Investigation of bioavailability and anti-pancreatic cancer efficacy of a self-nanoemulsifying erlotinib delivery system

ABSTRACT

Aims

A new self-nanoemulsifying drug delivery system (SNEDDS) was developed for erlotinib (Ert) oral delivery.

Materials and methods

A pseudo-ternary phase diagram for olive oil, Tween 80 and polyethylene glycol (PEG) 600 mixtures, was firstly constructed. Based on the data about Ert solubility and cytotoxicity of these components, a SNEDDS composed of 10% olive oil, 20% Tween 80 and 70% (V/V) polyethylene glycol 600 was selected for Ert loading (Ert-SNEDDS).

Results and conclusions

SNEDDS formed 31.2-nm droplets upon dilution in water, and Ert loading led to increment in the oil droplets to 83.9 ± 0.6 nm. Ert-SNEDDS represented a loading capacity and an entrapment efficiency of 22.7 ± 0.7 and 40.7 ± 0.5%, respectively. Ert release from Ert-SNEDDS was monitored in both a mixture of phosphate buffer saline and 0.5% Tween 80, and artificial gastric fluid. Ert-SNEDDS was orally administrated in rats, and the Ert plasma level was monitored over time to measure pharmacokinetic parameters. Ert-SNEDDS led to enhancement in the drug bioavailability and changed the release route of Ert. Ert-SNEDDS showed enhanced cytotoxicity toward ASPC-1 and PANC-1 cells, and half-maximal inhibitory concentration values were obtained and compared with free Ert. Ert-SNEDDS may be considered as an alternative route for oral Ert delivery.

1. Introduction

Despite advances in the last decade for treatment of cancer, pancreatic adenocarcinoma is one of the most fatal human malignancies and remains the fourth main cause of cancer death in the Western world [1], due to rapid metastasis, resistance to chemotherapy and radiotherapy, and high mortality after diagnosis [2,3]. Ideally, chemotherapeutic agents should include nontoxic components that synergistically act to eliminate cancer cells in a targeted manner. Several synthetic and natural compounds have been discovered as drugs or chemotherapy-combined drugs in preclinical experiments of pancreatic cancer [4–6]. Between the selective drugs that have been assessed, erlotinib (Ert), as a small anilinoquinazoline derivative, has been indicated for solid tumors treatment e.g., advanced pancreatic adenocarcinoma [7–9]. In up to 55% of human pancreatic cancers, epidermal growth factor receptor (EGFR) is excessive expressed [10,11], and Ert selectively hinders the trans-phosphorylation of tyrosine kinases in the EGFR domain of tyrosine kinase [12–14]. Inhibition of tyrosine kinase prevents angiogenesis, accelerates cell apoptosis [15,16], and efficiently suppresses proliferation of cancer cells. However, Ert is located in the Biopharmaceutical Classification System class II that is specified by high permeability and poor solubility. Ert has low bioavailability in oral form, mainly due to instability in gastrointestinal tract, limited solubility, and high first-pass metabolism [17]. The novel Ert delivery system would reduce its low bioavailability and solubility. Therefore, great efforts have been recently dedicated to development of new systems for effective Ert delivery [18].

At present, many nanostructures for drug carrying such as metals, metal oxides, hydrogels, liposomes, emulsions and proteins have been developed [16,19–30]. A lipid nanocarrier efficaciously enhanced the delivery of Ert into the skin in C57BL/6 mice with psoriasis disease [31]. In a study, the A549 cellular uptake of nanoparticles of hyaluronic acid- and serum albumin-modified Ert were facilitated due to the augmentation of the solubility of Ert in nanoparticulate formulation that subsequently decreased the value of the half-maximal inhibitory concentration (IC₅₀) of the nanoparticles in comparison with the free Ert [32]. In a study, oral bioavailability and solubility of Ert were improved using a solid self-emulsifying drug delivery system prepared using 5% Labrafil M2125CS, 65% Labrasol and 30% Transcutol as oil, surfactant, and cosurfactant, respectively. The formulation was spray dried with the solid inert carriers of dextran 40 and Aerosil® 200. Pharmacokinetic study of this formulation in vivo in rats indicated that the maximum concentration (C_max) and area under the curve (AUC) of Ert were significantly enhanced, compared to the pure Ert powder [33].

Among the promising strategies to increase the dissolution extent, rate of absorption and oral bioavailability of lipophilic drugs, great attention has been paid to self-micro and self-nanoemulsifying drug delivery systems (SMEDDSs and SNEDDSs) [21,34]. These systems are anhydrous mixtures of synthetic or natural oil, surfactant, cosurfactant and the lipophilic drug, which dispersed in the gastrointestinal (GI) environment with gentle agitation to form oil-in water emulsions in micro- and nano-sized ranges [34,35]. SNEDDSs improve the solubilization and absorption of drugs, prevent the cellular efflux mechanisms, and minimize the high first-pass metabolism effect of hepatic. Furthermore, retention of drugs from decomposition by the GI environment is attainable [24]. There are studies on loading of poor-soluble drugs in water into SNEDDSs such as cilostazol [36], rosuvastatin calcium [37], irbesartan [38], and cyclosporine [39]. Nevertheless, increment in the oral bioavailability and absorption of Ert by SNEDDSs has not been reported yet. The primary aims of the present study are to investigate a SNEDDS for Ert using olive oil, Tween 80 and polyethylene glycol (PEG) 600, and to determine the characteristics of oil droplet size, stability, in vitro anti-proliferative efficiency in pancreatic cancer cells and in vivo bioavailability.

2. Materials and methods

2.1. Materials

Ert was received from Arasto Pharmaceutical Chemicals Inc (Iran). Virgin olive oil, Tween 80, PEG 600, dimethyl sulfoxide (DMSO) and chloroform were purchased from Merck (Germany) or Scharlau (Spain). Methanol, trypan blue, high-performance liquid chromatography (HPLC)-grade acetonitrile and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were prepared from Sigma (USA). A Dulbecco’s modified eagle medium (high glucose, DMEM), fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid and penicillin G 10,000 unit mL⁻¹/streptomycin 10,000 μg mL⁻¹ solution were purchased from Gibco (USA). Pancreatic adenocarcinoma cells from human of ASPC-1 and PANC-1 were purchased from Pasteur Institute Cell Bank (Iran).

2.2. Construction of pseudo-ternary phase diagram of the SNEDDS components

Self-nanoemulsifying formulations that can emulsify under a mild agitation are determined by plotting ternary phase diagrams of olive oil, Tween 80 surfactant and PEG 600 cosurfactant. Ternary phase diagrams were built using the conventional titration technique. In brief, mixtures of the surfactant and cosurfactant will exact weight ratios were titrated with the oil that was added dropwise under vigorous stirring by a magnetic stirrer until turbidity was appeared in the mixture. Relative turbidity or phase separation and the ease of emulsions formation were visually identified. The components ratio was converted to weight percentage before plotting the phase diagram. The shaded area within the triangle diagram represents the regain of biphasic.

2.3. Determination of solubility of Ert in SNEDDS components

A study was performed to determine the solubility of Ert in the SNEDDS components. To determine the saturation solubility, we added a certain quantity of Ert (approximately 20 mg) to vials containing 1.0 mL of each component and shook it for 24 h at ambient temperature to dissolve the added drug. To separate the unresolved Ert, we centrifuged the vials at 15,000 rpm for 20 min. The supernatants were carefully removed from each vial and Ert concentrations in the supernatants were then determined by a UV-vis spectrophotometer from Rayleigh UV-2100 (China). The absorption coefficients for Ert in the solutions were already determined by measuring the absorbance values of standard Ert solutions prepared in the SNEDDS components. The values of solubility were described as mean ± standard deviation.

2.4. Preparation of Ert-loaded SNEDDS (Ert-SNEDDS)

Based on the data obtained from solubility study and the pattern of ternary phase diagram, the drug-loaded SNEDDS was firstly prepared by mixing the desired amounts of olive oil (10 μL), Tween 80 (20 μL) and PEG 600 (70 μL). Then, the resultant mixture was shaken for a period of 5 min until a clear solution was obtained. Subsequently, certain weighed amount of the drug was added and the blend was mixed for 10 min until a homogeneous blend was generated. The final Ert-SNEDDS was obtained by diluting this last 4-component mixture in 10-fold with distilled water with agitation. The formulations were stored at 25°C until used.

2.5. Determination of size of droplets and zeta potential of SNEDDSs

Size of droplets and zeta potential of BL-SNEDDS and Ert-SNEDDS were measured by dynamic light scattering (DLS) technique using a Zetasizer of Malvern (UK) with three replicated measurement.

2.6. Stability of SNEDDSs

Stability of the blank SNEDDS (BL-SNEDDS) and Ert-SNEDDS was weekly assessed both visually and by DLS. SNEDDSs were kept for at least 3 months.

2.7. Measurement of loading capacity and encapsulation efficiency of Ert

To measure loading capacity and encapsulation efficiency of Ert into SNEDDS, we added 1.0 mL chloroform to 2.0 mL Ert-SNEDDS prepared using 6.0 mg Ert, and then mixed it with a vortex mixture for 5 min. Then, it was kept until two phases were separated, and the organic phase was removed. The Ert content of the organic phase was measured by HPLC.

2.8. HPLC analysis

Quantitative analysis of Ert in various excipients was performed by HPLC on a Waters instrument (USA) equipped with a column of C18 reverse phase of 250 mm × 4.6 mm and a UV-vis detector working at 330 nm. The mobile phase consisted of a mixture of phosphate buffer saline (20 mmol L⁻¹, pH 7.4, PBS), acetonitrile and methanol in ratios of 50:30:20 (V/V) [40]. The mobile phase was already filtered by a filter with pore size of 0.22 μm and degassed by a bath sonicator. Samples with a typical volume of 60 μL were injected into the system using a Hamilton syringe. The mobile phase flow rate was selected as 1.0 mL min⁻¹ and the retention time was about 15 min. To construct a calibration curve, we prepared standard Ert solutions in the mobile phase with various concentrations of 31–1000 µg mL⁻¹. All the experiments were done in triplicate.

2.9. In vitro ert release from SNEDDS

Ert release profile in vitro was recorded by the method of dialysis bag. 5 mL Ert solution in water (primary dissolved in DMSO following by dilution in water, free Ert) or Ert-SNEDDS (1.0 mg mL⁻¹) was stuffed into a dialysis bag (10000 MWCO), and inserted in 25 mL PBS containing 0.5% Tween 80 (PBS/Tween 80) to mimic the sink conditions [41], or 25 mL artificial gastric fluid (AGF), as two release media. The release medium was rotated at 100 rpm and 37°C. Aliquots (500 µL) were withdrawn at different intervals of predetermined time (0.5, 1, 2, 3, 5, and 8 h) from the release medium, and the same volume from fresh medium was replaced. The concentration of Ert in the samples was analyzed using HPLC.

2.10. In vivo drug release

Male Sprague-Dawley rats weighting 250–300 g were employed for the pharmacokinetic studies. The animals were kept with standard food and water ad libitum in their cages of environmentally controlled (23 ± 1°C, 12/12 h dark/light cycles) for at least 1 week before including in the experiment. Food was removed 12 h before dosing and at all times, water was available. All experimental protocols were conducted after being approved by the Animal Ethics Committee of Shiraz University of Medical Sciences (19950). The animals were separated in a random manner into two different groups of seven-animal each: group 1 (control group) was treated with free Ert and group 2 (test group) received Ert-SNEDDS.

All the rats were administered with a dose of 30 mg kg⁻¹ Ert [42] (as free Ert or Ert content of Ert-SNEDDS), through oral gavage on the day of the experiment. Approximately 0.5 mL blood samples were obtained via heart puncture at 20, 40, 60, 120, 240, 369, 480 and 600 min after the drug administration and under light diethyl ether anesthesia. Three blood samples were collected for each time point and transferred into microcentrifuge tubes. The plasma was subsequently harvested from these samples by centrifugation (5000 rpm, 3 min), and stored at −20°C until analysis.

For preparation of plasma samples for determination of Ert contents by HPLC, the plasma proteins were separated by precipitation with addition of acetonitrile and then vortex mixing for 2 min. The tubes were then centrifuged at 15,000 rpm for 5 min and the supernatant was collected for HPLC analysis using optimized chromatographic conditions detailed above.

2.11. Cytotoxicity assay

Pancreatic adenocarcinoma cell lines of AsPC-1 and PANC-1 from human were cultured in DMEM containing 10% FBS and penicillin/streptomycin, and incubated at 37°C, 5% CO₂, 95% air [19,43–45]. After the cells were sufficiently proliferated, the MTT assay was done to assess the cytotoxicity effect of free Ert, BL-SNEDDS and Ert-SNEDDS on the cells. 1 × 10⁴ cells were seeded into each well of 96-well plates, and after the cell sticking, the plates were incubated with different concentrations of free Ert or Ert-SNEDDS for 48 h and 72 h in a triplicate manner. Each well was washed twice with PBS, and 20 μL MTT dye was added (1.0 mg mL⁻¹ dissolved in PBS). The plates were kept for 3 h in dark at 37°C, and then the medium of each well was removed and 100 μL DMSO was added. The absorbances were measured using a plate reader of BioTek (USA) at 570 nm. For data analysis, the percentage of cell viability was measured from the mean optical density of each well.

2.12. Histopathology analysis

Liver, intestine and spleen were taken out from the animals, fixed in a 10% formalin solution at room temperature for the period of 5 days. From these tissues, the sections were separated and stained using hematoxylin-eosin dye and evaluated by light microscopy.

3. Results

3.1. Pseudo-ternary phase diagram of the SNEDDS components

Figure 1 shows this ternary phase diagram. The area within the closed line is the biphasic region, and the outer ones are the monophasic region; upon formation of SNEDDS, a clear, transparent and isotropic phase is formed.

Figure 1.

A ternary phase diagram plotted for olive oil, Tween 80 and PEG 600. The axes are presented in weight ratios. The area within the closed line is the biphasic region, and the outer ones are the monophasic region. Point F shows the ratios of olive oil, Tween 80 and PEG 600 employed for preparation of BL-SNEDDS and Ert-SNEDDS.

3.2. Solubility evaluation of ERT in SNEDDS components

To select the ratios of olive oil, Tween 80, and PEG 600 to form Ert-SNEDDS, we determined the solubility of Ert in these components as well as the toxicity of them against the cell lines. Typical UV-vis spectra of Ert dissolved in olive oil, Tween 80 and PEG 600, and standard curves of Ert solubility in these components are presented in Supplementary Material S1. Based on these results, solubility of Ert was obtained as 0.022 ± 0.002, 3.81 ± 0.05 and 6.18 ± 0.04 mg mL⁻¹ in olive oil, Tween 80 and PEG 600, respectively.

3.3. Size of droplets and zeta potential of SNEDDSs

The diameter of the oil droplets, as one of determinant parameters that regulates the release and absorption rates of a drug, in both BL-SNEDDS and Ert-SNEDDS was measured by particle size analysis. The results are shown in Supplementary Material S2, and based on the results, mean droplet sizes were obtained as 31.2 ± 0.4 and 83.9 ± 0.6 nm with polydispersity indices (PIs) of 0.04 and 0.05, for BL-SNEDDS and Ert-SNEDDS, respectively. The values of zeta potential for BL-SNEDDS and Ert-SNEDDS were also measured to be −3.5 ± 0.3 and −19.3 ± 0.7 mV, respectively.

3.4. Stability evaluation of SNEDDSs

To verify the BL-SNEDDS as well as Ert-SNEDDS stability, they were stored at room temperature for more than two months, and no phase separation, sedimentation color change or any change in the physical properties were observed for this period of time. Diameter of the oil droplets as well as zeta potential were also weekly monitored by DLS. Within two months, irregular changes in the diameter of the oil droplets and zeta potentials of BL-SNEDDS and Ert-SNEDDS were observed with relative standard deviations of 4.3% and 4.8% for droplet size, and 3.3 and 3.2% for zeta potential, respectively.

3.5. Ert loading capacity and encapsulation efficiency measurement

For determination of loading capacity and encapsulation efficiency of Ert into SNEDDS and its release from Ert-SNEDDS both in vitro and in vivo, HPLC analysis of Ert was performed. A calibration plot was firstly prepared, as represented in Supplementary Material S3. Using the calibration plot, the following figures of merit were attained. i) determination concentration range: 31.3 to 1000 μg mL⁻¹; ii) equation of regression: y=(124 ± 2)×+(6922 ± 787), R² = 0.9994; iii) relative standard deviation for reproducibility of determination of 125 µg mL⁻¹ of Ert: 3.4%; iv) detection limit (calculated as 3×sdb/slp, where sdb is a standard deviation for the blank signal and slp is a slope for calibration plot): 4.5 µg mL⁻¹; v) quantitation limit (calculated as 10×sdb/slp): 15 µg mL⁻¹; and vi) relative standard deviation of inter-day and intra-day determinations of 125 µg mL⁻¹ of Ert: 3.8 and 3.3%, respectively.

Using HPLC, loading capacity and encapsulation efficiency of Ert into SNEDDS were obtained using the following formulas:

$$\mathrm{Loading}\mathrm{capacity}\mathit{\hspace{0.17em}}=\mathit{\hspace{0.17em}}\mathrm{loaded}\mathit{\hspace{0.17em}}\mathrm{Ert}\mathit{\hspace{0.17em}}\mathrm{mass}\mathit{\hspace{0.17em}}/\mathit{\hspace{0.17em}}\mathrm{SNEDDS}\mathit{\hspace{0.17em}}\mathrm{mass}$$ (1)

$$\mathrm{Entrapment}\mathit{\hspace{0.17em}}\mathrm{efficiency}\mathit{\hspace{0.17em}}=\mathit{\hspace{0.17em}}\mathrm{loaded}\mathit{\hspace{0.17em}}\mathrm{Ert}\mathit{\hspace{0.17em}}\mathrm{mass}\mathit{\hspace{0.17em}}/\mathit{\hspace{0.17em}}\mathrm{added}\mathit{\hspace{0.17em}}\mathrm{Ert}\mathit{\hspace{0.17em}}\mathrm{mass}$$ (2)

as 22.7 ± 0.7 and 40.7 ± 0.5%, respectively.

3.6. In vitro Ert release analysis

The profiles of in vitro Ert release of free Ert and from Ert-SNEDDS were recorded in two media of PBS/Tween 80 as well as AGF, as shown in Figure 2. Release profiles of free Ert and Ert-SNEDDS in both media represented similar patterns. Free Ert release represented a high rate of dispersion in the media, while Ert release from Ert-SNEDDS showed very slow rate at the initial times, followed by faster rates (burst release) at the extended period of time. This sigmoid behavior observed for the patterns of Ert-SNEDDS release indicated a slow release behavior for the nanoemulsions formulation due to the Ert entanglement in this formulation.

Figure 2.

In vitro release profiles (as cumulative amounts of Ert release) of free Ert (Free Ert-PBS/Tween 80 and Free Ert-AGF) and Ert-SNEDDS (Ert-SNEDDS-PBS/Tween 80 and Ert-SNEDDS-AGF) from PBS/Tween 80 (Free Ert-PBS/Tween 80 and Ert-SNEDDS-PBS/Tween 80) and AGF (Free Ert-AGF and Ert-SNEDDS-AGF).

3.7. In vivo drug release analysis

Ert concentration in the rat plasma was quantified to record its time profile upon gavage of free Ert or Ert-SNEDDS with a dose of 30 mg kg⁻¹. The results are shown in Figure 3. The pharmacokinetic variables of the orally administered free Ert and Ert-SNEDDS were measured, and summarized in Table 1. The statistical analysis indicated that the difference between Cmax for free Ert and Ert-SNEDDS was significant (p < 0.05).

Figure 3.

Mean Ert concentration in the plasma over time upon rat administration either with free Ert or Ert-SNEDDS with a dose of 30 mg kg⁻¹ to obtain the pharmacokinetic parameters.

Table 1.

Pharmacokinetic parameters of oral administered free Ert and Ert-SNEDDS.

Parameter	Free Ert	Ert-SNEDDS
Cmax/µg mL−1	105 ± 4	793 ± 3
Tmax/min	20	60
AUC0–10 /mg h mL−1	0.76 ± 0.08	2.75 ± 0.14

The data was obtained from the results presented in Figure 3 upon quantification of Ert concentration–time profiles in the rat plasma. C_max, T_max, and AUC_0–10 denote the maximum Ert concentration, the time taking for Ert to reach its maximum concentration after administration, and area under the Ert plasma concentration curve for 0–10 h, respectively.

3.8. Cytotoxicity analysis

The MTT assay was performed for cell proliferation evaluation of the cytotoxicity of free Ert and Ert-SNEDDS against pancreatic adenocarcinoma cells of PANC-1 and ASPC-1. Firstly, the cytotoxicity of the components of Ert-SNEDDS was evaluated after 24 h, and the results are shown in Figure 4. The Ert-SNEDDS components represented low toxicity, and the highest cytotoxic one was Tween 80. This was one of the reasons for selection of point F in the ternary phase diagram (vide supra). The viability of ASPC-1 and PANC-1 cells was assessed upon incubation with free Ert as well as Ert-SNEDDS for 48 and 72 h, and the results are presented in Figure 5. The cell viability depended on the free Ert as well as Ert-SNEDDS concentration, and the cell growth was more effectively inhibited by Ert-SNEDDS, compared to free Ert. Statistical analyses of the data showed that for both the cell lines, both free Ert and Ert-SNEDDS, and at all the concentrations, decrements in the cell viabilities in the presence of either free Ert or Ert-SNEDDS were significantly different with the viabilities of the related controls. In addition, at concentrations ≤50 µg mL⁻¹ and incubation time of 48 h, PANC-1 cell viabilities had no significant differences upon treatment with either free Ert or Ert-SNEDDS. Cell viability differences at other conditions had significant differences. Therefore, just at concentrations ≤50 µg mL⁻¹ and incubation time of 48 h, Ert-SNEDDS had not effectiveness over free Ert, and at the other conditions, Ert loading into SNEDDS led to enhancement in its efficacy. Values of IC₅₀ for free Ert and Ert-SNEDDS against ASPC-1 and PANC-1 cells at two times of incubation of 48 and 72 h were calculated, as reported in Table 2.

Figure 4.

Viabilities of ASPC-1 (Olive oil/ASPC-1, PEG600-ASPC-1 and Tween 80/ASPC-1) and PANC-1 (Olive oil/PANC-1, PEG600/PANC-1 and Tween 80/PANC-1) cells upon treatment with different concentrations (0 to 10 μg mL⁻¹) of olive oil (Olive oil/ASPC-1 and Olive oil/PANC-1), PEG 600 (PEG600-ASPC-1 and PEG600/PANC-1), and Tween 80 (Tween 80/ASPC-1 and Tween 80/PANC-1), as the Ert-SNEDDS components. The incubation time for all the components with both the cell lines was 24 h. Panels A, B and C are related to viabilities of the cells upon treatment with olive oil, PEG 600, and Tween 80, respectively.

Figure 5.

Viabilities of ASPC-1 (panel A) and PANC-1 (panel B) cells upon treatment with different concentrations (0 to 150 μg mL⁻¹) of free Ert (Free Ert/ASPC-1/48 h, Free Ert/ASPC-1/72 h, Free Ert/PANC-1/48 h, and Free Ert/PANC-1/72 h) and Ert-SNEDDS (Ert-SNEDDS/ASPC-1/48 h, Ert-SNEDDS/ASPC-1/72 h, Ert-SNEDDS/PANC-1/48 h, and Ert-SNEDDS/PANC-1/72 h). The incubation times for both free Ert and Ert-SNEDDS with both the cell lines were 48 (Free Ert/ASPC-1/48 h, Ert-SNEDDS/ASPC-1/48 h, Free Ert/PANC-1/48 h, and Ert-SNEDDS/PANC-1/48 h) and 72 (Free Ert/ASPC-1/72 h, Ert-SNEDDS/ASPC-1/72 h, Free Ert/PANC-1/72 h, and Ert-SNEDDS/PANC-1/72 h) h. “ns” denotes not significant difference, and *, ** and *** indicate highly significant (p < 0.001), very significant (p < 0.01) and significant (p < 0.05) difference, respectively.

Table 2.

Values of IC₅₀ for free Ert and Ert-SNEDDS against ASPC-1 and PANC-1 cells at two incubation times of 48 and 72 h.

Formulation	Cell line	Incubation time/h	IC50/μg mL−1
Free Ert	ASPC-1	48	131.9
Ert-SNEDDS	ASPC-1	48	32.1
Free Ert	ASPC-1	72	39.1
Ert-SNEDDS	ASPC-1	72	18.4
Free Ert	PANC-1	48	57.1
Ert-SNEDDS	PANC-1	48	33.1
Free Ert	PANC-1	72	29.1
Ert-SNEDDS	PANC-1	72	9.5

3.9. Histopathological analysis

Histopathological analysis of the liver, intestine and spleen of the rats after Ert-SNEDDS administration (Supplementary Material S4) revealed that the tissues of these organs remained normal, and Ert-SNEDDS did not affect them.

4. Discussion

For selection of SNEDDS components, different criteria were considered. These include biocompatibility, formation of fine oil droplets with a narrow diameter distribution, creation of a stable emulsion with uniform physical form and solubility of the drug in the SNEDDS components. As to biocompatibility, we choose virgin olive oil that contains many naturally occurring compounds and is used orally; Ert-SNEDDS was designed to be orally administrated. PEG is also a well-known biocompatible polymer with a huge application in biological studies. The intricate point for the biocompatibility can arise from the surfactant. From the surfactants, nonionic ones are more biocompatible with less toxicity effects, compared to the ionic ones. Besides, there should be an attempt to select the amount of the surfactant as low as possible. In order that dispersion of fine oil droplets in water with a narrow size distribution form, several parameters such as full miscibility of the oil, surfactant and cosurfactant, and the value of hydrophilic-to-lipophilic balance (HLB) of the surfactant are determinant. For the former, there is a need to know the miscibility behavior of these ingredients; therefore, we needed to plot a ternary phase diagram for these components. For the latter, HLB value of a surfactant should be larger than 10 [46], and this value for Tween 80 is equal to 15. Therefore, Tween 80 would be a good choice. To form a stable nanoemulsion with a thermodynamic stability (formation of a stable and visual single phase), the aforementioned parameters of miscibility and HLB value of the surfactant are important. The cosurfactant also plays a critical role in stabilizing the oil droplets to keep them dispersed in water. As for the drug solubilizing ability of the components, we should determine the solubility of Ert in each component, and use the phase diagram to select the best formulation (ratios of the oil:surfactant:cosurfactant) with component(s) (as better solvent(s)).

A pseudo-ternary phase diagram was plotted for mixtures of olive oil, Tween 80 and PEG 600 to recognize the ratios of these components that provided a single phase, and form spontaneous nanoemulsions upon adding to water.

The determined solubility of Ert in SNEDDS components showed that the Ert solubility is the highest in PEG 600. On the other hand, from the SNEDDS components, Tween 80 was found to have the highest cell toxicity (vide infra). Therefore, we selected a point (denoted as F in Figure 1) for preparation of SNEDDS that included 10 µL olive oil, 20 µL Tween 80 and 70 µL PEG 600 to attain a high Ert loading into SNEDDS keeping a low cytyotoxicity. Upon dilution in water (1:10), these volumes of the SNEDDS components give a thermodynamically stable nanoemulsion with a low free energy value containing a very low amount of surfactant and a very high ampunt of solubilizing Ert solvent. At a molecular point of view, PEG 600/Tween 80-stabilized nanodroplets of the oils carry Ert that is mainly dissolved in the PEG 600 layer around the droplets in water.

The stability evaluation of BL-SNEDDS and Ert-SNEDDS showed two months as the stability time of the formulations.

The values obtained from particle size analysis indicated that upon Ert loading, the oil droplets dispersed in water were distended, arising most probably from the interaction of PEG 600 and Ert that leads to turning over of PEG 600 molecules from the oil ones. In addition, PI values confirmed the monodispersity of the oil droplets in both BL-SNEDDS and Ert-SNEDDS. Also, the zeta potential with enhanced negative value for Ert-SNEDDS leads to decrement in the formulation absorption by the circulating proteins in the bloodstream [47].

The resultant pharmacokinetic parameters from monitoring of the of the rats’ plasma level indicated that Ert-SNEDDS delivered Ert with higher levels at the time span of the experiment (10 h) and created the highest Ert level at longer time, compared to free Ert with the same dose. The oral bioavailability of Ert in rats was enhanced by Ert-SNEDDS by 3.62 times, compared to free Ert. This enhanced absorption of Ert by SNEDDS can be related to enhancement in the drug solubility by the SNEDDS components, drug release from an increased interfacial surface area (surface of the oil droplets) [35], improved oral absorption and intestinal lymphatic transport via transcellular pathway by SNEDDS [48], while the surfactant and cosurfactant of SNEDDS can improve the drug permeability through cell membrane disturbance [49]. It should also be added that as a potential direction for future researches, an in vitro release study in AGF for 2 h followed by simulated intestinal fluid for 4 to 6 h (pH 7.4) can be set up for systemic circulation for 24 h, thus mimicking the oral conditions.

The IC50 values of free Ert and Ert-SNEDDS that obtained from cytotoxicity measurement, indicated that SNEDDS enhanced the Ert toxicity, and upon loading into SNEDDS, its uptake by the cancer cells was enhanced, its dose was reduced and its efficacy was enhanced.

Histopathological analysis confirmed limited side effects of Ert-SNEDDS. It should also be noted that considering Ert function and action, and the SNEDDS entity, kidney, bone marrow and lung organs can also be considered for histopathological analyses in future investigations.

5. Conclusion

SNEDDSs are developed as simple, biocompatible and low-cost drug nanocarriers mainly for water-insoluble drugs. Here, olive oil as an oral and low-cost oil, Tween 80 as a well-known surfactant with suitable HLB value, and PEG 600 as a biocompatible and low-cost cosurfactant formed a SNEDDS for Ert. BL-SNEDDS comprised uniform 31.2-nm droplets that enlarged upon Ert loading to 83.9 ± 0.6 nm. SNEDDS developed for Ert enhanced its efficacy by enhancement in its cell toxicity, bioavailability and in vivo plasma level, and change in the release patterns. Ert-SNEDDS has been introduced as a novel Ert carrier for oral administration. Other SNEDDS formulations comprising the components employed in this study with other ratios, or comprising other oral oils, surfactants and cosurfactants can be considered for further studies on the Ert delivery systems. On the other hand, SNEDDS presented in this study may be applicable for enhancement of the efficacy of other poor water soluble drugs.

Funding Statement

This paper was not funded.

Article highlights

• A pseudo-ternary phase diagram for olive oil, Tween 80 and PEG 600 was constructed.

• A SNEDDS composed of 10% olive oil, 20% Tween 80 and 70% (V) PEG 600 was selected for Ert loading.

• ERT-SNEDDS formed oil droplets of 83.9 ± 0.6 nm.

• Loading capacity and entrapment efficiency were 22.7 ± 0.7 and 40.7 ± 0.5%, respectively.

• Ert-SNEDDS led to enhancement in the drug bioavailability, and changed the release route of Ert.

• Ert-SNEDDS showed enhanced cytotoxicity toward ASPC-1 and PANC-1 cells.

Author contributions

Maryam Karimi: Methodology, Formal analysis, Investigation, Data Curation, Writing – Original Draft, Visualization

Rezvan Dehdari Vais: Methodology, Investigation, Visualization

Khashayar Karimian: Methodology, Investigation, Writing – Review & Editing

Alireza Parsaei: Methodology, Investigation, Writing - Review & Editing Hossein Heli: Conceptualization, Methodology, Validation, Resources, Writing - Review & Editing, Supervision, Project administration, Funding acquisition

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/20415990.2025.2466412