Materials

Tuftsin acetate was provided by Bachem AG (Bubendorf, Switzerland). Aminopeptidase N from porcine kidney, octanoic acid (caprylic acid), penicillin, streptomycin, phosphate buffered saline (PBS), trypsin and fetal bovine serum (FBS) were obtained from Merck KGaA (Darmstadt, Germany).

Isopropyl myristate, sodium lauryl sulfate (LS), 4-nitroaniline, 4-(2- hydroxyethyl)− 1-piperazineethanesulfonic acid (HEPES, ≥ 99.5%) and tris(hydroxymethyl)aminomethane (TRIS, ≥ 99.9%) were purchased from Carl Roth GmbH (Karlsruhe, Germany). 2-Octyldodecan- 1-ol was supplied by TCI (Eschborn, Germany). L-Leucine- 4-nitroanilide and 1-octanol were obtained from ThermoFisher GmbH (Kandel, Germany). Sodium docusate (AOT) and sodium N-octadecyl sulfate (OS) were provided by Alfa Aesar. FaSSIF/FaSSGF powder was obtained from Biorelevant (London, United Kingdom). Water for HPLC and D-glucose anhydrous (≥ 99.5%) were supplied by VWR (Linz, Austria). PEG- 35 castor oil (Kolliphor® EL), polyoxyethylene (20) oleyl ether (Brij® O20), polyoxyethylene (10) oleyl ether (Brij® O10), eugenol, citronellol, trifluoroacetic acid (TFA), acetonitrile, minimum essential medium eagle (MEM), resazurin sodium salt, Triton-X 100, potassium chloride, calcium chloride and sodium chloride were purchased from Sigma-Aldrich (Vienna, Austria).

Tuftsin quantification via HPLC

Tuftsin quantification was carried out using RP-HPLC, following a methodology previously described [16] with some modifications. The analytical setup comprised a Chromaster 5430 diode array detector, Chromaster 5310 column oven, Chromaster 5260 auto sampler, and Chromaster 5160 pump. The stationary phase featured a Waters XSelect® HSS C18 column (4.6 × 250 mm, 5 µm) (Vienna, Austria). A binary solvent system consisting of water and acetonitrile (96:4, v/v) with 0.1% (v/v) trifluoroacetic acid (TFA) served as the mobile phase. Isocratic elution was conducted at a flow rate of 1.2 mL/min over a duration of 10 min. The column temperature was set to 40 °C and the injection volume was 20 µL. Tuftsin was detected at a wavelength of 220 nm. A calibration curve covering a concentration range from 1 to 0.008 mg/mL was established (R2 > 0.99).

Preparation and characterization of SEDDS

For the preparation of SEDDS lipids, surfactants, and cosolvents resistant to lipase were chosen to ensure sufficient stability of the formulations. Excipients as listed in Fig. 1D were homogenized using a Thermomixer at 60 °C and 2000 rpm for 2 h to produce the SEDDS preconcentrates. Post-preparation, preconcentrates were emulsified in a concentration of 1% (v/v) in demineralized water or 5% (v/v) in 50 mM Tris buffer pH 6.5 for the characterization of size and polydispersity index (PDI). Additionally, the zeta potential was determined for samples emulsified in demineralized water. The resulting nanoemulsions were preheated to 37 °C and agitated using a thermomixer before measurement.

Data were obtained via dynamic light scattering (DLS) for size measurements and electrophoretic light scattering for zeta potential using the ZetaSizer Nano ZSP (Malvern Instruments in Worcestershire, United Kingdom). All samples were analyzed in triplicates at 37 °C.

Furthermore, the stability of the nanoemulsions was determined at a concentration of 1% (v/v) in biorelevant media, including fasted state simulated gastric fluid (FaSSGF) and fasted state simulated intestinal fluid (FaSSIF) [17], as well as in 20 mM HBS pH 6.5 and 50 mM Tris buffer pH 6.5. The size and PDI of the emulsions were monitored over a 4 h time period.

HIP-loaded SEDDS for degradation studies were prepared by first dissolving the HIP in SEDDS preconcentrates through agitation on a thermomixer at 37 °C and 2000 rpm overnight. The concentrations employed for SEDDS- 1, SEDDS- 2, and SEDDS- 3 were 20 mg/mL, 26 mg/mL, and 30 mg/mL, respectively. These concentrations were derived from the maximum solubility of HIP in SEDDS preconcentrates as described in the following chapter. Subsequently, SEDDS preconcentrates containing HIP were emulsified 5% (v/v) in 50 mM Tris buffer pH 6.5 or 20 mM HBS pH 6.5.

Hydrophobic ion pairing

Hydrophobic ion pairing was carried out to increase lipophilicity of tuftsin. To that end the peptide was dissolved in a concentration of 1 mg/mL in 0.01 M HCl. Counterions, namely AOT, OS, and LS, were dissolved in 1 mL 0.01 M HCl with molar ratios of 3:1, 4.5:1 and 6:1 corresponding to charge ratios of 1, 1.5 and 2 (counterion:tuftsin) [18, 19]. To form the complex, 500 µL of the counterion solution were added dropwise to 500 µL of the peptide solution using a Thermomixer (Eppendorf AG, Germany). The mixture was incubated at 25 °C while being agitated at 400 rpm for 1 h. Afterwards, the samples were centrifuged at 12,500 rpm for 15 min using a MiniSpin® (Eppendorf AG, Germany) [18, 20].

The resulting precipitate of tuftsin-counterion complex was washed twice with 0.01 M HCl and then lyophilized (Christ Gamma 1–16 LSC Freeze Dryer, Martin Christ Gefriertrocknungsanlagen GmbH, Germany). The obtained complex was stored at − 20 °C until further use.

Maximum solubility and payload of ion pair in SEDDS preconcentrates

In order to determine the maximum solubility of the hydrophobic ion pair (HIP) in SEDDS preconcentrates, the latter were added to an excess of HIP and the resulting samples were agitated for 24 h at 37 °C. Following this incubation, the samples were centrifuged at 12,500 rpm for 15 min to separate undissolved HIP and 10 µL aliquots were collected from the supernatant [18]. Before injection into the HPLC system, these aliquots were diluted in a ratio of 1:9 with methanol containing 0.1% (v/v) TFA to facilitate HIP dissociation and to determine the concentration of dissolved HIP. The weight of maximum dissolved HIP corresponds to the maximum payload (wt%) of HIP in SEDDS preconcentrates [21]. Therefore, the weight of each SEDDS preconcentrate was determined by weighing 1 mL aliquots using an analytical balance (Sartorius MSE225P- 100-DI, Göttingen, Germany). Payloads were calculated by Eq. (1):

$$payload\;\left(wt\%\right)= \frac\;\times 100$$

(1)

Log P1-octanol/water and log DSEDDS/release medium determination

The distribution coefficient (logP) of HIPs and tuftsin between 1-octanol and water was determined. HIPs were dissolved and tuftsin was dispersed in 1-octanol. Subsequently, an equal volume of water was added to the organic phase and the samples were incubated for 24 h at 37 °C with continuous agitation at 400 rpm using a thermomixer [22]. Following the incubation period, the samples were subjected to centrifugation at 12,500 rpm for 15 min to separate the phases. The aqueous phase was directly injected into the HPLC system, while the 1-octanol phase was diluted with a mixture of 0.1% (v/v) TFA in methanol at a 1:3 ratio. LogP values were calculated by using Eq. (2):

$$\text_=log\frac_}_}$$

(2)

The distribution of HIP between SEDDS and release medium was determined by quantifying the maximum solubility of HIPs in SEDDS preconcentrates and HEPES buffered saline (HBS) composed of 20 mM HEPES, 1 g/L glucose anhydrous, 136.7 mM NaCl, 5 mM KCl and 1 mM CaCl2. An excess amount of HIP was added to the respective medium and samples were agitated for 24 h at 37 °C [23]. After incubation, the samples were centrifuged at 12,500 rpm for 15 min and the concentration of tuftsin in the supernatant was determined. The calculation was performed using Eq. (3):

$$\text_=log\frac_}_}$$

(3)

Cell viability – resazurin assay

The potential cytotoxic properties of HIP-loaded SEDDS were investigated via resazurin assay at various SEDDS concentrations. Caco- 2 cells were seeded at a density of 5 × 104 cells per well in a 96-well plate and cultured for 72 h at 37 °C, with 95% humidity and 5% CO2 to reach confluency. The culture medium employed was Minimum Essential Medium (MEM), containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) and a penicillin/streptomycin solution (final concentration 100 units/0.1 mg/L) [24].

SEDDS were emulsified in sterile HBS at pH 7.4 at concentrations of 0.01%, 0.0125%, 0.025%, and 0.05% (v/v). A 0.1% (v/v) Triton-X solution in HBS served as a positive control, while sterile HBS applied to cells served as the negative control.

After removing MEM, cells were washed twice with prewarmed HBS. The cells were treated with SEDDS for a period of 4 h and 24 h, with an application volume of 100 µL. After the incubation period, cells were washed twice and 100 µL 0.1% (m/v) resazurin solution in HBS were added. After 2 h of incubation, aliquots of 100 µL were transferred into a black 96-well plate and fluorescence intensity was measured at an excitation wavelength of 540 nm and an emission wavelength of 590 nm with the Tecan Spark plate reader. Cell viability was calculated according following Eq. (4):

$$Cell\;viability\;\left(\%\right)= \frac \times 100\%$$

(4)

Degradation study by isolated aminopeptidase N

SEDDS were initially loaded with HIPs to determine the degradation of the incorporated peptide by isolated aminopeptidase N. HIPs were dissolved in preconcentrates of SEDDS at maximum solubility as previously mentioned at 37 °C and 2000 rpm overnight. Subsequently, the preconcentrates were emulsified in 50 mM Tris buffer pH 6.5, resulting in a final concentration of 5% (v/v). Tuftsin was dissolved in a concentration of 1 mg/mL in the same buffer. Aminopeptidase N suspension was diluted with 50 mM Tris buffer pH 6.5 to reach a final concentration of 0.63% (v/v) corresponding to 53.5 mU/mL of enzyme activity [25] and incubated for 15 min at 37 °C. To initiate the enzymatic reaction, samples and the enzyme solution were mixed in a 1:1 ratio and further incubated at 37 °C and 400 rpm for 4 h. At predetermined time intervals, 50 µl aliquots were withdrawn from each sample and an equal volume of methanol containing 2% (v/v) TFA was added to terminate the enzymatic reaction [26]. Following this, the concentration of tuftsin in the supernatant was quantified using HPLC.

Degradation study with rat intestine

Rat intestine was obtained freshly from male Sprague–Dawley rats (200–300 g) supplied by Janvier Labs (Saint Berthevin, France). The middle section of the small intestine was longitudinally dissected and thoroughly washed with HBS pH 6.5 to remove intestinal contents. The intestine was stored at − 20 °C in HBS until further use.

After thawing the small intestine was cut into 1 × 1 cm pieces that were placed in tubes containing 500 µL of HBS. SEDDS with dissolved HIP were emulsified in a concentration of 10% (v/v) in HBS. 500 µL of nanoemulsions were added to each tube to obtain a final concentration of 5% (v/v) SEDDS. Thereafter, samples were incubated on a thermomixer at 37 °C with continuous agitation at 400 rpm. Over a 4 h time period, 100 µL aliquots were withdrawn from each sample at predefined time intervals. To terminate the enzymatic reaction, an equal volume of methanol containing 2% (v/v) TFA was added to each aliquot. Subsequently, the concentration of tuftsin in the supernatant was quantified using HPLC.

Determination of aminopeptidase N enzyme activity

The enzymatic activity of rat intestine in the presence and the absence of SEDDS was determined. This was carried out to confirm enzyme activity after the freezing and thawing process and to assess any potential inhibitory effects of SEDDS on the enzyme activity of rat intestine.

L-Leucine- 4-nitroanilide was used as substrate, which undergoes hydrolysis by aminopeptidase N to liberate the colored product 4-nitroaniline. The substrate was dissolved in a concentration of 5 mM in 20 mM HBS pH 6.5. To determine the enzymatic activity of rat intestinal mucosa, 1 × 1 cm pieces were placed in 500 µL of 20 mM HBS pH 6.5 mixed with 500 µL of the substrate solution and then incubated at 37 °C with continuous agitation at 400 rpm for 4 h. Afterwards, 500 µL aliquots were withdrawn from each sample and diluted with methanol containing 2% (v/v) TFA before centrifugation at 12,500 rpm for 5 min. The absorbance of the supernatant (100 µL) was measured photometrically at a wavelength of 405 nm by Tecan Spark (Tecan Sales Austria GmbH, Austria) [27]. The concentration of the colored product was determined using a calibration curve with 4-nitroaniline in HBS in the concentration range of 69–2.15 µg/mL.

To investigate potential inhibitory effects of SEDDS on aminopeptidase N activity on rat intestinal mucosa, 1 × 1 cm pieces of rat intestinal mucosa were incubated with each 500 µL of HBS, substrate solution and 5% (v/v) SEDDS. After 4 h of incubation at 37 °C and 400 rpm agitation, aliquots were withdrawn from each sample and appropriately diluted with methanol containing 2% (v/v) TFA to dissolve all contents. After centrifugation at 12,500 rpm for 5 min, the amount of 4-nitroaniline in 100 µL of the supernatant was quantified at 405 nm. Enzyme activity was calculated in mU, assuming that 1 U converts 1 µmol substrate/min.

Furthermore, a possible inhibitory effect of SEDDS and excipients on aminopeptidase N activity was investigated using the isolated enzyme, following a method previously described with slight modifications [28]. Surfactant solutions were prepared in 20 mM HBS pH 6.5 in the same concentration as they are present in SEDDS. Either 5% (v/v) SEDDS or surfactant solution were mixed in 500 µL together with further 500 µL of a 41.6 mU/mL enzyme solution in 20 mM HBS pH 6.5. L-Leucine- 4-nitroanilide was added prior to SEDDS and surfactant solution in a final concentration of 5 mM.

A 5 mM substrate solution served as the positive control, representing 100% enzyme activity. The samples were incubated on a thermomixer for 1 h at 37 °C and 400 rpm agitation. To stop the enzymatic reaction, 1 mL of methanol containing 2% (v/v) TFA were added to each sample. After centrifugation at 12,500 rpm, the concentration of 4-nitroaniline in the supernatant was quantified. The inhibitory activity was calculated using the following Eq. (5):

$$Inhibitory\;activity\;[\%]=100-\left( \frac_}_}\right)\times100$$

(5)

Ex-vivo permeation study on rat intestinal mucosa

The amount of permeated peptide was determined via an ex vivo permeation study on freshly excised mucosa from Sprague–Dawley rats, following a previously established protocol with some modifications [29]. The rat intestine was carefully cleaned to remove its intestinal contents and then cut into 1.5 cm sections. These sections were fixed in Ussing chambers with a surface area of 0.64 cm2 and 1 mL of 20 mM HBS pH 6.5 was added to each acceptor chamber, while the donor chambers were filled with 1 mL of 5% (v/v) SEDDS in 20 mM HBS pH 6.5, facing the luminal side of the intestine.

The Ussing chambers were incubated in a water bath at 37 °C, and every 1 h, 100 µL aliquots were withdrawn from the acceptor chambers over a 4 h period. Withdrawn samples were replaced with pre-warmed HBS to maintain sink conditions. The concentration of permeated tuftsin in the collected aliquots was determined via HPLC and the apparent permeability coefficient [30]was subsequently calculated using the following Eq. (6):

$$_[cm ^]= \frac^\right] c \left[\mu g ^\right] t [s]}$$

(6)

where Papp is the apparent permeability coefficient, Q is the total amount of test substance permeated through the mucosa, A is the diffusion area of the Ussing chamber system, c is the initial concentration of tuftsin in the donor compartment and t is the total time of experiment.

Statistical design and data analysis

All experiments were performed at least in triplicate and results were presented as means ± standard deviation. Statistical data analysis was performed using the student t-test and the one-way ANOVA (GraphPad Prism 5) followed by Bonferroni correction with p ≤ 0.05 as the minimal level of significance.