Materials
Zein, cholic acid, and rhamnolipids R90 (90%) were purchased from Sigma-Aldrich (Shanghai, China). Liraglutide was from GL Biochem (Shanghai). Mucin (from porcine stomach) was from meilun bio (Shanghai, China). Sulfo-cyanine 5 NHS ester (Cy5, analytical grade) was from Lumiprobe (Maryland, USA). Fluorescein isothiocyanate (FITC) was from Tokyo Chemical Industry (Tokyo, Japan). Anthrone was from Aladdin (Shanghai, China). Chlorpromazine, genistein, amiloride, and 1, 1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) were from Beyotime Biotechnology (Shanghai, China). All cell culture reagents and materials were from Thermo Fisher Scientific (Shanghai, China). All other reagents were from Sinopharm Chemical Reagent (Shanghai, China). Total bile acid (TBA) kits were from Nanjing Jiancheng Bioengineering Institute.
Preparation and characterization of LIRA/CA complex
LIRA was dissolved in 20 mM pH 7.4 phosphate buffer. CA, RLs, and SDS were dissolved in deionized water separately and adjusted to pH 7.4. LIRA solution was mixed with cholic acid solution at a molar ratio of 1:18 or 1:36 (LIRA: CA). Similarly, LIRA solution was mixed with RLs or SDS solution at a molar ratio of 1:18 (LIRA: RLs/SDS). The final concentration of LIRA was kept at 1 mg mL− 1. The mixed solution was stirred at room temperature for 10 min under magnetic stirring.
The free LIRA and LIRA mixed with CA, RLs or SDS were loaded to the upper chamber of ultrafiltration spin columns (Millipore Amicon® Ultra, cutoff molecular weight: 50 kDa) and centrifuged at 12,000 rpm for 20 min, the amount of LIRA that is ultrafiltered across the membrane was analyzed by BCA assay. After lyophilization, FTIR spectra of LIRA, CA, physical mixture of LIRA and CA, and LIRA/CA Complex were recorded on a FTIR spectrometer (Nicolet 6700, Thermo Nicolet) using potassium bromide tableting.
Molecular docking by autodock vina
The structure of LIRA in aqueous solution was obtained from Protein Data Bank whose code was 4apd1. AutoDock Tools 1.5.7 were employed to add hydrogens to LIRA structure and to generate the input files of ligands with rotatable bonds. The grid box with dimentions of x: 80 Å, y: 45 Å and z: 50 Å, centering on x: − 0.675, y: − 0.006 and z: 0.348 was built to include the whole LIRA structure. Ligand-LIRA docking was calculated using Lamarckian Genetic Algorithm by Autodock Vina 1.1.2. [41, 42] Docking of various ligands produced the affinity values for comparison, the non-covalent interaction in the calculation comprises hydrogen bonds, hydrophobic force, Gaussian steric interactions, repulsion and torsion terms and the best docking conformation between ligand and LIRA with the highest binding affinity was extracted for analysis by PyMol software.
Fabrication and characterization of LIRA/CA complex-loaded zein nanoparticles
LIRA/CA Complex was prepared as described above. Zein was dissolved in ethanol/water (9:1, V/V). The above LIRA/CA Complex solutions and zein solution were mixed to form an ethanol/water stock solution. Subsequently, the RLs aqueous solution was added into the mixed solution under magnetic stirring and stirred at room temperature for 3 h before supplementing deionized water to the final volume. The final concentrations of LIRA, CA, zein and RLs were 0.5, 1.0, 5.0 and 0.8 mg mL− 1. Due to the hydrophobicity of zein, the LIRA/CA Complex-loaded zein NPs was obtained. The formulations that did not contain CA or RLs were prepared as a control.
Fluorescein isothiocyanate (FITC)-conjugated liraglutide (FITC-LIRA) was synthesized and the loading efficiency (LE) of LIRA was determined by ultrafiltration method as previously reported [20]. The ultrafiltered FITC-LIRA was analyzed by a microplate reader (Cytation, Biotek), CA was analyzed by total bile acid kits and RLs were quantified by colorimetric determination of sugars with anthrone sulfate [43]. The laser particle size analyzer (Anton Paar) was used to analyze hydrodynamic diameter (Dh) and ζ-potential. The Morphology was observed by TEM (CM120, Philips) and FESEM (Ultra 55, Zeiss).
The in vitro releases of LIRA from NPs were evaluated in the dialysis tubing (cutoff molecular weight 100 kDa, Spectrum Laboratories Inc.) as reported in our previous study [20]. The simulated gastrointestinal fluid was pH 2.0 HCl solution at 2 h and 10 mM pH 7.4 phosphate buffer during 2–48 h.
In vitro mucus penetration study
The mucus layer was simulated by the mucin solution of 10 mg mL−1. A transwell® plate (12-well, PET, 1.0 μm, Millipore) was used and the mucus layer thickness in the apical side (AP) was 0.6 mm. The FITC-LIRA or FITC-LIRA-loaded NP solution was gently added on the top of the mucus layer and incubated in the basolateral side (BL) with fresh HBSS at 37 °C. Solution in the BL was collected and analyzed at predetermined time intervals. The transportation rate is calculated by the ratio of FITC-LIRA in BL to the total.
Cytotoxicity measurements
Caco-2 cells were seeded in 96 well plates at a density of 5000 cells/well. After overnight incubation, the original medium was replaced with serum-free fresh medium containing gradient concentrations of NPs. After incubation at 37 °C for 48 h. The medium was removed and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added at a concentration of 0.5 mg mL− 1. The incubation continued for 4 h, then the MTT solution was replaced by dimethyl sulfoxide. After shaking for 10 min, the absorbance was measured at 490 nm.
Cellular uptake
To evaluate the uptake behavior, FITC-LIRA was used in the preparation of NPs. Caco-2 cells were seeded in 24 well plates at a density of 5 ⋅ 104 cells/well and allowed to adhere for 24 h. Then, free FITC-LIRA, LIRA/CA Complex, and FITC-LIRA loaded NPs (50 µg mL−1) were added. After 2 h incubation, cells were digested and resuspended in 250 µL Hank’s Balanced Salt Solution (HBSS). The fluorescence intensity in cells was measured by flow cytometer (FACS Calibur, BD).
The influence of CA and RLs on cell uptake was observed by CLSM. Briefly, Caco-2 cells were seeded in confocal dishes. After 24 h, NPs (50 µg mL−1) were added and incubated for 2 h. Then cells were fixed and cell membrane were stained with Dil after nucleus staining with DAPI.
To evaluate the endocytosis mechanism of NPs, Caco-2 cells in 24 well plates were prepared and measured with the same procedure as above, but pre-incubated with different endocytic inhibitors which were 10 µg/mL chlorpromazine (CPZ), 100 µM genistein and 10 µg/mL amiloride for 0.5 h before addition of NPs solutions.
Permeability in caco-2 cell monolayers
Caco-2 cell monolayers were constructed as previously reported. Briefly, Caco-2 cells were seeded in 24-well transwell® (PET, 1.0 μm, Millipore) at a density of 2 ⋅ 104 cells/well and cultured for 14−21 days. A transepithelial electrical resistance (TEER) over 500 Ω·cm2 was necessary. Before the addition of FITC-LIRA loaded NPs solutions (50 µg/mL), the medium in the apical side (AP) and basolateral side (BL) was replaced with pre-warmed HBSS and the cell monolayers were incubated for 30 min. At predetermined time intervals, the FITC-LIRA concentration in the BL solution was analyzed by a microplate reader. The apparent LIRA permeability (Papp) was calculated by
$${\text{P}}_{\text{a}\text{p}\text{p}}=\frac{\text{Q}}{\text{A}{\text{C}}_{0}\text{t}} (\text{n}=3)$$
where Q was the accumulative amount (ng) of FITC-LIRA in the BL, A was insert membrane growth area which was 0.33 cm2 in the 24-well transwell®, C0 was the initial FITC-LIRA concentration (ng/mL) in the AP and t was the duration time (s).
TEER values at different time points were also measured in the experiment [44]. To further validate the intactness of tight junctions, the Caco-2 cells on the microporous membrane of transwell® were cut down and incubated with the primary antibody against ZO-1 (1:400, ab150083, Abcam), the corresponding fluorescent secondary antibodies (1:400, 21773-1-AP, proteintech) were incubated after three washes.
The organ distribution of LIRA after oral administration
Healthy male C57BL/6 mice aged 6–8 weeks were purchased from the Animal Center of the Chinese Academy of Sciences (Beijing, China). The animal experiments were performed at Experimental Animal Center of School of Pharmacy of Wenzhou Medicine University, and were approved by Experimental Animal Ethics Committee of School of Pharmacy of Wenzhou Medicine University (ID Number: wydw2021-0140). We synthesized Sulfo-cyanine-5 NHS ester labeled LIRA (Cy5-LIRA) and Rhodamine B isothiocyanate-conjugated zein (RITC-zein) as reported [45], followed by the preparation of double fluorescence-labeled NPs. The mice were fasted overnight with free access to water and randomly divided into four groups. The solutions of LIRA/CA Complex, LIRA/CA@Zein/RLs, LIRA@Zein/RLs, and LIRA/CA@Zein were administrated orally at a dose of 0.4 mg kg−1. After 2, 6, 12, and 24 h, the mice were sacrificed and organs were taken out, washed, and imaged (In Vivo Xtreme, Bruker).
Oral pharmacodynamics evaluation in T2DM mice
The streptozotocin-induced T2DM mice were built as reported. [46] The T2DM mice with fasting blood glucose levels (BGL) higher than 16 mM were chosen to evaluate the hypoglycemic effect of formulations. The experiment was carried out in two batches. In the first batch, the mice were randomly divided into 4 groups and fasted for 10 h with free access to water, Free LIRA and LIRA/CA complex were orally administrated at a dose of 2 mg kg−1, respectively; LIRA solution was subcutaneously injected at a dose of 0.2 mg kg−1, saline was orally administered as a control. In the second batch, the mice were randomly divided into 5 groups and fasted as above. The mice in LIRA/CA@Zein/RLs, LIRA@Zein/RLs, and LIRA/CA@Zein groups (Control, p.o.) were administrated orally at a dose of 4 mg kg−1, and saline was administrated (Saline, p.o.) as control. In the Free LIRA group, LIRA solution (LIRA, s.c.) was subcutaneously injected at a dose of 0.4 mg kg−1. At predetermined time intervals, BGL was measured based on blood from the tail by a glucometer (ACCU-CHEK Active, Roche). Food was provided for 2 h at 12, 24, and 36 h post-administration, and water was available throughout the experiment. The oral pharmacological bioavailability (PA) of LIRA was calculated according to the following equation:
$$\text{P}\text{A} \left(\text{\%}\right)=\frac{{(\text{A}\text{A}\text{C}}_{\text{N}\text{P}\text{s}, p.o.}-{\text{A}\text{A}\text{C}}_{\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}})/{\text{D}\text{o}\text{s}\text{e}}_{\text{N}\text{P}\text{s}, p.o.}}{{(\text{A}\text{A}\text{C}}_{\text{L}\text{I}\text{R}\text{A}, s.c.}-{\text{A}\text{A}\text{C}}_{\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}})/{\text{D}\text{o}\text{s}\text{e}}_{\text{L}\text{I}\text{R}\text{A},s.c.}}\times 100\text{\%} (\text{n}=6)$$
where AAC was the area above the relative BGL-time curve.
Statistical analysis
All the data were presented as mean ± standard deviation (SD). Statistical significance was analyzed by one-way ANOVA analysis followed by Turkey post hoc tests, and P < 0.05 was regarded to be statistically significant.
