A Standardized Ex Vivo Porcine Oromucosal Model for Evaluating Peptide Fluxes

Our research develop a standardized ex vivo porcine buccal mucosal protocol to preserve tissue integrity and improve reliable measurement of peptide permeability over time. Our ex vivo study protocol is applicable in preclinical studies investigating peptide and small molecule permeation across porcine buccal and sublingual tissue models. To begin, obtain the pig cheek tissue in ice cold PBS at pH 7.4.

Using a scalpel, separate the buccal mucosae from the underlying tissue. Rinse the tissue three times with PBS and discard mucosae with visible surface damage. Then immerse the intact mucosae in PBS at 65 degrees Celsius for three minutes.

Using a size 5 watchmaker's forceps, separate the epithelium and underlying connective tissue from the smooth muscle. Gently dry the dissected oral mucosae on cellulose filter paper. Mount the dissected mucosal tissue in Franz diffusion cells with an exposed diffusion area of 1.2 square centimeters.

Equilibrate the tissue for 30 minutes with one milliliter of PBS at pH 6.8 in the donor compartment and 10 milliliters of PBS at pH 7.4 in the receptor compartment. After equilibration, replace the donor compartment's PBS solution with one milliliter of PBS containing a one-to-one ratio of GLP-1RA and GDC, each at 25 milligrams per milliliter. Continuously stir the receptor compartment at 300 revolutions per minute while maintaining 10 milliliters of PBS at pH 7.4 and 37 degrees Celsius.

Sample one milliliter from the receptor compartment every 60 minutes during the 180 minute long experiment. Take triplicate samples from the donor compartment at the start and end of the experiment. Replenish the chambers with one milliliter of PBS after each sampling.

At the end of the experiment, store the tissue in 10%formalin for histology, or homogenize the tissue to quantify the GLP-1RA peptide remaining in the tissue. To confirm the permeation-enhancing effect of GDC before film formulation, perform GLP-1RA permeability studies using Hilltop chambers loaded with the peptide enhancer solution. After confirming enhancer activity using Hilltop chambers, assess ex vivo permeability from GLP-1RA and GDC loaded mucoadhesive bilayer films mounted on porcine buccal tissues in Franz diffusion cells, or use 3D inserts.

For assessing epithelial barrier integrity, add fluorescein isothiocyanate-dextran 4000 or FD-4 at 0.05 millimolar to the donor compartment. Collect samples from the receiver compartment over three hours to assess tissue leakage. After three hours of the tissue integrity study, measure the FD-4 concentration using a plate reader.

To improve peptide detection sensitivity during film permeation studies, use pre-prepared GLP-1RA and GDC loaded buccal bilayer films. Obtain a 3D insert using an ultraviolet curable resin and mount the tissue into the insert. Position the porcine buccal mucosae across a window area of 1.4 square centimeters between the apical and basolateral chamber.

Place films of area one by one square centimeters on top of the tissue. After placing the film, pipette 0.5 milliliters of PBS at pH 6.8 into the apical chamber, and then pipette 1.5 milliliters of PBS at pH 7.4 into the basolateral chamber. Collect 0.2 milliliter samples from the receiver side at one hour intervals over three hours, and replace with equal volumes of fresh buffer after each sampling.

Measure the transepithelial electrical resistance using the chopstick electrodes placed in the donor and receiver sides of the inserts, and calculate the transepithelial electrical resistance using Ohm's law. To confirm porcine buccal and sublingual epithelial tissue integrity, use FD-4 at 0.05 millimolar as a fluorescent marker. After the three-hour experiment, quantify GLP-1RA permeation and residual tissue content using ultra-high performance liquid chromatography.

Repeat all permeation experiments three times and quantify the cumulative flux of GLP-1RA. After fixing the tissues in formalin for 48 hours, dehydrate them in 70%ethanol and embed them in paraffin. Section the tissues at a thickness of five micrometers using a microtome.

Next, stain the tissue sections with hematoxylin and eosin to examine their histological integrity using light microscopy at 4X magnification. Finally, image the slides and analyze them using a suitable image analysis software. GLP-1RA permeation increased when GDC was included at a one-to-one ratio as compared to controls without GDC.

When the GLP-1RA and GDC combination was incorporated into bilayer films, GLP-1RA permeation across isolated porcine buccal tissues was not quantifiable using the conventional Franz diffusion cell setup. Using 3D inserts, cumulative GLP-1RA permeation from buccal films over three hours was measurable and reached approximately 1.8%TEER values declined over time in the GDC added treatment groups and corresponded with increased GLP-1RA permeation. PBS control treated tissues showed no TEER changes, indicating that GDC was the primary contributor to the permeation-enhancing effect.

Histological assessment indicated that the buccal mucosae exposed to either an aqueous mixture or a bilayer film containing GLP-1RA and GDC maintained epithelial integrity. Only mild edema and superficial epithelial erosion were observed. Exposure to blank film did not produce visible tissue damage.

This protocol enables accurate measurement of drug permeation across mucosal tissues, supporting reliable preclinical absorption prediction and formulation development using large animal models. The main challenge is maintaining tissue integrity throughout epithelial isolation and the entire experimental procedure to ensure accurate and reproducible permeability measurements. Future studies can evaluate additional oromucosal peptide formulations using the standardized cross-laboratory ex vivo protocols to improve correlation with human in vivo absorption.