Glyceryl ester surfactants: Promising excipients to enhance the cell permeating properties of SEDDS

Accepted Manuscript Research paper Glyceryl ester surfactants: Promising excipients to enhance the cell permeating properties of SEDDS Forouhe Zahir-Jouzdani, Noemi Lupo, Martin Hermann, Felix Prüfert, Fatemeh Atyabi, Andreas Bernkop Schnürch PII: DOI: Reference:

S0939-6411(17)31161-X https://doi.org/10.1016/j.ejpb.2018.05.032 EJPB 12787

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Revised Date: Accepted Date:

9 October 2017 15 April 2018 26 May 2018

Please cite this article as: F. Zahir-Jouzdani, N. Lupo, M. Hermann, F. Prüfert, F. Atyabi, A. Bernkop Schnürch, Glyceryl ester surfactants: Promising excipients to enhance the cell permeating properties of SEDDS, European Journal of Pharmaceutics and Biopharmaceutics (2018), doi: https://doi.org/10.1016/j.ejpb.2018.05.032

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Glyceryl ester surfactants: Promising excipients to enhance the cell permeating properties of SEDDS Forouhe Zahir-Jouzdani1,2,3, Noemi Lupo1, Martin Hermann4, Felix Prüfert 1, Fatemeh Atyabi2, Andreas Bernkop Schnürch1,5,*

1

Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, Innsbruck, Austria

2

Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, 14174,

Iran 3

Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran Iran

4

Department of Anaesthesiology and Critical Care Medicine, Medical University Innsbruck, Innsbruck, Austria

5

Thiomatrix Forschungs-und Beratungs GmbH, Research Center Innsbruck, Trientlgasse 65, 6020 Innsbruck, Austria

*Corresponding Author: Andreas Bernkop-Schnürch, Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria Tel.: +43-512-507 58601 30 Fax: +43-512-507 58699 e-mail: [email protected]

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Abstract Aim: The aim of the study is the evaluation of the impact of glyceryl ester surfactants on cell permeating properties of SEDDS (self-emulsifying drug delivery systems). Methods: SEDDS containing the glyceryl ester surfactants polyglyceryl-3-stearate (TGlysurf9), polyglyceryl-5oleate (TGlysurf11.5) and glyceryl stearate citrate (TGlysurf12) were prepared and characterized regarding droplet size and zeta potential. Toxicity studies were performed on Caco-2 cells using resazuring assay. The formulations were loaded with fluorescein diacetate (FDA) and curcumin, and cell uptake studies on Caco-2 cells were performed. Cell uptake was visualized via real time live confocal microscopy. Cell permeability of the SEDDS was tested and trans-epithelial electrical resistance (TEER) measurements were performed. Furthermore, the anti-proliferative and anti-migration activity of curcumin loaded in the SEEDS was investigated. Results: The developed SEDDS (0.05% m/v) showed no cytotoxicity on Caco-2 cells after 3 h of incubation. Glyceryl esters-SEDDS showed a significant higher FDA and curcumin cell uptake than SEDDS without glyceryl ester surfactants (p<0.05). TGlysurf9-SEDDS showed thereby the most pronounced permeation enhancing properties. TEER remained constant during the permeation study. Curcumin loaded in TGlysurf9-SEDDS exhibited 1.9-fold higher anti-proliferative effect than curcumin loaded in SEDDS without glyceryl ester surfactants. Furthermore, curcumin loaded in glyceryl ester-SEDDS inhibited Caco-2 cells migration to a higher extent than unloaded curcumin and curcumin loaded in SEDDS without the glyceryl ester surfactants. Conclusions: Glyceryl ester surfactants and in particular polyglyceryl-3-stearate might be a promising excipient for the formulation of SEDDS exhibiting enhanced cellular uptake and permeation enhancing properties. Key words: Cell permeation enhancer, cell uptake, glycerol ester surfactants, SEDDS, curcumin. 2

1. Introduction In the last years, self-emulsifying drug delivery systems (SEDDS), lipid based formulations consisting of oil, surfactant and/or co-surfactant, are gaining more and more interest as drug delivery systems due to their capacity to enhance the dissolution rate and the bioavailability of poorly water-soluble and permeable drugs [1]. SEDDS form stable o/w nanoemulsions under gentle agitation [2]. Moreover, SEDDS showed potential as gene delivery system. However, the cellular uptake of SEDDS remains poor. To overcome this issue, Mahoom et al. incorporated into SEDDS the cell penetrating peptide TAT [3]. The optimized formulation showed 2.6-fold increased cell uptake efficiency versus the blank SEDDS. As SEDDS contain a considerable amount of surfactants, which are known for their permeation enhancing properties [4], further knowledge about their effect on the cell uptake and permeation enhancing properties is of special interest. Particularly, glyceryl ester surfactants are gaining more and more attention as possible alternative to PEG based surfactants, that might contain hazardous impurities such as 1.4dioxane and ethylene oxide depending on the sources [5]. Glyceryl ester surfactants are mainly composed by mono- or polyglycerol esterified with fatty acids of variable length. Due to their safe toxicological profile and their multifunctional structure they are widely present in food and cosmetic products as emulsifiers, dispersants, thickeners, solubilizers, spreading agents, lubricants or emollients [6]. However, to our knowledge studies regarding their effect on SEDDS cell permeating properties are missing. To address this issue, SEDDS containing glyceryl ester surfactants were developed and characterized regarding size and zeta potential. As illustrated in Fig. 1, polyglyceryl-3-stearate (TGlysurf9), polyglyceryl-5-oleate (TGlysurf11.5) and glyceryl stearate citrate (TGlysurf12) (Fig.1) were chosen as representative model surfactants. Cell uptake studies using FDA and curcumin as model drugs were performed on Caco-2 cells. Moreover, the

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cell uptake was qualitatively visualized via real time live confocal microscopy. Permeability studies were performed and the trans-epithelial electrical resistance (TEER) of cell monolayers was measured before and at the end of permeability studies. Furthermore, the anti-proliferative and anti-migration effect of curcumin loaded in glyceryl ester surfactant based formulations was investigated. 2. Materials and methods 2.1. Materials Polyglyceryl-3-stearate (TGlysurf9), polyglyceryl-5-oleate (TGlysurf11.5) and glyceryl stearate citrate (TGlysurf12) were obtained from Dermofeel®, Germany. Ethyl oleate, glycerol 85, triacetin, Tween 80, fluorescein di-acetate (FDA), Nile-red and curcumin were purchased from Sigma-Aldrich (Vienna, Austria). Caco-2 cells were obtained from European Collection of Cell Culture (ECACC, Health Protection Agency, Porton Down, Salisbury, Wiltshire, UK). All other chemicals were of analytical grade and purchased from commercial sources. 2.2. Methods 2.2.1 Preparation and loading of SEDDS Different formulations were prepared by homogeneously mixing ethyl oleate (30 % m/m), triacetin (10 % m/m), glycerol 85 (10 % m/m) and Tween 80 (25 % m/m). To this mixture, glyceryl

ester

surfactants

(polyglyceryl-3-stearate

(TGlysurf9),

polyglyceryl-5-oleate

(TGlysurf11.5) and glyceryl stearate citrate (TGlysurf12) were added at a final concentration of 25 % (m/m) under heating at 50°C. Moreover, different formulations containing polyglyceryl-3stearate were prepared by decreasing the concentration of TGlysurf9 from 25% to 5%. The formulations were stored at room temperature and examined regarding phase separation within 4

24 h. The formulations were diluted in water (1:100) and characterized regarding size and εpotential (Malvern, UK). FDA and curcumin were incorporated into the SEDDS as follows. FDA was dissolved in acetonitrile (25 mg/ml) and incorporated into the SEDDS at a final concentration of 0.2%, whereas 2 mg of curcumin were dispersed in 10 µl of ethanol and loaded at a final concentration of 5% under heating at 50°C. 2.2.2. Cell viability assay Cell viability was investigated on Caco-2 cell via resazurin assay being based on the reduction of the blue non-fluorescent resazurin to the red fluorescent resofurin by metabolic active cells [7]. Caco-2 cells were seeded in 24 well plates at a concentration of 5×105 cells/ml. The cells were maintained at 37 °C under 5% CO2 and 90% relative humidity in minimum essential medium (MEM, containing phenol red, Earls salts, 10% fetal bovine serum and 1% penicillin and streptomycin). After 48 h incubation, cells were washed twice with pre-warmed (37 °C) 100 mM phosphate buffered saline (PBS) pH 7.4 and incubated with Opti-Mem (serum reduced medium without phenol red) for 30 min. Thereafter, SEDDS were diluted 1:100 in demineralized water and added to the medium at a final concentration of 0.05 % (m/v). Opti-MEM and Triton-X 100 (4 % m/v) were used as positive and negative control, respectively. After 3 h of incubation, cells were washed twice with pre-warmed PBS and 250 μL of resazurin solution (2.2 µl in Opti-MEM without phenol red) was added to cells. After 2 h, the fluorescence was measured at 540ex nm and 590em nm (Tecan® Infinite M200 spectrophotometer, Austria). 2.2.3. Cell uptake study Caco-2 cells were seeded in 24-well plates in a final concentration of 5×104 cells/ml. Cells were incubated for 10 days at 37 °C under 5% CO2 and 90% relative humidity in minimum essential

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medium. Caco-2 cells were washed twice with pre-warmed PBS and incubated with Opti-Mem for 30 min. Afterwards, SEDDS formulations loaded with FDA or curcumin were added to cells at final concentration of 0.05 % (m/v). After 2 h of incubation, cells treated with formulation marked with FDA were washed with pre-warmed PBS and lysed by Triton-X 100 solution (2 % (m/v) in 5 M NaOH), whereas cells treated with formulations containing curcumin were lysed with Triton-X 100 (2 % (m/v) in water. Lysed cells were collected and centrifuged at 800 rpm for 4 min. The lysate without removing the formulations was used as 100%, whereas the lysate of the untreated cells was used as 0%. Fluorescent intensity (FI) of FDA and curcumin were measured at 485ex nm and 515em nm and 442ex nm and 515em nm, respectively. The cellular uptake efficiency was calculated as follows (Eq.1):

Where FIF and FI100 represent the lysate with and without removing the formulation and FI0 represents the lysate of the untreated cells. 2.2.4. Real-time live confocal microscopy Caco-2 cells were seeded in a concentration of 5x104cells/well in 8-well chambered cover glass slides ibidi μ-slide 8 well™ slides (Ibidi, Munich, Germany) in 300 µl MEM supplemented with 20% FCS. After 24 h the cells were washed with pre-warmed PBS and incubated with Opti-Mem for 30 min. SEDDS loaded with Nile-red (0.01 w/v %) were added to the cells at final concentration of 0.05 % (m/v). As control, images of untreated cells and of cells incubated with Nile Red suspended in Opti-Mem (5*10-6 m/v %) were taken. After 2 h incubation at 37°C, the formulations were removed and fresh Opti-Mem was added. The cells were analyzed with a spinning disk confocal system (UltraVIEW VoX; Perkin Elmer, Waltham, MA) connected to a 6

Zeiss AxioObserver Z1 microscope (Zeiss, Oberkochen, Germany). Images were acquired with the Volocity software (Perkin Elmer); using a 63x oil immersion objective with a numerical aperture of 1.4. Images shown are z-stacks of 20 planes with a spacing of 0.5 µm. 2.2.5. Permeation study Caco-2 cells were seeded onto the donor compartment of Transwell 12-well plates (0.4 µm pore size, 1.13 1.13 cm2 permeation area) at a density of 5x104 cells/ml. Cells were maintained at 37 °

C under 5% CO2 and 90% relative humidity in minimum essential medium (MEM, containing

phenol red, Earls salts, 10% fetal bovine serum and 1% penicillin and streptomycin) for 2 weeks replacing the medium every second day. Cells were washed twice with pre-warmed PBS and incubated with Opti-Mem for 30 min. SEDDS loaded with FDA were added to the cells at final concentration of 0.05% (m/v). At predetermined time points, 50 µl of samples were withdrawn from the acceptor compartment and replaced with equal volume of pre-warmed fresh medium. The samples were diluted 1:1 with 5 M NaOH to cleave of the acetate group from FDA. The amount of permeated FDA was calculated as mentioned above. To determine the integrity of tight junction, TEER (trans-epithelial electrical resistence) measurements were performed before, during and after the permeation assay using an epithelial voltohmmeter (EVOM, World Precision Instruments, Germany). 2.2.6. Determination of the effect of curcumin loaded in SEDDS on Caco-2 cells 2.2.6.1 Anti-proliferation assay Caco-2 cells were seeded in 24-well plates (5x104 cells/ml) and cultured for 10 days as mentioned above. Cells were washed with pre-warmed PBS and incubated for 30 min with OptiMem. Afterwards, SEDDS loaded with curcumin (5% w/w) were added to cells supernatant 7

medium at final concentration of 0.05 % (m/v). After 2 h, cells were washed twice with prewarmed PBS and incubated for 24 h with fresh Opti-Mem. To evaluate the anti-proliferative effect of curcumin, cells were detached from each well by trypsin treatment (100µg/well). Cells were centrifuged and resuspended in 1 ml of pre-warmed medium. They were stained with trypan-blue and counted using Invitrogen® cell counter. Viability of Caco-2 cells in each well was determined by the same instrument. 2.2.6.2 In vitro wound healing assay For this purpose, Caco-2 cells were seeded in 6 wells plate at final amounts of 105cells/ml and cultured for 5 days. At the day of experiment, Caco-2 cells were washed with pre-warmed OptiMem and incubated with curcumin loaded SEDDS (0.05% (m/v), blank SEDDS (0.05% (m/v) and 0.025 mg of curcumin dissolved in Opti-Mem containing 0.1% of ethanol. Caco-2 cells were incubated with formulations for 2 h. Thereafter, supernatant medium was removed again and cells were washed twice with pre-warmed PBS. The cell monolayers were scratched using a sterile yellow tip and incubated with pre-warmed OptiMem. Images of the scratched monolayers were captured at time 0 and 24 h with Motic AE31 inverted microscope equipped with Progres CF scan using an objective 20X. 2.3. Statistical data analysis The statistical data analysis was done using the software GraphPad Prism 5. The means were compared using the analysis of variance (ANOVA) (α=0.05). Dunnett´s test was used as post hoc multiple comparison test. Level of p≤ 0.05 was set for significant (*), p≤ 0.01 for very significant (**) and p≤ 0.001 (***) for highly significant. The results were expressed as the mean of at least three experiments ± standard deviation (SD).

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3. Results and discussion 3.1. SEDDS preparation and characterization As reported in Tab. 1, the developed formulations displayed sizes in the range of 30-200 nm with a narrow PDI and a zeta potential ranging between -7 and -30 mv. Particularly, SEDDS containing glyceryl ester surfactants were characterized by a higher average size and more negative zeta potential than SEDDS without glyceryl ester surfactants, with the exception of TGlysurf11.5 containing SEDDS, whose size was comparable with the one of the control formulation. Moreover, the size of SEDDS containing different concentrations of polyglyceryl-3stearate (TGlysurf9) decreased with the decreasing of the surfactant concentration. The incorporation of FDA and curcumin in the SEDDS did not have any significant impact on the size, PDI and zeta potential of the nanodroplets (Tab. 1 and 2). 3.2. Cell viability assay As surfactants are known for their solubilizing effect on the plasma membrane, cytotoxicity studies were performed to evaluated the toxicity of the developed formulation on Caco-2 cells over a period of 3 h [8]. According to the results, SEDDS at a concentration of 0.05 % (m/v) resulted in 90% cell viability (data not shown). No significant difference was observed between the formulations containing the glyceryl ester surfactants and the control, which further confirms the safety of glyceryl ester surfactants. The results were in agreement with previous studies showing no toxicity of SEDDS on Caco-2 cells within the range of dilution from 1:500 to 1:2000 [9, 10]. Therefore, this concentration was used in all the further studies.

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3.3. Cell uptake study

The cellular uptake of the glyceryl ester surfactants containing SEDDS and the control formulation was assessed by quantification of sodium-fluorescein formed by activation of the internalized FDA with NaOH and of the uptaken curcumin. As illustrated in Fig. 2 A, formulations TGlysurf9, TGlysurf11.5 and TGlysurf12 displayed 2.2-, 1.2- and 1.9- fold increased FDA uptake versus the control formulation, respectively. The same trend was observed in the curcumin uptake study (Fig. 2 B). The formulations TGlysurf9, TGlysurf11.5 and TGlysurf12 showed 1.6-, 1.13- and 1.4- fold increased curcumin uptake in comparison to the control, respectively. Moreover, SEDDS containing different amounts of polyglyceryl-3-stearate (TGlysurf9) showed a concentration depend uptake efficiency in both uptake experiments (Fig. 2 C and D). However, SEDDS containing a concentration of polyglyceryl-3-stearate below 20% (TGlysurf9 (10%) and TGlysurf9 (5%)) did not show significantly different uptake efficiency when compared with the control formulation. As the most promising glyceryl ester surfactants polyglyceryl-3-stearate (TGlysurf9) and glyceryl stearate citrate (TGlysurf12) are characterized by the same alkyl domain and by different degree of glycerol polymerization, it is likely to believe that the degree of glycerol polymerization plays a crucial role in the cell permeating properties of stearate based glyceryl ester surfactants. It has been proven that compounds having hydroxyl groups and di-esters in their structure promote a significant disorder in the phospholipid bilayer resulting in a higher fluidity of the membrane. Indeed, other hydroxylate surfactants as the PEG-based surfactant Solutol HS15 (Macrogol 15 hydroxy stearate) have shown promising properties as permeation enhancer. Solutol HS15 is the main component of the novel absorption formulation CriticalSorb, which is in clinical trials for the nasal delivery of human growth hormone. CriticalSorb has shown promising effect also as intestinal permeation 10

enhancer. The interaction of Solutol HS15 with the plasma membrane of Caco-2 cells was proven by observing an increase in the endocytosis rate of Caco-2 cells, which correlates with lipid asymmetry induced by the internalization of the surfactant into the plasma membrane [11]. Despite the higher degree of glycerol polymerization, polyglyceryl-5-oleate (TGlysurf11.5) exhibited less pronounced cell permeating properties. This can be ascribed to the presence of the double bond in the structure, which turns of the aliphatic chain conferring to the glyceryl ester surfactant a more bulky and lipophilic character. In a previous study, polyglycerol ester of fatty acids increased the accumulation of daunomycin in Caco-2 cells without inhibition of the efflux transporter P-gp. Therefore, the observed effect was ascribed to the promotion of cellular uptake of the drug from the apical side and not to the inhibition of the efflux. Moreover, it was shown that these surfactants did not affect the integrity of tight junctions [12]. However, to our knowledge their cell uptake enhancing properties in SEDDS were never evaluated. Recently, the cell permeating properties of SEDDS have been enhanced by incorporating the cell penetrating peptide TAT into SEDDS after conjugation with oleoyl chloride. The SEDDS-TAT formulation containing FDA as fluorescent marker showed 2.3- fold higher cell uptake versus the control formulation after 2 h incubation on Caco-2 cells [3]. The results obtained with the SEDDS-TAT formulation are close to those obtained with TGlysurf9 containing SEDDS. Moreover, glyceryl ester surfactants offer the advantage to be less toxic than TAT, whose high toxicity is due to its positive charge [13]. However, whereas the enhanced cell uptake characterizing the SEDDSTAT formulation might be due to interaction of the peptide TAT with the heparin sulfate domains distributed on the cell surface, the mechanism involved in the enhanced uptake shown by SEDDS containing glyceryl ester surfactants is unknown.

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3.4. Real-time live confocal microscopy The uptake efficiency of glyceryl ester surfactants omitting SEDDS (control) and glyceryl ester surfactants containing SEDDS (TGlysurf9, TGlysurf11.5 and TGlysurf12) was visualized via real time live confocal microscopy. All the formulations were stained with Nile- red before performing the study. Fig. 3 demonstrates that all the formulations were taken up by cells as shown by the intracellular fluorescence, whereas the uptake of Nile-Red suspended in Opti-Mem was negligible. SEDDS containing glyceryl ester surfactants showed the most prominent cellular uptake confirming the data of the quantitative study. 3.5. Permeation study In numerous studies, both negatively and positively charged SEDDS showed to possess promising cell permeating properties, which seem to be strictly correlated to their composition. Indeed, depending on the type and amount of surfactants used for the preparation of SEDDS, different permeation pathways were observed. In a previous study, the cell permeability of ßlactamase loaded in SEDDS composed by Lauroglycol FCC, Cremophor EL and Transcutol HP was enhanced of 24.9-fold without disruption of the tight junctions. Moreover, in the study nanodroplets have been found in the acceptor compartment providing evidence of transcellular transport of the SEDDS [14]. On the contrary, the cell permeability of curcumin encapsulated in SEDDS containing Labrasol, Gelucire 44/14, Vitamin E TGPS and PEG 400 was 6.3-fold improved via both transcellular and paracellular pathways [15]. Labrasol and Gelucire 44/14 have been reported to decrease the TEER of Caco-2 monolayers in a concentration dependent manner [15, 16]. The cell permeability of FDA loaded in SEDDS with and without glyceryl ester surfactants is reported in Fig. 4. TGlysurf9-SEDDS increased FDA permeation by 1.66-fold compared to the control. On the contrary, the glycerol ester surfactants based formulations 12

TGlysurf11.5 and TGlysurf12 did not significantly enhance the FDA permeation respect to the control. Moreover, TEER measurements revealed that the TEER remained constant during the permeation study in all the wells (data not shown), meaning that the formulations did not disrupt the integrity of tight junctions. Therefore, the observed enhanced FDA permeation is mainly due to the transcellular transport of the SEDDS through the cell monolayer. In a previous work, glycerol esters of acetoacetic acid have been used as adjuvants to promote the rectal absorption of insulin loaded in suppositories [17]. However, to our knowledge data regarding their effect on SEDDS permeation properties are not reported in literature. 3.6. Determination of the effect of curcumin loaded in SEDDS on Caco-2 cells 3.6.1 Anti-proliferation assay Curcumin is a hydrophobic polyphenol extracted from the herb Curcuma longa. Curcumin has been widely studied for its multiple pharmacological applications as anti-inflammatory, antioxidant and anti-carcinogenic agent [18]. However, its therapeutic effect is limited by its low water solubility and poor bioavailability. In this study, the anti-proliferation effect of curcumin loaded in SEDDS on Caco-2 cells was evaluated to further confirm the effect of glyceryl ester surfactants on the cell penetrating properties of SEDDS. The results of the anti-proliferation assay are reported in Fig. 5. Curcumin loaded in TGlysurf9 and TGlysurf12 SEDDS exhibited 1.9-and 1.4-fold higher anti-proliferative effect compared to the curcumin loaded in SEDDS without glyceryl ester surfactants (control). However, TGlysurf11.5 did not significantly improve the curcumin anti-proliferative effect in comparison to the control formulation. These outcomes are in accordance with the results of the uptake study showing the superior cell penetrating enhancer effect of TGlysurf9 in comparison to the other glycerol ester surfactants. Indeed in previous studies, the antiproliferative effect of curcumin on cancer cells has been 13

reported to be dose dependent [19]. Moreover, in a previous work curcumin loaded in SEDDS showed an enhanced neuroprotective effect [2]. 3.6.2. In vitro wound healing assay The in vitro wound healing assay is a straightforward and economical method to study cell migration in vitro. This method is based on the observation that, upon creation of a new artificial gap, so called "wound", on a confluent cell monolayer, the cells on the edge of the newly created gap will move toward the opening to close the "wound" until new cell–cell contacts are reestablished [20]. In numerous studies, the anti-migrating effect of curcumin on different cancer cells has been reported [19, 21]. Curcumin inhibits cell migration by arresting the cell cycle and by inducing apoptosis [22]. Therefore, a higher curcumin cell uptake correlates with a more efficient inhibition of cell proliferation and migration. After 24 h of incubation, curcumin loaded in SEDDS inhibited cell proliferation and wound closure to a higher extent than curcumin, whereas cells treated with unloaded SEDDS almost closed the wound (Fig. 6). Particularly, curcumin loaded in glyceryl ester containing SEDDS (TGlysurf9, TGlysurf11.5 and TGlysurf12) showed a more pronounced inhibitory effect than in control SEDDS. However, it was not possible to establish a rank order among the glyceryl ester containing SEDDS. The results are in accordance with those of the anti-proliferating assay and confirm the superior cell permeating properties of SEDDS containing glyceryl ester surfactants.

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4. Conclusion In this study, the cell permeating properties of different glyceryl ester surfactants incorporated in SEDDS was evaluated. According to the presented results, certain glyceryl ester surfactants might represent a promising class of surfactants for the development of SEDDS with enhanced cell permeating properties. Among the tested glyceryl ester surfactants, polyglyceryl-3-stearate (TGlysurf9) seems to be most promising. Indeed, SEDDS containing TGlysurf9 showed the highest cell uptake efficiency and permeation across Caco-2 cell monolayer. Moreover, curcumin loaded in TGlysurf9 containing SEDDS showed the most pronounced antiproliferating and anti-migration effect. However, further studies are necessary to confirm the potential of this class of surfactant as permeation enhancers and their application in the development of lipid based drug delivery systems with enhanced cell permeating properties. Acknowledgments This work was supported by Tehran University of Medical Science, Tehran-Iran.

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[16] X. Sha, G. Yan, Y. Wu, J. Li, X. Fang, Effect of self-microemulsifying drug delivery systems containing Labrasol on tight junctions in Caco-2 cells, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 24 (2005) 477-486. [17] T. Nishihata, S. Kim, S. Morishita, A. Kamada, N. Yata, T. Higuchi, Adjuvant effects of glyceryl esters of acetoacetic acid on rectal absorption of insulin and inulin in rabbits, Journal of pharmaceutical sciences, 72 (1983) 280-285. [18] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavailability of Curcumin: Problems and Promises, Molecular pharmaceutics, 4 (2007) 807-818. [19] K. Mehta, P. Pantazis, T. McQueen, B.B. Aggarwal, Antiproliferative effect of curcumin (diferuloylmethane) against human breast tumor cell lines, Anti-Cancer Drugs, 8 (1997) 470-481. [20] C.-C. Liang, A.Y. Park, J.-L. Guan, In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro, Nature protocols, 2 (2007) 329-333. [21] V.K. Sirohi, P. Popli, P. Sankhwar, J.B. Kaushal, K. Gupta, M. Manohar, A. Dwivedi, Curcumin exhibits anti-tumor effect and attenuates cellular migration via Slit-2 mediated down-regulation of SDF-1 and CXCR4 in endometrial adenocarcinoma cells, The Journal of nutritional biochemistry, 44 (2017) 60-70. [22] D. Liu, Z. Chen, The Effect of Curcumin on Breast Cancer Cells, Journal of Breast Cancer, 16 (2013) 133-137.

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Figures: Fig. 1: Chemical structure of polyglyceryl ester surfactants. Fig. 2 (A+B+C+D): Uptake efficiency of SEDDS with (TGlysurf9, TGlysurf11.5, TGlysurf12) and without (control) glyceryl ester surfactants loaded with FDA (A) and curcumin (B) after 2 h incubation onto Caco-2 cell monolayer. Uptake efficiency of SEDDS containing different concentration of polyglyceryl-3-stearate (TGlysurf9): Control (0%), TGlysurf9 (25%), TGlysurf9 (20%), TGlysurf9 (10%), TGlysurf9 (5%) loaded with FDA (C) and curcumin (D) after 2 h incubation onto Caco-2 cell monolayer. Indicated values are means ± SD of at least three experiments; * p<0.05, ** p<0.01,*** p<0.001 versus the control. Fig. 3: Single optical plane images of untreated cells (A), cells treated with suspended Nile-Red (B), cells treated with SEDDS without glyceryl ester surfactants (control (C) and with glyceryl ester surfactants TGlysurf9 (D), TGlysurf11.5 (E), TGlysurf12 (F). SEDDS are labelled with Nile-Red. Fig. 4: FDA permeation across Caco-2 cells monolayer loaded in SEDDS with TGlysurf9 (X), TGlysurf11.5 (□) and TGlysurf12 (○) and without (control) glyceryl ester surfactants. Indicated values are means of at least three experiments ± SD; * p<0.05, ** p<0.01,*** p<0.001 versus the control. Fig. 5: Antiproliferative effect on Caco-2 cells of curcumin loaded in SEDDS with TGlysurf9, TGlysurf11.5 and TGlysurf12 and without (control) glyceryl ester surfactants. Indicated values are means of at least three experiments ± SD; * p<0.05, ** p<0.01,*** p<0.001 versus the control. Fig. 6: Whitefield images (Objective 20X) of Caco-2 cell monolayer untreated and treated with curcumin, curcumin loaded in SEDDS with TGlysurf9, TGlysurf11.5 and TGlysurf12 and without (control) glyceryl ester surfactants. The images were taken immediately after the wound healing assay and after 24 h incubation with Opti-Mem.

18

19

20

21

22

23

24

25

26

27

Tab. 1: Characterization of FDA (0.2% m/m) loaded glyceryl ester surfactants SEDDS having been diluted 1:100 with demineralized water. Indicated values are the means ± SD of at least three experiments.

Nomenclature

Composition

50% Tween 80 30% Ethyl Oleate 10% Triacetin 10% Glycerol 85 25% Tween 80 TGlysurf9 25% Polyglyceryl-3stearate 30% Ethyl Oleate 10% Triacetin 10% Glycerol 85 TGlysurf9(20%) 30% Tween 80 20% Polyglyceryl-3stearate 30% Ethyl Oleate 10% Triacetin 10% Glycerol 85 TGlysurf9(10%) 40% Tween 80 10% Polyglyceryl-3stearate 30% Ethyl Oleate 10% Triacetin 10% Glycerol 85 TGlysurf9(5%) 45% Tween 80 5% Polyglyceryl-3stearate 30% Ethyl Oleate 10% Triacetin 10% Glycerol 85 25%Tween 80 TGlysurf11.5 25% Polyglyceryl-5-oleate 30% Ethyl Oleate 10% Triacetin 10% Glycerol 85 25% Tween 80 TGlysurf12 25% Control

Nanodroplet size (nm) 37.3 ± 0.9

Zeta potential Polydispersity (mV) index (PDI) -7.2 ± 2

0.2

219.2 ± 7.5

-16.5 ± 8.3

0.3

149.7 ± 1.0

-10.9 ± 0.7

0.5

140.5 ± 6.7

-10.1 ± 2.0

0.5

29.2 ± 0.1

-12.7 ± 1.4

0.5

47.9 ± 3

-19 ± 7.1

0.2

229.0 ± 3

-28 ± 6.3

0.3

28

Glycerylstearatecitrate 30% Ethyl Oleate 10% Triacetin 10% Glycerol 85

Tab. 2: Characterization of curcumin (5% m/m) loaded glyceryl ester surfactants SEDDS having been diluted 1:100 with demineralized water. Indicated values are the means ± SD of at least three experiments. Nomenclature

Nano-droplet

Zeta potential (mV)

size (nm)

Polydispersity index (PDI)

Curcumin/Control

40 ± 18

-6.4 ± 4

0.2

Curcumin/TGlysurf9

217 ± 60

-21 ± 5

0.3

45 ± 7

-30 ± 6

0.2

232 ± 60

-28 ± 8

0.3

Curcumin/TGlysurf11.5 Curcumin/TGlysurf12

29

GA

30