2.2. Methods

To activate carboxylic moieties of l-cysteine, 1.50 g of l-cysteine, 0.60 g of EDAC × HCl, and 0.36 g of NHS were stirred for 3 h in DMF. Subsequently, 0.50 g of low-molecular-weight CS was dissolved in 200 mL of demineralized water and the pH was adjusted to 2 using 1 M HCl. Activated l-cysteine was added to the CS solution and pH was adjusted to 5.5 using 1 M NaOH. After 1 h of stirring at room temperature, the mixture was transferred to a dialysis tube with a molecular weight cut-off of 10–20 kDa (Nadir dialysis membrane; Carl Roth, Karlsruhe, Germany). The reaction product was dialyzed against 10 L of 1 mM HCl, twice against 10 L of 1 mM HCl containing 1% NaCl, and again twice against 10 L of 1 mM HCl. The dialysis product was frozen at −80 °C and lyophilized (Gamma 1-16 LSC, Christ, Osterode, Germany). To ensure complete removal of uncoupled l-cysteine, a sample omitting EDAC and NHS was prepared as control following the same procedure.

S-protection with 6-MNA was achieved via a thiol/disulfide-exchange reaction according to a slightly modified protocol previously applied by Laffleur et al.^17,21 Briefly, 200 mg of CS-Cys was dissolved in 50 mL of a DMF/water mixture in a ratio of 7:3 and adjusted to pH 6.2 by the addition of 1 M HCl. After dissolving 50 mg of 6,6′-DTNA in 50 mL of DMF, the solution was added dropwise to the solution of CS-Cys. The mixture was stirred for 5 h at room temperature while maintaining the pH between 6.0 and 6.2 using 1 M NaOH. The product was transferred to a dialysis tube with a molecular weight cut-off of 10–20 kDa and dialyzed three times against a mixture of 3 L of demineralized water containing 1% NaCl and 0.5 L of DMSO. Subsequently, salt and DMSO were removed by dialyzing five times against 10 L of demineralized water. The final product was obtained after lyophilization.

To substitute 6-MNA with l-cysteine, 100 mg of l-cysteine was dissolved in 25 mL of demineralized water. The l-cysteine solution was added dropwise after the previous reaction between CS-Cys and 6,6′-DTNA was completed. The pH was maintained between 6.0 and 6.2 using 1 M NaOH and the mixture was stirred for a further 90 min. The product was dialyzed in the same way as CS-Cys-MNA. The final product was obtained after lyophilization.

All ¹H NMR measurements were performed on a “Mars” 400 MHz Avance 4 Neo spectrometer from Bruker Corporation (Billerica, MA, 400 MHz) in D₂O with the addition of 1% acetic acid-d₄.

FT-IR spectra were recorded on a Spectrum Two spectrometer (PerkinElmer, Beaconsfield, U.K.) using four scans at a resolution of 1 cm^–1 recorded from 4000 to 400 cm^–1. The depicted spectra are the mean of applied scans.

Ellman’s test was used to determine the degree of thiolation.²² Briefly, 0.5–1.0 mg of each polymer were hydrated in 250 μL of demineralized water. After dilution with 250 μL of 0.5 M phosphate buffer, pH 8.0, 500 μL of Ellman’s reagent containing 3 mg of DTNB in 10 mL of 0.5 M phosphate buffer, pH 8.0, were added. After 90 min of incubation, protected from light and at room temperature, the samples were centrifuged for 5 min at 13 400 rpm. Aliquots of 100 μL were transferred to a UV plate, and absorbance was measured at 450 nm using a Tecan Spark (Tecan, Grödig, Austria). Calibration curves were established using l-cysteine-HCl × H₂O (R² ≥ 0.99).

To quantify disulfide bonds, samples were hydrated in 350 μL of demineralized water and subsequently diluted with 150 μL of 0.5 M Tris buffer, pH 7.6. Disulfide bonds were reduced with an excess of sodium borohydride being dissolved in demineralized water at a concentration of 40 mg/mL. After discarding the unreacted sodium borohydride by adding 5 M HCl, the solution was neutralized using a 1 M phosphate buffer, pH 8.0. Ellman’s test was then conducted as described above.

To prove, the removal of uncoupled 6-MNA and quantify the 6-MNA immobilized on thiolated polymers, a test previously described by Lupo et al.²² was applied. First, 0.5–1.0 mg of each S-protected thiolated polymer was hydrated in 250 μL of demineralized water. After dilution with 250 μL of 0.5 M phosphate buffer, pH 8.0, 500 μL of a freshly prepared 0.2% (m/v) L-glutathione solution was added and incubated for 90 min protected from light at room temperature. To quantify the unbound 6-MNA, samples in the absence of L-glutathione were prepared in the same way. Aliquots of 100 μL were transferred to a UV plate and measured at 354 nm. Calibration curves were established using 6-MNA (R² ≥ 0.99).

SLN were prepared using an emulsification ultrasonication method.^23,24 Briefly, 500 mg of cetyl palmitate and 50 mg of egg lecithin were molten at 65 °C. To prepare labeled SLN, 5 mg of LGR was added to the lipid phase. As an aqueous phase, 200 mg of Pluronic F127 was dissolved in 10 mL of demineralized water. When applying coatings, 40 mg of polymeric coating material was also added to the aqueous phase. The aqueous phase was heated to 65 °C and added to the lipid blend. The mixture was pre-emulsified for 30 s by means of high shear homogenization at 27 000 rpm (IKA EuroTurrax T206, Staufen, Germany). The formed pre-emulsion was sonicated twice for 60 s, applying an amplitude of 80% with a Hielscher UP200H (Hielscher, Teltow, Germany). The resulting hot nanoemulsion was immediately transferred to an ice bath. After cooling, SLN were used without further purification.

When applying CS coating, 1% acetic acid (m/v) served as an aqueous phase. When applying coatings of CS-Cys and CS-Cys-Cys, demineralized water was used and in the case of CS-Cys-MNA, 0.5 M phosphate buffer was added with the pH adjusted to 8.0. SLN were subsequently prepared as described above.

The hydrodynamic diameter of SLN formulations expressed as Z-average and PDI were derived from the autocorrelation fit of the data obtained from dynamic light scattering (DLS) using the cumulant method. Therefore, SLN dispersions were diluted 1:100 in 10 mM PBS, pH 7.4, and transferred to disposable polystyrene cuvettes. Samples were measured using a He–Ne laser with a wavelength of 633 nm and a backscattering angle of 173°. ζ potential was measured via electrophoretic light scattering after diluting SLN dispersions 1:500 in demineralized water by applying the Smoluchowski relation. Samples were measured at a scattering angle of 12.8° with the aid of a dip cell (Malvern Instruments, Malvern, U.K.). Each replicate of both methods consisted of three consecutive runs and was carried out at 37 °C using a ZetaSizer Nano ZSP (Malvern Instruments, Worcestershire, U.K.). To determine storage stability, SLN formulations were measured following the same protocol after storage at 4 °C for 30, 90, and 180 days.

The shape and surface morphology were investigated using an energy filter transmission electron microscopy (EFTEM). Therefore, SLN dispersions were mounted on 200 mesh, Formvar/carbon-coated copper grids (Balzers Union, Liechtenstein), dried, and examined with a Zeiss Libra 120 (Carl Zeiss AG, Oberkochen, Germany). Images were obtained with a 2 x 2k high-speed camera (Troendle, Germany) and ImageSP software (Troendle, Germany). SLN formulations were diluted 1:10 with demineralized water before measurements.

Undiluted SLN dispersions and single components of each dispersion as bulk material were analyzed using PXRD. The samples were measured on a Mylar (6 μ) foil. The PXRD patterns were obtained using an X’Pert PRO diffractometer (PANalytical, Almelo, the Netherlands) equipped with a θ/θ coupled goniometer in transmission geometry, a Cu Kα_1,2 radiation source with a focusing 0.5° divergence slit and 0.02° Soller slit collimator on the incident beam side, a 2 mm antiscattering slit and 0.02° Soller slit collimator on the diffracted beam side mirror, and a solid-state PIXcel detector. The patterns were recorded at a tube voltage of 40 kV and a tube current of 40 mA, with a step size of 2θ = 0.013° with 80 s (components) or 400 s (SLN formulations) per step in the 2θ range between 2 and 40°.

Mucus was scraped off from the freshly excised porcine intestine, which was obtained from a local slaughterhouse. Intestinal segments that contained food residues, as well as mucus that appeared yellow, were discarded. The crude mucus was frozen at −20 °C until purification. To purify the collected mucus, the crude mucus was diluted 1–5 with 0.1 M NaCl solution and gently stirred for 1 h at 10 °C. After centrifugation at 10 400g and 4 °C (Sigma 3-18KS, Sigma Laborzentrifugen, Osterode am Harz, Germany) for 2 h, the supernatant and granular material on the bottom were discarded. Subsequently, the mucus was diluted with half of the volume of 0.1 M NaCl. Stirring and centrifugation were repeated as described above. The supernatant was removed and the purified mucus was stored at −20 °C until further use.

For the mean of single-particle tracking (SPT), an altered protocol was applied as described by Le-Vinh et al.²⁵ In brief, the fresh porcine small intestine was put on ice immediately after being collected. Then, it was rinsed with ice-cold 67 mM phosphate buffer, pH 6.7, containing 0.02% w/v sodium azide and a mix of protease inhibitors to remove the debris. The mucus was collected by gently scraping the epithelial surface of the jejunal segment of the intestine with a plastic scraper, collected in aliquots, and directly put on ice. The debris was further removed by extracting the mucus overnight at 16 °C under gentle stirring in 7 volumes of extraction buffer adjusted to pH 6.5 containing 10 mM sodium phosphate, 4 M guanidinium hydrochloride, 5 mM EDTA, 5 mM N-ethylmaleimide, and 0.02% (w/v) sodium azide. The precipitated material was collected by centrifugation for 30 min at 22 104g and 10 °C and re-extracted in the same manner with 10 volumes of the extraction buffer. After another centrifugation, the insoluble precipitate was collected and stored at −80 °C prior to use.

The stability of SLN formulations was investigated in simulated intestinal fluid (SIF), simulated gastric fluid (SGF), fasted-state simulated intestinal fluid (FaSSIF), and fed-state simulated intestinal fluid (FeSSIF). The SIF and SGF were prepared according to USP specifications. FaSSIF and FeSSIF were prepared according to the supplier’s manual. To determine stability, SLN were diluted 1:100 in each medium. After 4 h of incubation at 37 °C, SLN were analyzed via DLS as described previously.

To determine changes in the characteristics of SLN getting into contact with mucus, they were incubated with a dilution of purified mucus. Briefly, 100 mg of purified mucus was diluted in 1 mL of 10 mM PBS, pH 7.4. Equal volumes of SLN dispersions and mucus dilution were incubated at 37 °C for 4 h. The mixtures were diluted 1:50 with 10 mM PBS, pH 7.4, before measuring via DLS.

To further investigate the interaction of mucus and SLN formulations, rheological measurements were conducted. Therefore, 500 μL of purified mucus and 500 μL of undiluted SLN dispersion were gently mixed using a spatula. After 4 h of incubation at 37 °C, samples were transferred to a Haake Mars plate-plate rheometer (Thermo Scientific, Vienna, Austria). Strain sweep measurements were conducted at a frequency of 1 Hz, whereas frequency sweep measurements were conducted at a shear rate of 0.1 Pa.

To evaluate the diffusion characteristics of SLN in purified porcine intestinal mucus, SPT was employed. LGR-labeled SLN were diluted 1:500 with 10 mM PBS, pH 7.4, to yield a lipid content of 0.01% (w/v). Subsequently, SLN dilution was added to 30 μL of purified mucus in an amount that would yield a final concentration of 3% (mix 1) or 20% (v/v) (mix 2), respectively. Subsequently, the mixture was gently stirred using a pipette and equilibrated for 30 min at room temperature. To perform SPT experiments, 5 μL of the mucus–SLN mixture was inserted into a custom-made imaging chamber and placed onto the microscope stage. The sample was left on the microscope stage for a further 5 min so that the mucus–SLN mixture in the imaging chamber could reach equilibrium from the motion of handling. SLN diffusion was measured by tracking the positions of the labeled SLN using an sCMOS camera(Hamamatsu digital camera {"type":"entrez-nucleotide","attrs":{"text":"C11440","term_id":"1536511","term_text":"C11440"}}C11440, ORCA-ash 4.0, Japan) mounted on an inverted wide-field microscope (Dmi8, Leica, Germany) with a 63Ö/1.2NA objective, appropriate filters, and with an attached Lumencor Spectra × fluorescence illumination system (Olympus). For each sample, 90 image sequences were acquired with LASX software (Leica) at a temporal resolution of 10 ms to obtain at least 100 frames of particle trajectories.

To obtain particle trajectories, the image sequences were analyzed using the feature point detection and tracking algorithm of the ParticleTracker ImageJ plugin²⁶ and the ImageJ-Matlab extension.²⁷ Trajectories longer than 30 frames were analyzed using a custom-written Matlab program. A minimum of 400 trajectories was assessed for each formulation. The coordinates of SLN centroids were used to determine the time-averaged mean-squared displacement (MSD).

Particle position at time t is referred to as r(t), whereas τ is the time lag. From the ensemble-averaged MSD, the generalized time-independent diffusion coefficient and the dimensionless anomalous exponent were determined via a linear fit of the log transformation of eq 2.(28)

Anomalous diffusion can be efficiently estimated when an ensemble of trajectories is available.²⁹ The particle mobility was further evaluated via calculation of the effective diffusion coefficient D_e for a time scale of τ = 0.5 s.

D_e was normalized to the mean diffusion coefficient D_w measured in demineralized water. D_w was determined via a weighted linear fit to the ensemble-averaged MSD. Particles were considered immobile when ⟨Δr²(τ_0.5 s)⟩ was less than 13 nm, which is below the tracking resolution at a time lag of 0.5 s. Particles displaying an MSD below their diameter at that time scale were ranked as hindered, and particles were considered diffusive when D_e/D_w was approximately 1.²⁸

Mucus diffusion assay was conducted as previously described by Akkus et al.³⁰ with slight modifications. Briefly, silicon tubes with an inner diameter of 30 mm were cut into pieces of 5 mm in length. Tubes were filled with 150 μL of purified mucus and closed with a silicone plug on one end. Subsequently, 50 μL of labeled SLN dispersion was deposited on top of the mucus. The tube was closed on the other end and subjected to horizontal rotation with 50 rpm (IKA RM 18, IKA, Staufen, Germany). After 24 h of rotation at 37 °C, the tubes were frozen at −80 °C. Frozen tubes were cut into slices of 2 mm in length. Each slice was placed in 500 μL of DMF to extract LGR. The tube and undissolved parts were separated from the dye solution by means of centrifugation for 5 min at 12 000g. LGR was measured via fluorescence at λ_ex = 570 nm and λ_em = 610 nm.

The porcine intestinal mucosa was lengthwise opened and cut into pieces of 2 × 5 cm². Each piece was glued on a half-cut 50 mL falcon tube. After depositing 200 μL of labeled SLN dispersion on the mucosa, they were incubated horizontally for 10 min. Subsequently, each mucosa was mounted at an angle of 45° and rinsed with 10 mM PBS, pH 7.4 using a flow of 1 mL/min at 100% relative humidity. At predetermined time points, the amount of LGR that was washed off was measured. Therefore, the collected PBS was diluted 1:5 in DMF to extract LGR. After 120 min, LGR that remained on the mucosa was extracted by washing the mucosa with 10 mL DMF. All collected samples were measured via fluorescence at λ_em = 570 nm and λ_ex = 610 nm.

When two sets of data were compared with each other, Student’s t-test was applied. For the comparison of more than two data sets, one-way analysis of variance (ANOVA) and Bonferroni post hoc test were applied. GraphPad Prism 5 software was used for all statistical analyses.