a narrow size distribution and high drug entrapment efficiency. The drug release of ETP-NLCs exhibited a sustained behavior, which made it an ideal vehicle for drug delivery. ETP-NLCs could significantly enhance in vitro cytotoxicity and in vivo antitumor effect against SGC7901 cells and gastric cancer animal model compared to the free drug. It could be concluded that the resulting NLCs might be a promising nanomedicine for the treatment of gastric carcinoma.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.