Intestinal permeation enhancers enable oral delivery of macromolecules up to 70 kDa in size (1)
The small intestine is highly absorptive to small molecules and products of food digestion such as di- and tri-peptides, but intact peptides and proteins (>1 kDa and 5–10 nm in diameter) are unlikely to cross the epithelium into systemic circulation.
Permeation enhancers can either improve transcellular transport by fluidizing the cell membranes or paracellular transport by rearranging the tight junctions. FITC-dextrans cross the intestinal epithelium mainly by paracellular transport, but they have the potential for transcellular transport if the size is appropriate and the concentration gradient of dextran across the epithelium is sufficiently high to drive transport across this biological barrier.
In vitro permeability experiments
Monolayers were transferred into transport buffer (HBSS with 12.5 mM glucose and 25 mM HEPES) and allowed to equilibrate at 37◦C for one hour before the permeability experiments began. Negative control wells were maintained in the transport buffer for the duration of the experiment. The FITC-dextrans were dissolved in a transport buffer at a concentration of 10 mg/mL along with PPZ or SDC at concentrations of 0.1% (v/v) and 0.05% (w/v), respectively. These solutions were applied to the apical side of the Caco-2 monolayers and incubated at 37◦C on a temperature-controlled shaker. At 1 and 2 h after treatment began, samples were taken from the basolateral side of the monolayers. The fluorescence of these samples at 490/520 nm was measured using a BioTek Synergy2 plate reader. Individual calibration curves were made for each combination of dextran size and treatment and used to convert fluorescence measurements into the amount of FITC-dextran transferred across the monolayer, which was used to calculate the permeability of FITC-dextran across the monolayer:
Oral gavage of FITC-dextrans and permeation enhancers
Mice were fasted for 12 h with free access to water, after which a baseline blood sample was collected from the submandibular vein. Flexible plastic oral gavage needles were used to administer all oral gavages in these experiments. Mice were gavaged with 10 µL/g of solutions containing PBS, 0.6% (v/v) PPZ, or 2% (w/v) SDC and one of the five different-sized FITC-dextrans. In the oral gavage experiments, the dose of FITC-dextran was kept constant at 200 mg/kg (20 mg/mL). Blood was collected 3 h after dosing. Blood was collected and analyzed as described in the previous section. For the experiment using sodium bicarbonate, mice received 100 µL of 10% sodium bicarbonate solution 15 min prior to the FITC-dextran/permeation enhancer solution.
PPZ caused a nearly 10-fold increase in permeability for FD4 and a 2-fold increase for FD10. However, it did not significantly increase the permeability of the larger FITC-dextrans (40 and 70 kDa).
13 mg/kg PPZ (0.5% (v/v)) was not effective at increasing the amount of FD4 absorbed into the bloodstream after intestinal injection, but 65 mg/kg PPZ (3% (v/v)) caused a statistically significant 10-fold increase. Similar to in vitro experiments, PPZ increased the absorption of FD4 and FD10 but was ineffective for the two larger sizes of FITC-dextran.
After oral gavage of solutions containing FITC-dextrans with PPZ or SDC, only SDC increased intestinal permeability. In contrast, PPZ did not improve the absorption of any of the FITC-dextrans. The pre-treatment with sodium bicarbonate significantly improved the permeation enhancing action of PPZ.
A common concern with the use of permeation enhancers is that the epithelium will be permeabilized so much that pathogens such as lipopolysaccharide (LPS) and bacteria are able to cross into systemic circulation. It should be noted that LPS is >100 kDa and bacteria are 1–2 µm in their smallest dimension, making them much larger than the largest model macromolecule used for this work.
1-phenylpiperazine: PPZ increases intestinal paracellular (between cell) permeability by affecting the tight junction protein complexes that hold the epithelial cells together. In contrast, sodium deoxycholate: SDC increases transcellular (through cell) permeability by fluidizing the lipid membrane of the epithelial cells, enabling the diffusion of macromolecules across the monolayer.
The dimensions of the paracellular space are reported as being on the order of 5–10 Å in diameter for the pores formed by tight junction proteins and 100 Å in diameter for the entire space between the epithelial cells. The hydrodynamic diameters of FITC-dextrans increase with molecular weight, from FD4 with a reported diameter of 28 Å up to 120 Å for FD70. It is plausible that tight junction loosening may not be an effective mechanism for delivering very large molecular cargos.