Nanostructured lipid carriers loaded with tributyrin as an alternative to improve anticancer activity of all-trans retinoic acid

Abstract

Objectives

All-trans retinoic acid (ATRA) is one of the most successful examples of differentiation agents and histone deacetylase inhibitors, such as tributyrin (TB), are known for their antitumor activity and potentiating action of drugs such as ATRA. Nanostructured lipid carriers (NLC) represent a promising alternative to the encapsulation of lipophilic drugs such as ATRA. This study aimed to develop, characterize, and evaluate the cytotoxicity of ATRA-TB-loaded nanostructured lipid carriers (NLC) for cancer treatment.

Methods

The influence of in situ formation of an ion pairing between ATRA and a lipophilic amine (benethamine; BNT) on the characteristics of NLC (size, zeta potential, encapsulation efficiency) was evaluated. Tributyrin (TB), a butyric acid donor, was used as a component of the lipid matrix. In vitro activity on cell viability and distribution of cell cycle phases were evaluated for MCF-7, MDA-MB-231, HL-60, and Jurkat cell lines.

Results

The presence of the amine significantly increased the encapsulation efficiency of ATRA in NLC. Inhibition of cell viability by TB-ATRA-loaded NLC was more pronounced than the free drug. Analysis of the distribution of cell cycle phases also showed increased activity for TB-ATRA-loaded NLC, with the clear effect of cell cycle arrest in G₀/G₁ phase transition. The presence of TB played an important role in the activity of the formulation.

Conclusion

Taken together, these findings suggest that TB-ATRA-loaded NLC represent a promising alternative to intravenous administration of ATRA in cancer treatment.

1. Introduction

The concept of differentiation therapy emerged in the 1970s as a promising and revolutionary approach to the treatment of acute myeloid leukemia (AML). All-trans retinoic acid (ATRA) is one of the most successful differentiation agents, being used in the treatment of patients with acute promyelocytic leukemia (APL), a subtype of AML [1]. Many studies have shown that ATRA is effective against other types of cancer such as human multiple myeloma, liver, and breast carcinomas. ATRA has also been investigated as a chemopreventive agent in the treatment of oral leukoplakia [2, 3].

ATRA exerts its actions after binding to the nuclear receptors, retinoic acid receptors (RAR), and retinoid X receptors (RXR), which regulate a variety of genes involved in cell proliferation and differentiation. Generally, ATRA can block the cell cycle in the G₁ phase, causing cell proliferation inhibition and apoptosis [4]. In clinical trials, ATRA has been given to cancer patients by oral administration. However, the bioavailability of oral ATRA has been considered low and quite variable [5]. Moreover, continuous treatment with oral ATRA has been associated with progressive reduction of plasma concentrations, probably due to the induced cytochrome P-450-dependent metabolism [6]. Whereas these factors limit the clinical use of oral ATRA, an intravenous formulation could circumvent this problem.

The poor aqueous solubility of ATRA, a hydrophobic drug with an octanol/water partition coefficient log of 4.6, can represent an obstacle for intravenous administration [7]. Lipid nanocarriers such as nanoemulsions, solid lipid nanoparticles (SLN), and, more recently, nanostructured lipid carriers (NLC) have been studied as alternatives to enable intravenous administration of ATRA [8,9,10].

NLC have been developed to circumvent the limitations offered by the polymorphic transitions observed in the lipid matrix of SLN. NLC are prepared from a mixture of liquid and solid lipids that maintain a solid state at the end of preparation [11]. The presence of liquid lipid increases the number of imperfections in the solid lipid matrix, allowing greater accommodation for the drug and decreasing its expulsion over time [12]. In recent years, some studies have been developed using NLC loaded with ATRA for the treatment of cancer [10,13].

Usually, ATRA encapsulation in these nanocarriers is low. We previously reported that the in situ formation of an ion pairing among ATRA, a lipophilic acid, and lipophilic amines provides an interesting alternative to increase drug encapsulation in SLN [14]. Recently, a SLN formulation was developed and evaluated by Carneiro and colleagues [15] using the formation of an ion pairing between ATRA and a lipophilic amine, benethamine (BNT), with potential application for the cancer treatment.

On the other hand, histone deacetylase inhibitors, such as tributyrin (TB), are known for their antitumor activity and potentiating action of other drugs such as ATRA [16,17]. Moreover, the lipophilic nature of TB enables its use as a component of the oily core of nanoemulsions [18] and related nanosystems. In this sense, this work aimed to develop a new innovative formulation of NLC loaded with ATRA and TB as an alternative for the treatment of cancer. The influence of in situ formation of an ion pairing between ATRA and BNT on the characteristics of NLC was evaluated. Also, in vitro anticancer activity was investigated against different cancer cell lines: breast cancer and leukemia cells.

2. Materials and methods

2.1 Materials

All-trans retinoic acid (ATRA) and Compritol^® 888 ATO (glyceryl behenate, mixture of mono-, di-, and triacylglycerols of behenic acid [C₂₂]) were kindly provided by BASF (Ludwigshafen, Germany) and Gattefossé (Lyon, France), respectively. Super Refined Tween 80^™ (Polysobarte 80) was provided by Croda Inc (Edison, USA). Tributyrin and benethamine (BNT; N-Benzyl-2-phenethylamine) were purchased from Sigma-Aldrich (Saint Louis, USA). Hydrogenated soybean lecithin (Lipoid S 75-3) was purchased from Lipoid (Ludwigshafen, Germany). For in vitro studies, the Roswell Park Memorial Institute medium (RPMI) 1640 was obtained from Sigma-Aldrich (Saint Louis, USA); Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), staurosporine, and propidium iodide were purchased from Life Technologies (Carlsbad, USA). The following cancer cell lines were used: Jurkat (immortalized line of T lymphocyte) was provided by Gustavo Amarante-Mendes (São Paulo University, São Paulo, Brazil); MCF-7 (human breast adenocarcinoma), MDA-MB-231 (human breast adenocarcinoma, derived from metastatic site), and HL-60 (acute promyelocytic leukemia) cells were purchased from American Type Culture Collection (ATCC) (Manassas, USA). All other chemicals were of analytical grade.

2.2 Preparation of NLC

NLC were prepared by the hot melting homogenization method using an emulsification-ultrasound as previously described [19]. The composition of the formulations was based on previous studies [15], but with some modifications. Briefly (batch 10 mL), the oily phase, composed of Compritol^® 888 ATO (150 mg), tributyrin (50 mg), Tween 80^™ (50 mg), Lipoid S 75-3 (50 mg), ATRA (5 mg), and the aqueous phase were heated separately to 85°C. Next, the aqueous phase was gently dropped onto the oily phase with constant agitation at 8000 rpm in an Ultra Turrax T-25 homogenizer (Ika Labortechnik, Germany). This emulsion was immediately submitted to the high-intensity probe sonication (20% amplitude) for 10 minutes, using a high-intensity ultrasonic processor (CPX 500 model; Cole-Palmer Instruments, USA). The pH of the NLC was adjusted to 7.0 with a solution of 0.01 M HCl (Digimed DM 20, Brazil). Considering the intended use of NLC (intravenous administration), Tween 80^™ and Lipoid S 75-3 were selected as surfactants. The influence of the in situ formation of an ion pairing between ATRA and the lipophilic amine BNT was investigated (ATRA/BNT molar ratio was 1/2).

2.3 Particle size analysis

The mean particle diameter of NLC in the dispersion was determined by unimodal analysis through dynamic light scattering (DLS) using a Zetasizer Nano-ZS90 (Malvern Instruments, UK), at a fixed angle of 90° and at 25 °C. The NLC dispersions were diluted in distilled and filtered water (cellulose ester membrane, 0.45 μM, Millipore, USA). The data reported were particle size, evaluated as the intensity obtained from three repeat measurements, and the polydispersity index.

2.4 Zeta potential

Zeta potential measurements were carried out by the electrophoretic light scattering determination using a Zetasizer Nano-ZS90 (Malvern Instruments, UK), at 90° and 25°C. Before the measurements, NLC dispersions were diluted in distilled and filtered water (cellulose ester membrane, 0.45 μM, Millipore, USA). All measurements were performed in triplicate.

2.5 Drug-encapsulation efficiency

Encapsulation efficiency (EE) for ATRA in NLC was determined according to the method previously described [19]. This method was based on the determination of ATRA concentration in the NLC (before and after filtration; cellulose ester membrane, 0.45 μM, Millipore, USA) as well as in the external aqueous phase of the NLC, which was estimated by ultrafiltration method (Amicon^® 100 k, Millipore, USA) using a 100 kDa molecular weight cut-off membrane.

Briefly, an aliquot of the NLC dispersion was dissolved in tetrahydrofuran and later diluted in a mixture of acetonitrile:distilled water:phosphoric acid (80:19.9:0.1). This mixture keeps the ATRA in solution (dissolved), but causes lipid precipitation. This dispersion was filtered in a 0.45 μM Millex HV filter (Millipore, USA) and analyzed by high performance liquid chromatography (HPLC).

The HPLC system consisted of a Waters 515 HPLC Pump (Milford, USA), a Waters 717 Plus Auto-sampler, and a photodiode array detector (Waters 2996). A C18 reverse-phase column (125 mm of length, 4 mm of width, and particles of 5 μm) (LichroCart 125–4, Merck, Germany) was used. The mobile phase was a mixture of acetonitrile, distilled water, and phosphoric acid (80:19.9:0.1). The detection was carried out at 340 nm, with a flow rate of 1.0 mL/minute and 20 μL of sample. The five-point (0.25, 0.5, 1.0, 2.0, and 5.0 μg/mL) linear regression analysis resulted in the following linear equation: y = −2435 + 137200x (r = 0.9998).

Considering that the ATRA concentration found in the aqueous phase was negligible, EE was calculated using the following formula: EE (%) = (filtered ATRA/total ATRA) × 100.

2.6 Polarized light microscopy

The presence of ATRA crystals was evaluated by polarized light microscopy (Zeiss Axio Imager.M2, Carl Zeiss, Oberkochen, Germany). The samples were prepared in microscope slides (undiluted), utilizing a proper software (ZEN lite 2012, Carl Zeiss, Oberkochen, Germany). The microscope was equipped with an AxioCam digital camera (Model ERc 5S, Carl Zeiss, Oberkochen, Germany).

2.7 Transmission electron microscopy

Morphological examination of the NLC was performed with a transmission electron microscope (Tecnai G2-12, Spirit Biotwin, 120 kV, FEI, USA) using a negative staining method. The sample was prepared by placing a drop of NLC (dilution 1/50 in double-distilled water) onto a copper grid coated with carbon film. The excess droplets were removed with a filter paper. After three washings with water, the samples were stained with 2% (w/v) uranyl acetate in ethanol. The grid was dried at room temperature and then observed by TEM.

2.8 Differential scanning calorimetry

Differential scanning calorimetry (DSC) analyses were performed using a differential scanning calorimeter (DSC 2910, TA Instruments, USA). For DSC measurements, a scan rate of 10 °C/min was used at a temperature range of 0–250 °C, under nitrogen purge (50 mL/min). Lyophilized NLC were obtained using a freeze-drier (E-C Apparatus, USA) connected to an E2M18 vacuum pump (BOC Edwards, United Kingdom), after rapid freezing of the NLC preparation into liquid nitrogen. The samples were lyophilized for 24 h at a temperature of −45 °C. After freeze-drying, the lyophilized samples were placed directly in aluminum pans for DSC analyses. The ion pairing ATRA-BNT was prepared separately according to the following protocol: briefly, ATRA and BNT (molar ratio 1:1) were solubilized with a freshly prepared solution of methanol and chloroform (1:1). The reactional mixture was kept under agitation for 6 hours at room temperature and protected from light exposure. Then, solvents were evaporated in a rotary evaporator (R-215, Büchi, Switzerland) utilizing a vacuum pump (V-700, Büchi, Switzerland) and heating bath (B-491, Büchi, Switzerland) at 45 °C. The residue was collected and stored at – 20 °C prior to analysis.

2.9 Cell cultures

Cancer cell lines MCF-7 (human mammary adenocarcinoma), MDA-MB-231 (human mammary adenocarcinoma), HL-60 (human acute promyelocytic leukemia), and Jurkat (human acute T cell leukemia) were cultured in DMEM or RPMI medium containing fetal bovine serum (10%), 200 mM glutamine, and antibiotics (100 μg/mL streptomycin and 100 UI/mL penicillin). All cultures were kept in a humidified incubator with 5% CO₂ at 37 °C.

2.10 Analysis of cell viability

Cell proliferation was measured by MTT assay based on the reduction of tetrazolium salt to formazan crystals by living cells. Briefly, aliquots containing 7.0 × 10³ (MCF-7), 2.5 × 10³ (MDA-MB-231), 9 × 10³ (HL60), or 1.8 × 10⁴ (Jurkat) cells/well were seeded into 96-well plates. After 24 hours of incubation at 37 °C and 5% CO₂, freshly prepared solutions of free ATRA and ATRA-loaded NLC were added to the wells (ATRA concentration ranged from 0.5 to 50 μM). Free ATRA was dissolved in DMSO (33.3 mM) prior to dilution. Blank NLC (without ATRA) was diluted in the same way as ATRA-loaded NLC to simulate the same range of ATRA concentration. After 48 hours of incubation at 37 °C and 5% CO₂, 20 μL of the 5 mg/mL MTT solution was added to each plate. Plates were incubated at 37 °C for 4 hours, and then the medium was replaced by 200 μL of 0.04 M HCl solution in isopropanol. Cell viability was estimated by measuring the rate of mitochondrial reduction of MTT, determined by evaluating the absorbance of the converted dye at a wavelength of 595 nm. Absorbance values of the wells in which the cells were maintained in medium alone were considered as 100% of cell viability. Control groups included treatment with DMSO (negative control) and staurosporine (positive control). Cell viability was found to be 100% after treatment with negative control (DMSO 0.5%), while staurosporine was effective in promoting cell-growth inhibition. Data were expressed as percentage of cell viability compared to the control (mean ± standard deviation [SD]). At least three independent experiments were performed.

2.11 Subdiploid DNA content and cell cycle analysis

A flow-cytometric DNA fragmentation assay was employed as a quantitative measure of subdiploid content and phases of the cell cycle [20]. Aliquots containing 5.0 × 10⁴ (MCF-7, MDA-MB-231 and HL-60) or 1.0 × 10⁴ (Jurkat) cells/well were seeded into 24-well plates and incubated for 24 hours at 37 °C and 5% CO₂. After incubation, cells were treated with free ATRA, ATRA-loaded NLC, or blank NLC for 48 hours at 37 °C and 5% CO₂ (ATRA concentration was 25 μM). After this time, cells were centrifuged at 200 g for 5 minutes at room temperature, and the culture medium was aspirated off. The pellet was gently resuspended in 300 μL of hypotonic fluorochrome solution containing 0.5% Triton X-100 and 50 μg/mL propidium iodide. Cells were incubated in the dark at 4 °C for 4 hours and analyzed with a Guava^® EasyCyte^™ 6-2L Base System cytometer (Millipore, USA). Data analysis was performed with FlowJo^™ 7.6.5 (Tree Star Inc, Ashland, USA) to determine percentages of subdiploid content and phases of the cell cycle.

2.12 Data analysis

Statistical analyses were carried out using one-way ANOVA followed by Tukey’s test. For all analyses, the difference was considered statistically significant when P value was less than 0.05.

3. Results

3.1 Characterization of NLC

The main characteristics of blank NLC and ATRA-loaded NLC prepared with lipophilic amine BNT are listed in Table 1. The addition of ATRA (NLC A, 171 ± 18 nm) and ATRA+BNT ion pairing (NLC-B, 154 ± 4) increased the mean particle size in comparison to blank NLC (131 ± 4 nm). Mean particle sizes and PI for NLC A (171 ± 18 nm and 0.40 ± 0.11, respectively) were higher than those observed for NLC-B (154 ± 4 nm and 0.24 ± 0.03). This increase is directly related with ATRA crystals present in its external aqueous phase of NLC-A (Figure 1).

Table 1.

Characterization of blank NLC and ATRA-loaded NLC (NLC-A and NLC-B) for PCS diameter, polydispersion index and zeta potential.

		ATRA-loaded NLC
Parameter	Blank NLC	NLC-A	NLC-B
		0% BNT	0,1% BNT
Mean diameter (nm)	131 ± 4a	171± 18b	154 ± 4
Polydispersion index	0.25 ± 0.03	0.40 ± 0.11b	0.24 ± 0.03
Zeta potential (mV)	− 38 ± 4	− 33 ± 5	−43 ± 7

Note: Data are shown as mean ± SD (n = 3).

^aSignificant difference compared to NLC-A and NLC-B (p<0.05);

^bSignificant difference compared to blank NLC and NLC-B (p<0.05).

Abbreviations: BNT, benethamine; ATRA, all-trans retinoic acid; NLC, nanostructured lipid carrier.

Figure 1.

Negatively charged particles were obtained for blank NLC (−38 ± 4 mV) and ATRA-loaded NLC (NLC-A, −33 ± 5 mV, and NLC-B, −43 ± 7 mV). The presence of BNT did not change the surface charge of NLC (compare NLC-A and NLC-B). This can be attributed to its low potential for interfacial adsorption [15].

The EE for ATRA in NLC without BNT increased from 15 ± 2% (NLC-A) to 79 ± 3% for NLC with BNT (NLC-B) (Figure 1). Therefore, BNT promoted a significant increase of EE for ATRA in NLC prepared with tributyrin as a component of the lipid matrix. This increase in the EE could be due to the formation of an ion pairing between ATRA and BNT, which increases the lipophilic properties of the drug, making its incorporation into the lipid matrix easier (Figure 1). The presence of ATRA crystals was investigated in the NLC presenting low EE low (without BNT) and high EE (with BNT) (insert Figure 1). ATRA crystals were clearly present in the external phase of NLC without BNT. However, these crystals were absent in NLC with BNT (NLC-B). These findings are in agreement with our previous observations that showed the role of the lipophilic amines (stearylamine and BNT) in increasing ATRA drug-loading capacity in SLN [15,19].

3.2 Transmission electronic microscopy (TEM)

The NLC image obtained by TEM is shown in Figure 2. Blank NLC had nearly spherical shapes with little variability in shape and size (approximately 100 nm). These values of particle size were lower those determined by DLS. In fact, DLS measures the hydrodynamic diameter of the particles and this could explain these differences.

Figure 2.

3.3 Differential scanning calorimetry

The data of DSC analyses are shown in Figure 3. Compritol^® 888 ATO, ATRA, and the ATRA+BNT ion pairing presented the following endothermic melting point peaks: 75 °C, 187 °C and 114 °C respectively (Figure 3A). The melting point peak for the ion pairing was lower than that observed for the pure drug and this suggests that a different crystalline structure was formed from pure ATRA. The melting points for ATRA and the ion pairing were absent in the ATRA-loaded NLC (Figure 3B) and only the Compritol^® 888 ATO peak is shown, suggesting that ATRA is integrated in the lipid. On the other hand, it was also possible to note a slight decrease in the melting point of the lipid matrix for the ATRA-loaded NLC in comparison to blank NLC (from 76 °C to 74 °C), as well as a gradual broadening of this peak (Figure 3B). These modifications can be attributed to the incorporation of the ion pairing (ATRA+BNT) in the lipid matrix of the NLC.

Figure 3.

3.4 Cell viability studies

MTT assay was used to evaluate whether cytotoxicity of ATRA was affected by loading in NLC. Cancer cell lines MCF-7, MDA-MB-231, HL-60 and Jurkat were incubated with free ATRA, blank NLC (unloaded) or ATRA-loaded NLC and analyzed for their viability. The data obtained, expressed as cell viability (percentage), are shown in Figure 4.

Figure 4.

For MCF-7 cells, blank NLC showed no inhibitory activity up to an ATRA concentration of 10 μM. Thereafter, a reduction in cell viability to about 80% at the highest concentration was observed. This activity can be attributed to the TB, present in the NLC matrix, which can act as a butyric acid donor, causing the observed reduction in cell viability. In fact, an NLC containing caprylic/capric triglycerides as liquid lipid instead of TB was evaluated and this formulation had no effect on the cell viability (data not shown). The activity of free ATRA was low regardless of the concentration with cell viability of around 80%. The cytotoxicity of ATRA-loaded NLC was significantly higher than that observed for the free RA and blank NLC, reducing the cell viability to approximately 60% at 50 μM.

Leukemic cells (HL-60 and Jurkat) were more sensitive to ATRA, while the MDA-MB-231 cells were the least sensitive. In fact, free ATRA showed negligible cytotoxic effect in MDA-MB-231 cells at all evaluated concentrations. In contrast, cytotoxic activity for ATRA-loaded NLC was significantly higher than that observed for free ATRA. Unexpectedly, however, differences between ATRA-loaded NLC and blank NLC were negligible. Taken together, these data suggest that activity observed for NLC with or without ATRA could be attributed the release of butyric acid from TB, which was added into lipid matrix of the NLC.

For HL-60 cells, the activity for free ATRA and ATRA-loaded NLC was dose-dependent with the formulation showing cytotoxicity higher than that observed for free ATRA mainly at lower drug concentrations. For example, the cell viability at 0.5 μM was 60 ± 8% and 80 ± 4% for ATRA-loaded NLC and free ATRA, respectively. For Jurkat cells, cell viability after treatment with free ATRA was around 80% at most concentrations tested, with higher activity at 50 μM (54 ± 2%). For blank NLC, cell viabilities were around 70% and a greater effect was observed at 50 μM (50 ± 2%). The activity for ATRA-loaded NLC was significantly higher than that observed for free ATRA and blank NLC with a much greater effect at 50 μM (23 ± 2%). The cytotoxic activity of ATRA-loaded NLC was clearly improved in comparison to free ATRA and this was attributed to the butyric acid released by hydrolysis of TB.

3.5 Subdiploid DNA content and cell cycle analysis

Flow cytometry studies were performed in order to determine whether the improvement of the cytotoxic activity of ATRA after incorporation into NLC was associated with alterations in DNA fragmentation and/or distribution of the cell cycle phase. The data are summarized in Table 2. Representative histograms of cell cycle distribution after cell staining with propidium iodide are shown in Figure 5.

Table 2.

Effects of different treatments on DNA fragmentation and cell-cycle phases distribution of the breast cancer (A; MCF-7 and MDA-MB-231) and leukemic cell lines (B; HL-60 and Jurkat).

(A)
		Cell Cycle Distribution (%)
Sample	Subdiploid	G0/G1	S	G2/M
		MCF-7
Control	NS*	70.9 ± 3.8	10.9 ± 0.4	17.8 ± 3.6
Free ATRA	NS	77.7 ± 1.8	6.96 ± 0.5	15.3 ± 1.3
Blank NLC	NS	80.9 ± 0.5	6.6 ± 0.1	12.5 ± 0.4
ATRA-loaded NLC	NS	84.9 ± 0.6a	4.6 ± 0.2	10.0 ± 1.0
		MDA-MB-231
Control	NS	70.7 ± 1.3	12.0 ± 0.1	17.2 ± 1.5
Free ATRA	NS	60.1 ± 1.2	12.1 ± 0.2	27.5 ± 0.9
Blank NLC	NS	76.3 ± 0.7	9.4 ± 1.3	13.9 ± 0.6
ATRA-loaded NLC	NS	85.7 ± 1.2b	4.5 ± 0.3	9.5 ± 1.1

(B)
		Cell Cycle Distribution (%)
Sample	Subdiploid	G0/G1	S	G2/M
		HL-60
Control	NS*	77.5 ± 1.1	9.8 ± 2.5	12.5 ± 1.3
Free ATRA	NS	94.5 ± 0.8	2.9 ± 1.2	2.6 ± 0.4
Blank NLC	NS	82.7 ± 2.3	7.3 ± 1.1	10.1 ± 1.2
ATRA-loaded NLC	NS	98.5 ± 0.1b	0.4 ± 0.1	1.2 ± 0.1
		Jurkat
Control	NS	60.7 ± 8.5	20.2 ± 4.9	19.1 ± 3.5
Free ATRA	NS	65.7 ± 2.3	17.6 ± 1.2	16.8 ± 1.0
Blank NLC	NS	73.0 ± 0.3	12.4 ± 0.8	14.6 ± 0.6
ATRA-loaded NLC	NS	78.6 ± 2.5a	11.0 ± 0.9	10.5 ± 1.6

^*Non-significant (less than 1.0%).

Notes: Cancer cells were treated with ATRA-loaded NLC, free ATRA and unloaded (blank) NLC, as indicated in the methods section. Cell-cycle distributions were determined after 48 hours of treatment. The data are representative of three independent experiments. Mean ± SD is shown.

^aSignificant difference compared to Control and free ATRA (p<0.05);

^bSignificant difference compared to Control, free ATRA and blank NLC (p<0.05);

Abbreviations: ATRA, All-trans retinoic acid; NLC, Nanostructured lipid carriers; SD, standard deviation.

Figure 5.

The increase in subdiploid DNA content for all treatments (blank NLC, free ATRA, and ATRA-loaded NLC) was negligible in comparison to control since its value was less than 1% for all cell lines evaluated (Table 2). Nevertheless, the effects of ATRA encapsulation in NLC became more evident when analyzing the distribution of cell cycle phases. For MCF-7 cells, the percentage of cells in the G₀/G₁ phase for ATRA-loaded NLC (84.9 ± 0.6%) was increased (p<0.05) when compared to control and free ATRA (70.9 ± 3.8% and 77.7 ± 1.8%, respectively). Moreover, the increase in the G₀/G₁ phase was followed by a significant decrease in the percentage of cells in the S and G₂/M phases, as shown in Table 2 and illustrated in Figure 5. The effect of blank NLC was similar to that observed for free ATRA, with a significant increase (p<0.05) in the number of cells in G₀/G₁ compared to control. As suggested for the MTT assay, the effect of this formulation can be attributed to the presence of TB.

For MDA-MB-231 cells, it was observed that free ATRA did not arrest the cells in G₀/G₁. In contrast, blank NLC caused a significant increase of cells in G₀/G₁ (76.3 ± 0.7%) compared to control (70.7 ± 1.3%) and free ATRA (60.1 ± 1.2%). The effect of ATRA-loaded NLC (85.7 ± 1.2%) was even more pronounced than that observed for blank NLC. In agreement, the decrease in the S phase for ATRA-loaded NLC (4.5 ± 0.3%) was also significantly higher than that observed for blank NLC (9.4 ± 1.3%) and this suggests cell cycle arrest in G₀/G₁, as occurred for MCF-7 cells.

The most intense effect of cell cycle arrest in G₀/G₁ was observed for HL-60 cells. Free ATRA had a significant effect (94.5 ± 0.8%) compared to control (77.5 ± 1.1%). Blank NLC also caused an increase in G₀/G₁ (82.7 ± 2.3%), but lower than that observed for free ATRA. For ATRA-loaded NLC, almost 99% of the cells were in the G₀/G₁ phase. These data are in agreement with those observed in the MTT assay, which, in concentration of 20 μM (close to that used in flow cytometry), ATRA-loaded NLC and free ATRA caused important reduction in cell viability. The decrease in the S and G₂/M phases for ATRA-loaded NLC was statistically significant compared to the control. Free ATRA also showed a significant decrease in the S phase (2.9 ± 1.2%), which was revealed to be lower than that observed for the treatment with ATRA-loaded NLC (0.4 ± 0.1%).

For Jurkat cells, free ATRA had no significant differences in comparison to control for any of the cell cycle phases. Blank NLC showed a small but significant increase in the percentages of cells in G₀/G₁ compared to control. The ATRA-loaded NLC provided a significant increase in G₀/G₁-phases in comparison to the control and free ATRA.

4. Discussion

The aim of this study was to develop and evaluate the in vitro cytotoxic activity of NLC loaded with ATRA and TB. The EE for ATRA in NLC was significantly increased after the addition of the lipophilic BNT (from 15 ± 2% to 79 ± 3 %, Figure 1). Therefore, BNT promoted a significant increase of EE for ATRA in NLC. These findings are consistent with our previous observations [15,19]. In agreement, the data of DSC revealed the absence of melting point peaks for ATRA and ion pairing in ATRA-loaded NLC suggesting that ATRA was integrated in the lipid. The ion pairing-induced modifications in the lipid matrix of the ATRA-loaded NLC suggest a larger number of defects, allowing greater encapsulation for the ATRA in NLC.

Cytotoxic effects against four important tumor cell lines were evaluated. With respect to cell viability studies, a clear advantage was observed when ATRA-loaded NLC was compared to free ATRA and blank NLC for the MCF-7 line. The results obtained for free ATRA in MCF-7 cells were comparable with findings previously described [15,21]. The accumulation of MCF-7 cells in G₀/G₁ for ATRA-loaded NLC was accompanied by a reduction in the frequency of cells in the S and G₂/M phases. These findings are consistent with previous observations, which showed that treatment of MCF-7 cells with ATRA induces cell cycle arrest in G₀/G₁ before inducing apoptosis [22,23].

Remarkably for MDA-MB-231 cells, it was suggested that the activity of ATRA-loaded NLC and blank NLC results from the release of butyric acid from TB hydrolysis. In fact, free ATRA showed negligible activity against MDA-MB-231 cells confirming the intrinsic resistance of these cells to ATRA. Estrogen-independent cell lines, such as MDA-MB-231, are known for their low expression of RARα, which justifies its resistance to the effects of ATRA [24]. Evaluating the data of distribution of cell cycle phases presented in Table 2, it was clearly shown that ATRA-loaded NLC had the most pronounced effect in promoting cell cycle arrest in G₀/G₁. The differences between blank NLC and ATRA-loaded NLC in the MTT assay were insignificant and this can be related to the lower sensitivity of this technique compared to flow cytometry. On the other hand, profiles in Figure 5 for MDA-MB-231 cells treated with free ATRA or loaded in NLC revealed a peak shift in comparison with the control group and this can be attributed to some degree of aneuploidy (DNA content different from 2n). Aneuploidy has been described by several authors and is a common finding in tumor cells and other organisms, being characterized by irregular number of chromosomes [25]. As described by Cappello et al. 2014 [26], aneuploid MDA-MB-231 cells were identified in flow cytometry analysis with fluorescence intensity higher than diploid cells (G₀/G₁) but lower than G₂/M cells. This phenomenon was also observed for pluripotent stem cells undergoing differentiation induced by ATRA [27].

An ATRA-sensitive (HL-60) and an ATRA-resistant leukemic cell line (Jurkat) were evaluated in the present study. ATRA-loaded NLC showed a significant accumulation in the Go/G₁ phase when compared to free ATRA and the control for both cells, with a more pronounced effect in Jurkat cells. These data are in agreement with those obtained in studies of cell viability. ATRA resistance has been related to various events, including increased catabolism and genetic alterations [28]. The protection against increased metabolism may represent one of the advantages offered by ATRA encapsulation in NLC. In Jurkat cells, ATRA resistance is associated with the negativity of HTLV-1 (Human T cell Leukemia Virus Type 1) [29]. The differences between ATRA-loaded NLC and blank NLC were not significant in Jurkat cells, suggesting that the presence of TB, as a butyric acid donor, had an essential role in the activity of this formulation. These findings are in agreement with those previously published showing significant cytotoxic activity of butyric acid against Jurkat cells [30]. Taken together, the data suggest that ATRA-loaded NLC represent a novel approach that is potentially significant in the field of ATRA resistance.

It is noteworthy that for all cell lines evaluated, none of the treatments induced significant cell death, with values of subdiploid DNA content less than 1%. These findings are not unexpected, as several studies have shown that one of the main activities of ATRA is the cell cycle arrest in the G₀/G₁ phase, followed by differentiation and/or apoptosis [31]. In leukemic cells, for example, it is well known that treatment with inducers of differentiation, such as ATRA, makes the cells become resistant to apoptosis during the early stages of differentiation. Apoptosis occurs in such cases as a later event constituting an alternative fate for cells that for some reason are not able to resume the normal differentiation program [32].

Finally, TB as an oily core of a nanoemulsion provided the dual function of solubilizing the celecoxib and potentiating its cytotoxic activity [18]. In addition, the cytotoxicity of ATRA-loaded TB nanoemulsion against hepatic or colonic cancer cells was higher than that of free ATRA [17]. However, in this study, it is difficult to separate the effects of the combination of ATRA and TB from those of drug encapsulation, and data concerning drug encapsulation and comparison with blank nanoemulsion were not reported. In the present study, we reported for the first time a remarkable increase in anticancer activity of ATRA-TB-loaded NLC in comparison with free ATRA against leukemic cells using a formulation with high drug encapsulation.

5. Conclusion

In summary, an NLC formulation loaded with ATRA, a lipophilic acid, and a lipophilic amine (BNT) was designed and evaluated. It was possible to obtain high encapsulation efficiency for ATRA in NLC. Moreover, ATRA-loaded NLC promoted enhanced in vitro cytotoxic activity when compared to the free ATRA. TB, present in the lipid matrix of NLC, played an important role in the activity of the formulation. These findings suggest that the ATRA-loaded NLC is a promising alternative for the administration of ATRA in the treatment of cancer.

Key issues.

• This study aimed to develop and evaluate the in vitro cytotoxic activity of nanostructured lipid carriers (NLC) loaded with all-trans retinoic acid (ATRA) and tributyrin (TB). TB is a histone deacetylase inhibitor known for its antitumor activity and potentiating action of drugs such as ATRA.

• The lipophilic amine BNT promoted a significant increase of encapsulation for ATRA in NLC.

• Cytotoxic effects against four important tumor cell lines were evaluated. A clear advantage was observed when ATRA-loaded NLC was compared to free ATRA for the MCF-7, MDA-MB-231, HL-60 and Jurkat cell lines.

• For ATRA-resistant cells such as MDA-MB-231 and Jurkat cells, it was suggested that the activity of ATRA-loaded NLC results from the release of butyric acid from TB hydrolysis.

• The effects of treatments in MDA-MB-231 with ATRA-loaded NLC are probably related to some degree of aneuploidy (DNA content different from 2n).

• Flow cytometry assay was employed as a quantitative measure of subdiploid DNA content and phases of the cell cycle. ATRA-loaded NLC showed a significant accumulation in the Go/G₁ phase when compared to free ATRA for all cell lines.

• None of the treatments induced significant cell death, with values of subdiploid DNA content less than 1%. These findings are not unexpected, once ATRA-induced cell cycle arrest in the G₀/G₁ phase is usually followed by differentiation and/or apoptosis.

• A remarkable increase in anticancer activity of ATRA-TB-loaded NLC in comparison with free ATRA was obtained against leukemic cells using a formulation with high drug encapsulation.