In vitro evaluation of tumor targeting ability of a parenteral enoxaparin-coated self-emulsifying drug delivery system

Journal of Drug Delivery Science and Technology 53 (2019) 101144

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In vitro evaluation of tumor targeting ability of a parenteral enoxaparincoated self-emulsifying drug delivery system

T

Simona Giarraa, Noemi Lupob, Virginia Campania, Alfonso Carotenutoa, Laura Mayola, Giuseppe De Rosaa, Andreas Bernkop-Schnürchb,∗ a b

Department of Pharmacy, University of Naples Federico II, D. Montesano 49, 80131, Naples, Italy Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020, Innsbruck, Austria

ARTICLE INFO

ABSTRACT

Keywords: Enoxaparin Palmitoyl chloride Self-emulsifying drug delivery system Parenteral administration Tumor targeting

Enoxaparin (Enox) has been previously proposed as targeting ligands for tumor, due to the binding of the extracellular tumor matrix (ECM)components overexpressed in tumor tissues. Thus, in the present work we investigated the possibility to develop a parenteral Enox-coated self-emulsifying drug delivery system (SEDDS) for tumor targeting. Enox was chemically conjugated to palmitoyl chloride (PC) and then used to coat SEDDS. The formulations exhibited a size of 91–102 nm, and showed good stability in serum albumin and in plasma. Moreover, negligible hemolytic activity was found in the case of SEDDS and Enox-coated SEDDS. Furthermore, formulation coated with Enox showed a higher uptake on human breast adenocarcinoma and human epithelial colorectal adenocarcinoma cell lines, compared to uncoated SEDDS. The study provided the proof-of-principle to propose SEDDS for further investigation as novel candidate for tumor targeting by parenteral route of administration.

1. Introduction Novel cancer therapies based on active targeting to malignant cells represent a promising approach to overcome the numerous and serious side effects being associated with conventional chemotherapeutic drugs. The active targeting is achieved by decorating drug delivery systems with specific ligands having high affinity toward surface-receptors and/or antigens over-expressed in tumor tissues [1–8]. In this context, several studies demonstrated that low molecular weight heparin (LMWH), obtained by chemical or enzymatic depolymerization of unfractionated heparin (UFH), exhibit anticancer properties against different types of tumors [9–11]. The antitumor activity is related to their ability to bind various extracellular targets involved in the metastasis formation and over-expressed in many tumor tissues [12–14]. Moreover, LMWH can also inhibit the activity of P- and L-selectins being involved in cell-cell interactions, and of fibroblast growth factor (FGF) as well as vascular endothelial growth factor (VEGF), involved in angiogenesis process [15–18]. In particular, Enox has attracted great attention as tumor-active targeting ligand, due to its high binding affinity towards fibrinogen-derived products and angiogenic growth factors, which are over-expressed in the stroma of some tumors but not

in healthy tissues [19–22]. On the other hand, SEDDS are encountering a growing interest as drug delivery systems. They are based on a mixture of lipids, surfactants and co-solvents that, once in aqueous medium, under gentle agitation, spontaneously form a transparent and kinetically stable nanoemulsion [23–25]. Apart from their oral use, SEDDS have also attracted attention for the parenteral administration of hydrophobic drugs, particularly because of their physical stability, low costs of manufacturing and ease scale-up [26–28]. The aim of this work is to propose a novel use of SEDDS. The basic idea was firstly to propose SEDDS for intravenous administration, also demonstrating the possibility to functionalize this delivery system (until now exclusively used for oral administration) for tumor therapy. To this aim, Enox was used as potential active targeting ligand. In particular, thanks to the high-affinity binding of Enox to several components of the extracellular tumor matrix (ECM), we investigated the possibility to develop Enox-coated SEDDS as novel carrier for systemic administration and targeting tumors. It is worthy of note that Enox is used in this study only as model targeting ligand to provide a proof-of concept that can be translated to many other ligands. Because its hydrophilicity, unmodifiedEnox is not suitable for decorating SEDDS surface. For this reason, we chemically modified Enox with the fatty acid palmitoyl

Corresponding author. E-mail addresses: [email protected] (S. Giarra), [email protected] (N. Lupo), [email protected] (V. Campani), [email protected] (A. Carotenuto), [email protected] (L. Mayol), [email protected] (G. De Rosa), [email protected] (A. Bernkop-Schnürch). ∗

https://doi.org/10.1016/j.jddst.2019.101144 Received 26 January 2019; Received in revised form 6 June 2019; Accepted 4 July 2019 Available online 05 July 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.

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chloride (PC), in order to obtain the Enox-Palmitoyl (E-Pa) conjugate. The resulting amphiphilic conjugate could locate its hydrophobic fatty acid ester chain into the lipid core of nanoemulsion, while exposing the hydrophilic Enox on SEDDS surface. Then, the SEDDS were characterized in terms of size, ζ potential, protein adsorption, stability in plasma and hemolytic activity. Finally, in vitro cellular uptake of Enox-coated SEDDS was evaluated on two different cancer cell lines and compared with the uncoated SEDDS formulation with promising initial results. Thus, this study cannot provide a medicinal product for a specific application but only the proof of principle to foster further studies (encapsulation of different anticancer drugs, in vivo studies on specific tumor models).

solution and 10.5 M potassium hydroxide solution were added to 500 μL of about 1.2 mg/mL of sample solution in water and left to react for 15 min. Then, 750 μL of 3 M hydrochloric acid solution was added to the resultant mixture and the pH was adjusted to 1.1–1.5 with NaOH. Afterwards, 250 μL of 0.37 M iron (III) solution in 0.1 M hydrochloric acid and 125 μL of isopropyl alcohol were added to the sample solution and left to react for 10 min, thus obtaining a red to purple solution. The used reagents were kept at 4 °C until use. All steps were performed in ice bath at a temperature around 1 ± 0.1 °C. The ester concentrations were evaluated by measuring the absorbance at λ = 520 nm using a calibration curve of L-cysteine ethyl ester hydrochloride in the concentration range of 1.25–20 mg/mL (r2 = 0.995).

2. Materials and methods

2.5. Size exclusion HPLC (SEC-HPLC) for E-Pa quantification

2.1. Materials

The amount of unreacted Enox and of different E-Pa conjugates in the reaction mixture was quantified by SEC-HPLC following a method previously described by Matanović et al., but with some modifications [31]. The Chromaster Hitachi HPLC-systems was equipped with 5430 diode array detector, 5310 column oven, 5260 autosampler and 5160 pump. Analyses were performed on a BioSep-SEC-S 2000 column (300 × 7.80 mm, 5 μm) at a detection wavelength of 215 nm using a mobile phase composed of 1 mg/mL L-arginine solution (pH arranged to 6.5 with 0.1 M HCl). The flow rate was 1 mL/min, the injection volume was 90 μL and the temperature was set at 40 °C.

Peceol™ (glycerylmonooleate), and Labrafil M 1944 (oleoyl polyoxyl-6 glycerides) from Gattefossé (France). Enox (average MW 4500 Da), Palmitoyl chloride (PC), Cremophor EL (polyoxyl-35 castor oil), Transcutol® (diethylene glycol monoethyl ether), Fe (III) chloride, L-cysteine ethyl ester hydrochloride, toluidine blue, sodium chloride (NaCl), Triton X-100, fluorescein diacetate (FDA), sodium hydroxide (NaOH),tetrahydrofuran (THF), bovine serum albumin (BSA), dimethyl sulfoxide (DMSO) and n-hexane were obtained from Sigma-Aldrich (Austria). L-Arginine from Alfa Aesar GmbH (Germany), hydroxylamine hydrochloride from Acros Organics (USA) and potassium hydroxide (KOH) from Gatt-Koller (Austria) were used. Fetal bovine serum (FBS), phosphate buffer saline (PBS), minimum essential medium (MEM) and penicillin/streptomycin were purchased from Stricker (Austria). OptiMEM and RPMI medium were obtained from Gibco (Austria). All solvents, chemicals and media were of analytical grade and used as received. ®

2.6. Determination of Enox acylation by solution NMR investigation NMR spectra were acquired in D2O (Aldrich, Milwaukee, USA). NMR spectra were recorded with Varian UnityINOVA 500 MHz spectrometer. {1H-13C}-HSQC was optimized for 1JC-H = 135 Hz [32]. HSQC experiments were recorded using states-TPPI procedure for quadrature detection [33]. In the 2D experiments the time domain data consisted of 2048 complex points in t2 and 400-512 fids in t1 dimension. The relaxation delay was kept at 1.2 s. All the NMR spectra were acquired at T = 40 °C. The spectra were calibrated relative to residual HDO as internal standard (δ 4.65 ppm) for proton and external CH3OH (δ 49.5 ppm) for carbon-13. All spectra were processed with NMRPipe [34].

2.2. Uncoated SEDDS preparation Uncoated SEDDS were prepared as previously described by Zupančič et al. with some modifications [29]. Briefly, 20% Peceol, 40% Cremophor EL, 30% Labrafil-1944 and 10% Transcutol were gently mixed by vortex and kept at room temperature until use. 2.3. Synthesis of E-Pa conjugate

2.7. Enox-coated SEDDS preparations and characterization

The E-Pa conjugate was obtained through ester bond formation between the highly reactive acid halide PC and Enox hydroxyl groups; the reaction involves HCl release. Briefly, 500 μL of PC dissolved in tetrahydrofuran were added dropwise to 500 μL of Enox aqueous solution (500 μg/mL), at different molar ratios (Enox:PC 1:1; 1:50; 1:100; 1:150; 1:200). The resultant solution was mixed on a vortex for 2 min and then included on a thermomixer (Eppendorf), at 37 °C and 700 rpm for 1 h. Afterwards, the temperature was set at 25 °C and the sample was shaken at 700 rpm overnight to allow the evaporation of the organic solvent. The obtained aqueous phase, containing the E-Pa conjugate, was centrifuged at 13000 rpm for 20 min (Minispin, Eppendorf) to remove the unreacted PC, lyophilized (Christ Gamma 1-16 LSC Freeze dryer) and stored at 4 °C until use. Bromophenol blue (0.03% w/ v) was added to the resultant solution to test the progression of the reaction by measuring the pH of the different prepared conjugates.

The lyophilized E-Pa conjugates were dissolved in DMSO, added to the SEDDS pre-concentrate (at a final concentration of 1 mg/g) and included on a thermomixer at 25 °C and 700 rpm overnight, to allow the conjugate intercalation. Afterwards, the formulation was purified by dialysis membrane (cut-off 16000 Da) in water for 4 h. The mean droplet diameter and the ζ-potential of the uncoated formulation and of the Enox-coated SEDDS nanoemulsions were measured with a dynamic light scattering (Malvern, UK) by diluting the SEDDS pre-concentrate in physiologic solution at a final concentration of 1% (w/v) at room temperature. 2.8. SEDDS stability studies The adsorption of proteins on SEDDS surface is a critical issue for their in vivo stability after intravenous injection. The interaction with serum proteins and the possibility of self-aggregation were evaluated by monitoring the mean size and polydispersity index of the uncoated formulation (1 in the following) and of the corresponding Enox-coated formulation (1+E-Pa1:200 in the following) in serum albumin (1% w/v in water) and in plasma (diluted 1:100 in 20 mM phosphate buffer, pH 7.4), at final concentration of 1% (w/v), at 37 °C up to 4 h. ζ-potential stability analyses were also performed in water at 37 °C up to 4 h.

2.4. Evaluation of the degree of Enox-OH substitution by iron (III)/ hydroxylamine assay The degree of Enox-OH substitution was evaluated indirectly through iron (III)/hydroxylamine assay, a quantitative colorimetric method for carboxyl-ester determination previously described by Kakáč et al. [30]. Briefly, 250 μL of 15% (w/v) hydroxylamine hydrochloride 2

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2.9. Quantification of the amount of E-Pa1:200 on SEDDS surface by toluidine blue assay

well were seeded to a 24-well plate and incubated with 500 μL of different SEDDS concentrations (0.5 w/v, 0.25 w/v and 0.1 w/v) in optiMEM for 4 h. Untreated cells were used as negative control. After 4 h the formulations were removed and cells were washed twice with sterile PBS (pH = 7.1) and incubated with 500 μL of resazurin solution (44 μM) in white medium for 2 h. After incubation, the absorbance of the supernatant was measured by fluorometric assay (Microplatereader, TECAN Infinite M200; λex = 540 nm, λem = 590 nm). Results were calculated trough the following equation (1):

The amount of E-Pa1:200 conjugate on SEDDS surface was evaluated indirectly by toluidine blue colorimetric assay exploiting the capability of toluidine blue to bind the free sulfate groups of Enox [35]. Briefly, during the 1+E-Pa1:200 purification process, aliquots of the external water phase, containing the E-Pa1:200 conjugate released from the SEDDS, were withdrawn at predetermined time points and kept at 4 °C. Afterwards 300 μL of toluidine blue solution (25 mg in 500 mL 0.01 M HCl, containing 0.2% w/v NaCl) was added to 200 μL of each aliquot and left to react in a thermomixer at 25 °C and 700 rpm for 30 min. Then, 300 μL of n-hexane were added to the resultant solution, shortly mixed and the absorbance of the unreacted toluidine blue in the aqueous phase was measured at λ = 631 nm (Microplate-reader, TECAN Infinite M200). The amount of E-Pa1:200 conjugate on SEDDS surface was determined by subtracting the amount lost during the purification from the loaded one. The linearity of the response was assessed in the 7.8–125 μg/mL E-Pa1:200 concentration range (r2 = 0.996).

Cell viability (%) = (Fluorescence of treated cells-fluorescence of blank control)/ (Fluorescence of negative control-Fluorescence of blank control) X 100 (1) where blank control is the fluorescence of only medium without cells whereas negative control is the fluorescence of the untreated cells. 2.13. In vitro cellular uptake studies Fluorescent SEDDS were prepared by adding 20 μL of FDA solution (25 mg/mL in ACN) to 500 mg of formulations 1 and 1+E-Pa1:200. Approximately 1 × 105 MDA-MB-231 and 2 × 105 Caco-2 cells/well were seeded to a 24-well plate. Afterwards, cells were incubated with 0.25% (w/v) of formulations in opti-MEM. After 4 h of incubation, cells were washed twice with prewarmed sterile PBS (pH = 7.1), lysed with 5 M NaOH containing 1% (w/v) Triton-X and re-incubated for 30 min. The lysed cells without the removal of the formulations after 4 h of incubations were used as positive control (100% uptake), whereas untreated cells were used as negative control (0% uptake). At the end of the incubation time all samples were transferred in 2 mL tubes, centrifuged (13000 rpm, 5 min) and the intensity of fluorescence of the lysates were measured by fluorimeter (λex = 485 nm, λem = 555 nm). Results were expressed through the following equation (2):

2.10. In vitro hemolysis assay and sterility test The in vitro hemolysis assay is a fast and reliable test for evaluating the damage and lysis of erythrocytes cytoplasmic membrane. It was carried out with a method previously described by Mahmoud et al., but with some modifications [36]. Briefly, human blood was diluted with NaCl 0.9% (w/v) (1NaCl:10human blood) and then 0.2% (w/v) of uncoated and E-Pa1:200 coated formulations were added, mixed at 600 rpm for 30 s by vortex and incubated in a shaker bath at 37 °C for 4 h. The negative control (0% hemolysis) was obtained by diluting 10:1 human blood in NaCl 0.9% (w/v), whereas the positive control (100% hemolysis) was prepared by adding triton-X 0.5% (v/v) in diluted human

Uptake (%) = Fluorescence of lysate*/ Fluorescence of positive control × 100 blood with NaCl 0.9% (w/v). Afterwards, the samples were withdrawn, placed on ice for 2 min to quench erythrocyte lysis and centrifuged (3000 rpm for 5 min) to separate the supernatant from the pellet, consisting of intact red blood cells. The obtained supernatant was centrifuged another time (3000 rpm for 5 min) and the content of hemoglobin was measured at λ = 540 nm. To verify the possibility to sterilize SEDDS by filtration, the formulationswere diluted in NaCl (0.9% w/v) at a final concentration of 1% (w/v). The droplet mean diameter and polydispersity index were measured before and after filtration trough cellulose acetate filter (0.2 μm).

* with removing formulation

2.11. Cell cultures

3.1. Synthesis and characterization of E-Pa conjugate

Human breast adenocarcinoma MDA-MB-231 cell line, was cultured as monolayer in culture flask using RPMI medium, supplemented with 10% (v/v) FBS, 2 mM L-glutamine and penicillin/streptomycin solution (100 units/0.1 mg/L). Human epithelial colorectal adenocarcinoma Caco-2 cell line, obtained from European Collection of Cell Culture (ECACC, Health Protection Agency, Porton Down, Salisbury, Wiltshire, UK) was cultured as monolayer in culture flask using MEM medium supplemented with 10% (v/v) heat inactivated FBS and penicillin/ streptomycin solution (100 units/0.1 mg/L). Both cell cultures were maintained at 95% humidity and 37 °C in 5% CO2 atmosphere.

The E-Pa conjugate was obtained by covalently attaching PC to the hydroxyl groups of Enox, through an ester bond formation, as schematically represented in Scheme 1. A simple acylation reaction was carried out, thus allowing to get a water-soluble E-Pa conjugate in the aqueous phase of the reaction [37,38]. This approach was considered advantageous especially for formulations that could be proposed for further clinical and industrial development. Acronyms and the molar ratios of the different components used for the preparation of conjugates are summarized in Table 1. The acylation reaction released 1 mol of HCl per mole of formed esters. The HCl release led to a pH change in the solution, which was used to evaluate the progression of the reaction by adding bromophenol blue to the final solution. As expected, when increasing PC content, the pH value shifted from ∼5.3 (only Enox) to ∼ 1.8 (E-Pa1:200 conjugate) (data not shown). In order to evaluate the degree of Enox-OH substitution, iron (III)/hydroxylamine colorimetric assay was performed.

2.14. Statistical data analyses Statistical data analyses were performed using Student's t-test for comparison of two paired groups, assuming p˂0.05, p < .01 and p˂0.001 as levels of significance. All experiments were performed in triplicate and the results are reported as the means ± SD. 3. Results and discussion

2.12. In vitro toxicity assay The in vitro potential toxicity effect of the formulations 1 and 1+EPa1:200 were evaluated by resazurin assay using two different cell lines. Briefly approximately 1 × 105 MDA-MB-231 and 2 × 105 Caco-2 cells/ 3

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Scheme 1. Representation of synthetic reaction between Enox-PC and chemical structures of Enox, PC and E-Pa conjugate. Table 1 Acronyms, composition and degree of Enox-OH substitution (%), measured by iron (III)/hydroxylamine assay, of the different E-Pa conjugates. Conjugates Acronym

Enox (mol)

PC (mol)

Degree of Enox-OH substitution (%)

E-Pa1:1 E-Pa1:50 E-Pa1:100 E-Pa1:150 E-Pa1:200

1 1 1 1 1

1 50 100 150 200

0.000 45.26 62.10 70.54 84.38

± ± ± ± ±

0.61 3.70 2.32 1.15 2.13

The assay is based on the reaction between the ester groups with hydroxylamine to form a hydroxamic acid, which then reacts with ferric ion forming a spectrophotometrically visible chelate ferric hydroxamate complex. In this way, it was possible to determine the degree of Enox-OH groups reacted with the acyl halide PC. Results shown in Table 1 confirmed that the degree of Enox-OH substitution progressively increases with increasing amounts of PC being present at the coupling reaction, resulting in a maximum of 84.38% substitution for EPa1:200 conjugate. Moreover, SEC-HPLC analysis was performed to investigate the yield of the conjugation reaction. A single peak was found in Enox SEC-HPLC chromatogram at the retention time of 8.5 min, while no peaks were observed in the PC chromatogram. Differently, in the chromatograms of E-Pa1:50, E-Pa1:100 and E-Pa1:150 a second peak appeared at retention time of about 11.6 min, ascribable to the conjugate formation. In detail, the area of the peak attributed to the conjugate increased with increasing PC concentration, while the area of the peak at 8.5 min decreased. In the chromatogram of E-Pa1:200 only the peak at retention time 11.6 min was observed, thus suggesting that using E/PC molar ratio of 1:200, 100% of the Enox was converted into E-Pa conjugate. Representative chromatograms of Enox and E-Pa1:200 conjugate are illustrated in Fig. 1(a). The concentration (expressed as %

Fig. 1. Representative SEC-HPLC chromatograms of Enox and E-Pa1:200 conjugate (a) and concentrations (% v/v) of E-Pa conjugates and unreacted Enox in function of Enox/PC molar ratios (b), measured by integrating the area of the peaks acquired by SEC-HPLC chromatograms.

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conjugate. On the other hand, SEDDS showed a polydispersity index ranging from 0.190 to 0.255, suggesting that even due to the inclusion of E-Pa conjugates, SEDDS remained homogeneous in size. Moreover, the E-Pa1:200 conjugate additions resulted in a decrease of ζ potential from −4 mV to −11 mV. This could be ascribed to the amphiphilic properties of the conjugate that could locate its hydrophobic fatty acid ester chain into the lipid core of nanoemulsion, while exposing the hydrophilic and negatively charged Enox on SEDDS surface. 3.3. SEDDS stability studies The adsorption of proteins on the surface of nanocarriers is considered a crucial issue for their in vivo stability following administration. After injection, nanovectors are rapidly eliminated from the systemic circulation since they are able to recognize and adsorb serum proteins on their surface [39]. This could promote their fast aggregation rendering them easily recognizable for macrophages of endoplasmic reticulum and resulting in their subsequent elimination and liver accumulation, where they may cause side effects, and/or vessel occlusion [40,41]. Nanocarriers surface properties play therefore a key role in the opsonisation process since a more negative ζ potential can prevent aggregation through electrostatic stabilization. Table 2 shows a decrease of the ζ potential values obtained by adding E-Pa conjugates to the formulation. This can be reasonably ascribed to the negative charge of Enox, presumably present on the droplet surface. In line with this, the lowest ζ potential was observed in case of the conjugate with the highest content in PC, namely of E-Pa1:200, for which a more efficient intercalation between E-Pa fatty acid chains and the lipid phase of the droplets can be hypothesized. Therefore, it was considered for further studies. The mean diameter and the polydispersity index, before and after adding E-Pa1:200, were monitored in serum albumin and in human plasma at 37 °C. As it can be observed in Table 3 (a and b), the formulation was stable in presence of E-Pa1:200 conjugate up to 4 h. The inclusion of the E-Pa conjugate did not significantly affect the SEDDS stability in serum albumin and in human plasma. As for size and polydispersity index, also the ζ potential value of 1+E-Pa1:200 formulation was stable for 4 h at 37 °C (data not shown). Based on these results, it might be assumed that the prepared SEDDS, independently from the presence of E-Pa conjugate, should be stable in the systemic circulation.

Fig. 2. 1H NMR spectra of Enox (a) and E-Pa1:200 (b) in D2O solution, 500 MHz, at 40 °C.

v/v) of the different E-Pa conjugates obtained as well as of the unreacted Enox, measured by integrating the area of the peaks acquired by SEC-HPLC chromatograms, are reported in Fig. 1(b). Acylation of Enox was also confirmed by solution NMR investigation. 1D proton and 2D HSQC spectra were acquired both for Enox and E-Pa1:200 conjugate. 1H NMR spectra (Fig. 2 a,b) clearly show many variations upon the acylation step. In particular, the well distinguishable signal at 6.03 ppm attributable to the H4 of the ΔU2S (4,5-unsaturated 2-O-sulfated uronic acid residue) is down-field shifted at 6.35 ppm in agreement with the acylation of the vicinal 3-OH group. Signals at 1.31 and 0.90 ppm can be assigned to the acyl chain protons. Those signals appear very broad likely because the acyl chains form the hydrophobic core of micelles or other aggregate types hence resulting in a fast relaxation time of the corresponding signals. The HSQC spectra (Fig. 3 a,b) confirm that acylation of enoxaparin did occur. In fact, diagnostic methine signals at 5.35–5.10 ppm for the proton and 6870 ppm for the carbon appeared in the HSQC spectrum of the palmitoylated enoxaparin which are compatible with acylated CH groups. All together these results confirm the formation of the E-Pa conjugate via an ester bond between the fatty acid PC and the hydroxyl groups of Enox.

3.4. Quantification of the amount of E-Pa1:200 on SEDDS surface by toluidine blue assay It has been reported that the amount of heparin on the surface of nano-devices significantly influence their in vivo performance [35]. In this study, it was possible to reach a maximum of E-Pa conjugate concentration of 1 mg/g (E-Pa conjugate/SEDDS). When adding a concentration higher than 1 mg/g, increase in droplet size and instability of the dispersion was observed. The amount of E-Pa on SEDDS surface was indirectly evaluated by toluidine blue assay. Results showed a loss of 11% of E-Pa1:200 conjugate after 10 min from the preparation in the external water phase during the purification step. Afterwards, a plateau was reached suggesting no further release of conjugate up to 4 h (Fig. 4). This result demonstrated that 89% of E-Pa1:200 conjugate initially used in the preparation remains associated to the SEDDS and, due to the hydrophilicity of Enox, reasonably on the surface of SEDDS.

3.2. Preparation and characterization of uncoated and Enox-coated SEDDS Uncoated SEDDS formulation 1 after preparation was mixed with EPa conjugates prepared with different Enox/PC molar ratios, in order to obtain various Enox-coated SEDDS as summarized in Table 2. Both uncoated and Enox-coated SEDDS were diluted in physiologic solution (NaCl 0.9% w/v) at a final concentration of 1% (w/v), resulting in a clear nanoemulsion suitable for subsequent administration intravenously. Mean diameter, polydispersity index and ζ potential measurements of the prepared formulations are summarized in Table 2. The mean diameter of the SEDDS droplets was influenced by the addition of conjugates to formulations, as well as by the type of E-Pa

3.5. In vitro hemolysis assay and sterility test One of the key requirements for a preparation intended for parenteral administration is its hemocompatibility. The results of the

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Fig. 3. 1H-13C HSQC spectra of Enox (a) and E-Pa1:200 (b) in D2O solution at 40 °C.

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hemolysis assay showed for both uncoated and 1+E-Pa1:200-coated SEDDS a hemolysis lower than 2% generally regarded as non-toxic level (Fig. 5) [42]. Furthermore, another key requirement for systemic administration is the possibility to sterilize the formulation. Thus, both formulations were diluted in physiologic solution and filtered using 0.22 μm cellulose acetate filters. The size as well as the polydispersity index of both formulations did not undergo a significantly change after filtration (data not shown). These data indicated that it is possible to obtain a sterile formulation by filtration, thus avoiding the high-temperature autoclave sterilization procedure, which may result detrimental for the product [43]. All together these results support that the prepared formulations possess the key requirements for parenteral administration.

Table 2 Size and polydispersity index in physiologic solution (NaCl 0.9% w/v) and ζ potential in deionized water of formulations at 0 times, before and after addition of different E-Pa conjugates. Form.

d (nm)t=0

PI

1 1+E-Pa1:50 1+E-Pa1:100 1+E-Pa1:150 1+E-Pa1:200

91.1 ± 1.1 94.1 ± 0.3 92.3 ± 1.2 99.0 ± 0.3 102 ± 0.1

0.25 0.21 0.19 0.20 0.23

ζ potential (mV)t=0

t=0

± ± ± ± ±

0.1 0.1 0.1 0.1 0.1

−4.04 −6.74 −7.73 −7.29 −11.2

± ± ± ± ±

0.3 0.5 0.3 0.2 0.1

Table 3 Size stability and polydispersity index of formulations, with and without EPa1:200, in serum albumin (a, 1% w/v) and in plasma (b, 1:100) at 0 time and after 4 h at 37 °C. Form.

Viability of MDA-MB-231 and Caco-2 cells in the presence of uncoated and 1+E-Pa1:200-coated SEDDS was assessed via resazurin assay. Cells were incubated with formulations at different concentrations (0.1, 0.25 and 0.5% w/v). As illustrated in Fig. 6 a) concentration of 0.5% w/ v is clearly toxic on MDA-MB-231 cells. Indeed, formulation 1 and 1+E-Pa1:200 at 0.5% w/v showed a cell growth equal to 66.1% and 44.9% respectively on MDA-MB-231 cells whereas 95.1% and 95.2% respectively on Caco-2 cells. More in detail, formulation 1 is significantly different at concentrations of 0.1% w/v and 0.25% w/v compared to formulation 1 at a concentration of 0.5% w/v (***) on MDA-MB-231 cells. Moreover formulation 1+E-Pa1:200 is significantly different at concentrations of 0.1% w/v and 0.25% w/v compared to formulation 1+E-Pa1:200 at a concentration of 0.5% w/v (***) on MDAMB-231 cells. Not significantly differences were found by comparing both formulations 1 and 1+E-Pa1:200 at concentration of 0.1% w/v with 0.25% w/v and each other. Furthermore, not significantly differences were observed by comparing both formulations 1 and 1+EPa1:200 at all analyzed concentrations and each other on Caco-2 cells. All experiments were performed in triplicate and the results are reported as the means ± SD (Student's t-test, *p < .05,**p < .01,***p < .001). Therefore, as the maximum concentration of 1+EPa1:200 evaluated in this study, with not toxic effect on both cell lines analyzed, was 0.25 %v/w, the latter was chosen to perform the cell uptake study. It must be taken into account that any formulation, after injection into the body, will be diluted by intravascular fluids. Thus, 0.25% v/w would be a concentration suitable for future in vivo studies. Cell uptake of uncoated and Enox-coated SEDDS, loaded with fluorescein diacetate (FDA) was evaluated via cell uptake studies on both MDA-MB-231 and Caco-2 cell lines by measuring fluorescence derived from hydrolytic cleavage of FDA with NaOH. As shown in Fig. 7 (a and b) the uptake of 1+E-Pa1:200 coated SEDDS was found to increase 1.36-fold and 1.86-fold on MDA-MB-231 and Caco-2 cells, respectively, compared to uncoated formulation. It is well known that several components of the extracellular tumor matrix (ECM), such as different cytokines and grow factors are over-expressed in different kind of tumors exhibiting binding domains for UFH and its derivatives [44]. Based on these considerations the presence of sulfate motif of Enox seems to be crucial for the interactions between the ECM binding domains and Enox coated delivery systems. As previously described, the acylation reaction between Enox and PC to form E-Pa conjugate, leads to an ester bond formation between the fatty acid and the hydroxyl groups of Enox. By this way, it was possible to preserve the sulfate groups, as confirmed by toluidine blue assay and consequently the Enox binding activity. Moreover, it has been found that heparin and its derived products bind fibroblast growth factor receptor 1 (FGFR-1) overexpressed on different tumor cells. Finally UFH conjugates can also bind several drug trasporters of the ATP-binding cassette (ABC) family thus reverting multidrug resistance in cancer cells. Thus, different

BSA 37 °C d (nm)t=0

PIt=0

d (nm)t=4h

PIt=4h

1 1+E-Pa1:200

113.1 ± 8.41 111.8 ± 1.34

0.21 ± 0.08 0.23 ± 0.01

114.1 ± 4.88 106.7 ± 4.46

0.16 ± 0.04 0.21 ± 0.01

Form.

PLASMA 37 °C d (nm)t=0

PIt=0

d (nm)t=4h

PIt=4h

108.3 ± 1.07 103.4 ± 4.31

0.23 ± 0.01 0.20 ± 0.01

111.8 ± 1.81 110.1 ± 3.11

0.24 ± 0.05 0.24 ± 0.01

1 1+E-Pa1:200

3.6. In vitro cell studies

Fig. 4. Amount (%) of E-Pa1:200 on 1+E-Pa1:200 coated SEDDS surface, evaluated by toluidine blue assay during the purification process, up to 4 h.

Fig. 5. Hemolysis (%) of formulations 1 and 1+E-Pa1:200 (0.2% w/v), after 4 h of incubation in human blood (diluted 1:10 with NaCl 0.9% w/v) at 37 °C.

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Fig. 6. Cell growth (%) of MDA-MB-231 cells (a) and Caco-2 cells (b) treated with formulations 1 (black bar) and 1+E-Pa1:200 (white bar)at different concentrations (0.1,0.25 and0.5% w/v), after 4 h of incubation. Results are expressed as means of three independent experiments ± SD (Student's t. test, ***p < .001).

successfully prepared with an easily upscalable method. This was achieved by firstly preparing an amphiphilic conjugate able to direct the spontaneous exposition of Enox moieties on SEDDS surface. The obtained formulations showed good stability in serum albumin and in plasma with a negligible hemolytic activity. The in vitro cell uptake studies showed that the decoration of SEDDS with Enox increases the internalization efficiency on both MDR-MB-231 and Caco-2 cells, when compared with uncoated formulation. These results highly encourage the investigation of Enox-coated SEDDS in future studies as carrier for parenteral t delivery and targeting of anticancer drugs. Declarations of interest None.

Fig. 7. Uptake efficiency (%) of formulations 1 and 1+E-Pa1:200 (0.25% w/v), after 4 h of incubation upon MDA-MB-231 cells (a) and Caco-2 cells (b). Results are expressed as means of three independent experiments ± SD (Student's t. test,**p < .01).

Funding This research did not receive any specific grant from funding agencies in the public, commercial, ornot-for-profit sectors.

interactions could be responsible of the enhanced internalization of the 1+E-Pa1:200-coated SEDDS into cancer cells. Results indicate that the presence of the Enox on SEDDS surface increase cell uptake on both MDA-MB-231 and Caco-2 cell lines.

Acknowledgments The authors thank Abitec for donating as gift Capmul® PG8, Gattefossé for Peceol™ and Labrafil® M and Professor Ira-Ida Skvortsova (EXTRO-lab, Department for Therapeutic Radiology and Oncology, Medical University Innsbruck) for MDA-MB-231 cell lines.

4. Conclusions In the present study, Enox-coated SEDDS for tumor targeting were Annex Section Materials and methods

Heating-cooling cycle: SEDDS and 1+E-Pa1:200 were exposed to three cycles heating and cooling between 4 and 45 °C for 48 h [45–48]. Centrifugation tests: The formulations stable under heating-cooling cycle were then centrifuged for 30 min at 3500 rpm [45,47]. Freeze-thaw cycle: SEDDS were finally exposed to three freeze-thaw cycles between −21 °C and 25 °C for 48 h [45–48]. Robustness to dilution SEEDS and 1+E-Pa1:200 formulations were diluted with 0.9% p/v NaCl solution for 25, 50, 100, and 250 times followed by evaluation of their particle size [45–48]. After the experiments, samples were observed for physical change, separation, cracking and coalescence. For the robustness to dilution possible changes of the droplet size were also checked. All the tests were done in triplicate (n = 3).

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Results The results of the thermodynamic tests are summarized in Table 1. SEDDS and 1+E-Pa1:200 formulations passed the stress tests since both formulations were found stable after heating and cooling cycles, centrifugation, and freeze-thaw cycles. Indeed, no sign of alteration i.e. phase separation, turbidity, precipitation was observed suggesting that formulations resist to mechanical solicitations and critical temperatures. Table 1

Thermodynamic stability studies of 1 and 1+E-Pa1:200 formulations (n = 3). Thermodynamicstabilitystudies Form

Centrifugationcycle

Heating-coolingcycle

Freeze-thaw cycle

clear passed clear passed

clear passed clear passed

Aspect 1 1+E-Pa1:200

clear passed clear passed

This test was carried out to simulate the dilution process in the in vivo conditions. In order to imitate SEDDS gradual dilution after intravenous administration, a saline solution (NaCl 0.9% p/v) was used. Samples did not show any phase separation or precipitation suggesting that SEDDS were robust to all dilutions. Moreover, SEDDS particle size remained stable after dilutions (Table 2). Table 2

Robustness to dilution test of SEDDS and ENOX-SEEDS (n = 3). The variations of the averages are shown as SD. Form

1 1+E-Pa1:200

size (nm) ± SD After preparation

1:20

1:50

1:100

1:250

82.3 ± 3.14 105.2 ± 8.34

89.8 ± 8.6 106.7 ± 1.55

89.50 ± 0.58 117.7 ± 3.11

88.8 ± 3.46 118.3 ± 0.63

89.7 ± 0.36 119.6 ± 0.84

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