Nanostructured lipid carriers based temozolomide and gene co-encapsulated nanomedicine for gliomatosis cerebri combination therapy

Abstract

Background: Co-delivery of gene and anticancer drug into the same cancer cells or tissues by multifunctional nanocarriers may provide a new paradigm in cancer treatment. In this study, nanostructured lipid carriers (NLCs) were constructed as multifunctional nanomedicine for co-delivery of enhanced green fluorescence protein plasmid (DNA) and temozolomide (TMZ).

Methods: TMZ- and DNA-loaded NLCs (TMZ/DNA-NLCs) were prepared. Their particle size, zeta potential, gene-loading capacity (GL) and drug encapsulation efficiency (EE) were evaluated. In vitro cytotoxicity study TMZ/DNA-NLCs was tested in U87 malignant glioma cells (U87 MG cells). In vivo gene transfection and anti-tumor efficacy of the carriers were evaluated on mice bearing malignant glioma model.

Results: The optimum TMZ/DNA-NLCs formulations with the particle size of 179 nm and with a +23 mV surface charge; got 91% of GL and 83% of EE. The growth of U87 MG cells in vitro was obviously inhibited. TMZ/DNA-NLCs also displayed the highest gene transfection efficiency and the best antitumor activity than other formulations in vivo.

Conclusion: The results demonstrated that TMZ/DNA-NLCs were efficient in selective delivery to malignant glioma cells. Also TMZ/DNA-NLCs transfer both drug and gene to the gliomatosis cerebri, enhance the antitumor capacity and gene transfection efficacy. Thus, TMZ/DNA-NLCs could prove to be a superior co-delivery nanomedicine to achieve therapeutic efficacy and this report could be a new promising strategy for treatment in malignant gliomatosis cerebri.

• Combination therapy
• gene therapy
• gliomatosis cerebri
• nanostructured lipid carriers

Introduction

Gliomatosis cerebri (GC) is one of the most common primary brain tumor and may occur in patients of any age (Orza et al., 2013; Lee et al., 2014). Malignant gliomas are responsible for ∼11 000 patient deaths per year (Shapiro & Shapiro, 1998). Current therapy includes surgical intervention, radiotherapy and chemotherapy with temozolomide (TMZ) (Messaoudi et al., 2014). The standard of care for patients diagnosed with high-grade malignant glioma includes postoperative TMZ adjuvant to radiation (Buie & Valgus, 2012). However, this therapeutic strategy is associated with considerable toxicity and limited efficacy. Median survival is 14 months and the percentage of patients living for 5 years or more is <10% (Stupp et al., 2009).

To improve intracranial delivery, chemotherapeutic concentrations may be increased or the blood–brain barrier (BBB) temporarily opened. While such techniques increase the killing of tumor cells, they also result in an increase in normal tissue toxicity (Bernal et al., 2014). On one hand, gene therapy is recognized to be a novel method for the treatment of cancers and could overcome multi-drug resistance which is one of the most important side effects for chemotherapeutic agents (Podolska et al., 2012). On the other hand, co-delivery systems simultaneously transporting gene and antitumor drug into the same tumor cells or tissues by multifunctional nanocarriers may provide a new paradigm in cancer treatment, owing to their low toxicity and high therapy efficiency (Bao et al., 2014). Several works have been reported on gene and drug co-delivery for cancer therapy (Dong et al., 2013; Liu et al., 2013; Zheng et al., 2013; Ma et al., 2014).

So far, attempts have been made to simultaneously deliver genes and drugs into cancer cells, using polymeric nanoparticles (Liu et al., 2014), micelles (Morishima & Sato, 2014), nanoemulsions (Lee et al., 2014), dendrimers (Kaneshiro & Lu, 2009) and lipid nanoparticles to bring about satisfactory therapeutic efficiency (Tiwari & Pathak, 2011). In addition, many studies have shown that nano-sized delivery systems can reduce toxicity of drugs/genes (Shi et al., 2013; Taratula et al., 2013; Han et al., 2014). It has been reported that lipid nanoparticles improve the drug absorption and bioavailability due to their diameter and absorption enhancing effect of lipids (Hejri et al., 2013). Nanostructured lipid carriers (NLCs) represent an improved generation of lipid nanoparticles (Yuan et al., 2013). They are produced by controlled mixing of solid lipids with spatially incompatible liquid lipids, leading to a specific nanostructure to accommodate drugs/genes, and thus achieve higher loading capacity (Muller et al., 2002; Saupe et al., 2005).

Accordingly, we developed a TMZ and gene-loaded NLCs (TMZ/DNA-NLCs) and examined in mice bearing glioma xenografts. This system was expected to achieve stable gene and drug loading capacity, effectively reduce the growth of glioma xenografts and extend survival of tumor bearing animals.

Materials and methods

Materials

TMZ was kindly provided by Laimei Pharmaceutical Co., Ltd. Stearic acid, dimethyldioctadecylammonium bromide (DDAB), polyoxyethylene bisamine (H₂N-PEG-NH₂, molecular weight (MW) 3350), soybean phosphatidylcholine (SPC) and Tween-80, (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO). Distearoyl phosphatidylethanolamine (DSPE) was purchased from Avanti Polar Lipids (Alabaster, AL). COMPRITOL® 888 ATO (888 ATO) was generously provided by Gattefossé (Paramus, NJ). Polyoxyl castor oil (Cremophor ELP) was donated by BASF (Ludwigshafen, Germany). Injectable soya lecithin was obtained from Shanghai Taiwei Pharmaceutical Co., Ltd (Shanghai, China). The plasmid DNA encoding the enhanced green fluorescence protein (pEGFP) was provided by Zhejiang University (Zhejiang, China). Lipofectamine™ 2000, Quant-iT™ PicoGreen® dsDNA quantitation reagent was obtained from Invitrogen by Life Technologies (Carlsbad, CA). All other chemicals were of analytical grade or higher.

Animals and cells

U87 malignant glioma cells (U87 MG cells) were obtained from the American type culture collection (Manassas, VA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Fisher Chemicals, Fairlawn, NJ) in a 5% CO₂ fully humidified atmosphere.

BALB/c nude mice (5–6-week-old, 18–22 g) were purchased from the Shanghai Slack Laboratory Animal Co., Ltd. All animal experiments complied with the Animal Management Rules of the Ministry of Health of the People's Republic of China.

Preparation of NLCs

TMZ/DNA-NLCs, TMZ-NLCs, DNA-NLCs and blank NLCs were prepared by following steps.

NLCs were prepared by solvent diffusion method (Table 1). In brief, the lipid dispersion was composed of 888 ATO, Cremophor ELP and SPC at a ratio of 2:1:1 (w/w/w). Injectable soya lecithin and TMZ were dissolved in 1 mL of dimethyl formamide (DMF) and added to the lipid dispersion with heating at the temperature of 80–85 °C to form the lipid phase. Aqueous phase was prepared by dissolving DNA, Tween-80 and DDAB in 10 mL of water. This aqueous solution was then stirred and heated to 30 °C. The lipid phase was rapidly injected into the stirred aqueous phase (800 rpm) at 30 °C, and the resulting suspension was then dissolved with Milli-Q water and then dialyzed against Milli-Q water for 24 h to get the TMZ/DNA NLCs. TMZ-NLCs and DNA-NLCs were prepared using the same method using the single agent of TMZ or DNA. Blank NLCs were prepared using the same method without adding TMZ and DNA.

Table 1. NLCs composition.

Lipid phase	888 ATO/Cremophor ELP/SPC	Soya lecithin	TMZ
Amount (mg)	200/100/100	100	100
Aqueous phase	DNA	Tween-80	DDAB
Amount (mg)	100	20	10

Preparation of Lipofectamine™ 2000/DNA

Lipofectamine™ 2000/DNA formulation were prepared according to the manufacturer's instructions for Lipofectamine™ 2000. Briefly, 100 µL of pEGFP (1 µg/µL) was mixed with 200 µL of Lipofectamine™ 2000 by vortexing for 30 s. The mixture was incubated for 30 min at room temperature to facilitate the formation of the Lipofectamine™ 2000/DNA (Kong et al., 2012).

Characterization of the NLCs

The mean particle size, size distribution and zeta potential of TMZ/DNA-NLCs, TMZ-NLCs, DNA-NLCs and blank NLCs were determined by using the Malvern Zetasizer Nano ZS (Malvern Instrument Ltd., Worcestershire, UK). The average particle size was expressed as volume mean diameter.

PicoGreen-fluorometry assay was used to determine the gene-loading capacity (GL) of TMZ/DNA-NLCs, and DNA-NLCs, by measuring the fluorescence and comparing with the supernatant from TMZ-NLCs, and blank NLCs. GL was calculated according to the linear calibration curve of pEGFP: GL (%) = (total amount of pEGFP–the amount of free pEGFP)/(total amount of pEGFP) × 100.

The drug encapsulation efficiency (EE) of TMZ/DNA-NLCs, and TMZ-NLCs formulations were measured by using the HITACHI P-4010 inductively coupled plasma mass spectrometry (ICP-MS) (Hitachi Ltd, Kyoto, Japan). Briefly, 5 mL NLCs were centrifuged (16 000 rpm and 4 °C for 30 min) separately, and the supernatants were then determined using the ICP-MS. The EE was expressed as the percentage of the amount of TMZ encapsulated in the NLCs to the total amount of TMZ initially used. EE were calculated as EE (%) = concentration of (total TMZ−free TMZ)/concentration of total TMZ × 100.

In vitro cytotoxicity study

The cytotoxicity of TMZ/DNA-NLCs was tested in U87 MG cells using the MTT assay (Choi, 2014). Briefly, cells were seeded in a 96-well plate at a density of 3000 cells/well and allowed to adhere for 24 h prior to the assay. Cells were exposed to various concentrations of free TMZ solution, blank NLCs, TMZ-NLCs, DNA-NLCs and TMZ/DNA-NLCs, respectively. Culture medium was used as a blank control. After 48 h of incubation, MTT solution (5 mg/mL) was added to each well and the cells were incubated for another 4 h. Cellular viability was assessed according to the MTT manufacturer's procedures and the absorbance at 570 nm was measured using a microplate reader (Model 680, Bio-Rad Laboratories Inc., Philadelphia, PA). Cells without the addition of MTT reagents were used as a blank control. The drug concentration causing 50% inhibition (IC₅₀) was calculated using the CalcuSyn software (Biosoft, Ferguson, MO) (Casagrande et al., 2013).

In vivo therapeutic studies

pEGFP was employed as the model gene to examine transfection efficiency of TMZ/DNA-NLCs. BALB/c nude mice were housed at a temperature of 25 ± 2 °C and a relative humidity of 70 ± 5% under natural light/dark conditions for one week before dosing. Then the mice were inoculating subcutaneously (s.c.) in the right armpit with U87 MG cells suspended in PBS for 24 h for the preparation of malignant glioma bearing animal models. Mice were then divided into seven groups (six mice per group). The TMZ/DNA-NLCs, TMZ-NLCs, DNA-NLCs, Lipofectamine™ 2000/DNA, naked DNA solution (300 µL pre-injection), free TMZ solution and 0.9% sodium chloride solution were prepared and then injected intravenously into the mice via the tail vein (i.v.).

In vivo gene transfection analysis

After 48 or 72 h after i.v. injection, mice were sacrificed and the tumor tissue samples were taken out. The tumor tissues were homogenized by pressing the samples through a 30 μm cell mesh with the plunger of a 5 mL syringe. Erythrocyte lysis buffer was then added during homogenization to lyse the red blood cells. The homogenates were washed with PBS containing 0.5% bovine serum albumin and then filtered. The cells were finally obtained after centrifugation (3000 rpm and 4 °C for 10 min) and were seeded into 48-well plates in 1 mL of DMEM with 10% FBS. The quantitative study of the transfected cells was carried out as follows: The cells were washed with 1 mL of PBS and were detached with trypsin/EDTA. The supernatant was discarded and re-suspended with 300 μL of PBS and added into the flow cytometry to quantitate the amount of U87 MG cells that have been successfully transfected.

In vivo anti-tumor efficacy

The initial day of i.v. administration was defined as Day 0, and administration was then repeated once every 3 days over a 15-day therapeutic period. The body weights of mice and tumor sizes were also measured tumor growth was determined by caliper measurement every 3 days. The measurements were taken in two perpendicular dimensions and tumor volumes (mm³) were calculated by applying the formula (L × W²)/2, where L is the longest dimension and W is the dimension perpendicular to L (Sarisozen et al., 2014). The antitumor efficacy of each formulation was evaluated by tumor inhibition rate, and was calculated using the following formula: tumor inhibition rate (%) = (W_c–W_t)/W_c × 100. W_t and W_c represent the mean tumor weight of the treated and control groups, respectively.

Statistical analysis

Quantitative data were presented as means ± standard deviation (SD). Statistical significance was analyzed using the Student's t-test or one-way ANOVA with the p < 0.05 indicating significance.

Results

Characterization of NLCs

The mean particle size, size distribution, zeta potential, GL and EE of TMZ/DNA-NLCs, TMZ-NLCs, DNA-NLCs and blank NLCs were characterized and summarized in Table 2. The TMZ/DNA-NLCs has a size of 179 nm, with a potential of +23 mV. The GL and EE of TMZ/DNA-NLCs was 91 and 83%, respectively.

Table 2. Characterization of different vectors.

Formulations	Blank NLCs	DNA-NLCs	TMZ-NLCs	TMZ/ DNA-NLCs
Particle size  (nm)	70.2 ± 1.6	145.2 ± 1.6	153.5 ± 1.9	178.9 ± 2.7
Size distribution  (PDI)	0.11 ± 0.03	0.13 ± 0.02	0.15 ± 0.04	0.16 ± 0.05
Zeta potential  (mV)	+48.6 ± 4.1	+33.1 ± 3.9	+40.6 ± 3.5	+22.8 ± 2.8
GL (%)	N/A	N/A	91.9 ± 1.7	90.8 ± 1.5
EE (%)	N/A	85.4 ± 3.2	80.5 ± 2.8	82.7 ± 2.5

In vitro cytotoxicity

Figure 1 showed the cytotoxicity of TMZ/DNA-NLCs and other vectors in U87 MG cells at different concentrations after 48 h of incubation. Free TMZ solution and NLCs formulations inhibited the growth cells over the studied concentrations and the toxicity conformed to a concentration-dependent pattern. Both TMZ/DNA-NLCs and TMZ-NLCs showed significantly higher cytotoxicity than free TMZ solution (p < 0.05). The IC₅₀ values of TMZ solution, and TMZ/DNA-NLCs were 2.56, and 0.65 µM, respectively. The IC₅₀ value of TMZ/DNA-NLCs was 4-fold over TMZ solution in reducing viability of malignant glioma cells, accounting for the highest antitumor activity.

Figure 1. Cell viability of different vectors at various concentrations.

In vivo gene transfection

The in vivo transfection efficiencies of TMZ/DNA-NLCs, DNA-NLCs, TMZ-NLCs, Lipofectamine™ 2000/DNA and naked DNA solution were evaluated in tumor bearing BALB/c nude mice after 48 and 72 h of transfection. The quantitative study results of the transfected U87 MG cells were illustrated in Figure 2. Significantly higher transfection efficiency was observed on TMZ/DNA-NLCs and DNA-NLCs than other formulations at both 48 and 72 h post-transfection (p < 0.05).

Figure 2. Flow cytometric analysis of EGFP positive U87 MG cells.

In vivo anti-cancer therapy

The in vivo antitumor efficiency was evaluated in U87 MG solid tumors in mice. As shown in Figure 3, although tumor growth was suppressed to some extent after administration of free TMZ solution, while in contrast, tumor growth was significantly inhibited when NLCs formulations were administered intravenously. The most obviously tumor regressions were observed in the TMZ/DNA-NLCs group, the tumor growth was prominently inhibited, which attained only 512 mm³ on 15 days, while in saline-treated group, tumor volume grew rapidly to 1605 mm³ during 15-day therapeutic period. The tumor volume of TMZ/DNA-NLCs and free TMZ solution treated groups reached to 588 and 857 mm³, respectively. These results indicate that tumor growth was significantly inhibited by NLCs formulations (p < 0.05). At 15 days of administration, the tumor inhibition rates of tumor bearing mice treated with TMZ/DNA-NLCs and free TMZ were 69 and 21% compared with control. TMZ/DNA-NLCs inhibited tumor growth 3.3 times higher than that treated with free TMZ solution.

Figure 3. In vivo anticancer efficiency of different formulations.

Discussion

In the present study, we would like to introduce novel NLCs for the co-encapsulation of DNA and TMZ to achieve combination therapy of malignant glioma.

pEGFP has been used widely for in vitro and in vivo cell tracking, with the advantage of can be recognized without any further staining (Leath & Straughn, 2013). However, naked DNA would be degraded rapidly be nucleases, show poor cellular uptake and low transfection efficiency (Yu et al., 2014). Therefore, NLCs were applied as carriers for the loading of TMZ and DNA: TMZ was melted in the lipid dispersion and DNA was dissolved in the aqueous phase, NLCs were prepared by the emulsification-solvent diffusion method (Yu et al., 2009; Das et al., 2012). It is reported that the positive surface charge and proper particle size of nanocarriers were important for efficient gene and drug delivery, so it was expected that TMZ/DNA-NLCs with the size of ∼180 nm and +23 mV could obtain for efficient cellular endocytosis (Bothun et al., 2011).

The cytotoxicity of all formulations confirm to the concentration-dependent manner (Figure 1). MTT assay results indicated that TMZ/DNA-NLCs showed significantly higher cell inhibition than free TMZ solution (p < 0.05). The IC₅₀ value of TMZ/DNA-NLCs was 4-fold over TMZ solution in reducing viability of malignant glioma cells, accounting for the highest antitumor activity. The results indicated that enhanced permeability and retention (EPR) could appear effect in highly fenestrated tumor endothelial cells, uptake of NLCs by U87 MG cells might be enhanced by across the cell membrane via endocytosis or direct penetration may enhance intracellular drug accumulation (Yuan et al., 2013). For blank NLCs, cell viabilities were over 80%, implying there was no cytotoxicity of blank NLCs.

In order to investigate the transfection efficiency and antitumor activity of TMZ/DNA-NLCs in vivo, a mouse model of malignant glioma using U87 MG cells was constructed. Significantly higher transfection efficiency was observed over TMZ/DNA-NLCs than other formulations at both 48 and 72 h post-transfection (p < 0.05) (Figure 2). Taken together, the major mechanism accounting for the superiority of NLCs may be the positive surface charge of NLCs having high electrostatic interaction with the negatively charged tumor surface, excellent compliance of the NLCs to the cell membranes that could mediate the intracellular gene delivery via both endocytic and non-endocytic pathways.

The in vivo antitumor efficiency of TMZ/DNA-NLCs was assessed via measuring the mean tumor volume (mm³) and the inhibition of tumor growth in tumor bearing mice. The anti-tumor studies suggested that TMZ/DNA-NLCs showed significantly higher ability in reducing the tumor volume than free TMZ (p < 0.05) (Figure 3). At 15 days of administration, the tumor inhibition rates of tumor bearing mice treated with TMZ/DNA-NLCs and free TMZ were 69 and 21% compared with control. TMZ/DNA-NLCs inhibited tumor growth 3.3 times higher than that treated with free TMZ solution. The results of in vivo gene expression and anticancer drug delivery efficiency were very impressive, confirming that the TMZ/DNA-NLCs dramatically improved antitumor efficiency, and enhanced gene transfection efficacy without bring about more toxicity. The modified NLCs formulation could be a promising system for the co-delivery of TMZ and DNA against malignant glioma. This kind of nanomedicine could be applied for the loading of other drugs and genes for different types of GC therapy.

Conclusion

To co-deliver drug and gene efficiently to tumor cells, FA modified co-encapsulated pEGFP and TMZ-loaded NLCs were fabricated. The well-formed TMZ/DNA-NLCs formulation exhibited small particle size, reasonable positive charges and high cancer cell inhibition capacity in vitro. TMZ/DNA-NLCs provided clear advantages and significantly increased the in vivo gene transfection efficiency and anti-tumor efficacy. The effect of tumor-targeted and co-delivery ability of this NLCs formulation might be an effective combined tumor therapy strategy for treatment in malignant GC.

Declaration of interest

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