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. 2022 Dec 19;15(12):1585. doi: 10.3390/ph15121585

Table 3.

Studies on the permeation enhancement mechanism of sodium caprate (C10).

Experimental
Model		 Proposed
Mechanism of Action
(Rationale)		 Evidence Reference
Caco-2 cell monolayer TJ modulation TEM
  • -

    TJ dilatation was observed after treatment with 13 mM C10.

Fluorescence microscopy
  • -

    It is observed that the peri-junctional F actin ring disbands over time, which corresponds to the permeation enhancement timeline of 14C-Mannitol.

 [97]
Caco-2 cell monolayer TJ modulation TEM
  • -

    TJ dilatation was observed after treatment with 10 mM C10.

  • -

    Frequency of dilatations after exposure to C10 was more than 40% (much higher than C8 and C12).

Fluorescence microscopy
  • -

    F actin rings were shown to be disbanded after exposure to C10.

 [74]
Caco-2 cell monolayer TJ modulation
(PLC activation and CaM-dependent contraction of actin filament)		 Intracellular Ca2+ measurement (Fluorometric Ca2+ analyzer)
  • -

    C10 increased the intracellular Ca2+ regardless of the presence or absence of extracellular Ca2+ (0.05%–0.25% of C10, especially at 0.25%).

Inhibitor treatment for PLC-CaM signaling
  • -

    Treatment with KN-62 reduced the permeation-enhancing effect of C10 on FD-4K.

  • -

    Treatment with KN-62 and W7 inhibited the C10-induced TEER reduction effect.

 [38]
Ex vivo Ussing chamber (Rat ileum) TJ modulation TEM
  • -

    C10 (10 mM) caused dilatations in 34% of the visualized TJ regions.

Comparison of patterns with Cytochalasin B
  • -

    Dose–response curve (C10: 2.5–10 mM, CytB: 60–300 µM) for transepithelial potential difference and [51Cr]EDTA Papp are similar.

 [99]
Human airway
epithelial cell		 TJ modulation
(Ca2+-independent mechanism and
direct effect on the TJ protein)		 Fluo-4 Ca2+ assay
  • -

    30 mM C10 induced a rapid increase in Ca2+ from ER and returned to control levels by 300 s after exposure.

The effect of BAPTP-AM, La3+, and thapsigargin
  • -

    The addition of BAPTP-AM (intracellular Ca2+ chelator) did not affect the decrease in the TEER value of C10.

  • -

    The addition of La3+ (membrane Ca2+-ATPase inhibitor) and thapsigargin (ER Ca2+-ATPase inhibitor) that maintained an increase in intracellular Ca2+, did not affect the decrease in the RT value of C10.

The effect of signaling inhibitor
  • -

    Each treatment of U7, 48/80, H7, W7, and KN62 did not affect the reduction of the RT value of C10.

Immunofluorescent Labeling and Confocal microscopy
  • -

    JAM and actin were clearly redistributed after exposure to C10 (ZO-1 did not change).

  • -

    The redistribution was not blocked by BAPTA-AM.

  • -

    Claudin-1 and -4 were redistributed immediately after C10 treatment.

 [100]
  • -

    MDCK cell monolayer

  • -

    Lipid raft isolation study

 Membrane perturbation
(Lipid raft disruption)
TJ modulation
(Displacement of specific TJ proteins)		 Western-blot analysis
  • -

    Claudin -4,-5, and occludin were displaced from the lipid raft.

 [101]
  • -

    HEK-293 Cell expressing claudin-5-YFP, MDCK-2-Cell expressing Flag-claudin-5

  • -

    Ex vivo mouse brain capillary

 TJ modulation
(by reducing the membranous claudin-5 amount and the F-actin content)		 Immunofluorescent Labeling and Confocal microscopy
  • -

    Claudin-5-YFP homophilic interaction was lost and fragmented by treatment with 5, 7.5, and 10 mM C10 for 20 min, and recovered after removal of C10.

  • -

    Treatment with C10 yielded no significant change in CellMask, a membrane-inserting dye. (Compared to MβCD, which is a membrane disruptor.)

Immunoblotting
  • -

    7.5 mM C10 reduced membranous claudin-5 and intracellular F-actin in TJ-containing MDCK-2-Cells and in brain endothelial cells. (There was no change in ZO-1.)

  • -

    Claudin-5 was found to be displaced from the triton-insoluble (lipids) fraction of MDCK cells after treatment with C10.

 [102]
  • -

    HT-29/B6 Cell

 TJ modulation
(by reversible removal of tricellulin from the tricellular TJ)		 Two-path impedance spectroscopy
  • -

    A significant decrease in paracellular resistance was observed by C10, although transcellular resistance was not significantly changed.

Immunofluorescent Labeling and Confocal microscopy
  • -

    C10 specifically induced a decrease in tricellulin and claudin-5 signals, which was reversible after washout.

Localization of Sulfo-NHS-SS-biotin Permeation sites
  • -

    Biotin signal was detected only within or below tricellular cell contacts (not detected in bicellular cell contacts).

 [103]
  • -

    Caco-2 cell monolayer

  • -

    Ex vivo Ussing chamber (Rat colon)

  • -

    Rat intestinal

instillation		 Membrane perturbation Quantitative real-time PCR and gene expression microarrays
  • -

    After exposure to 8.5 mM C10 for 60 min, IL-8, an inflammatory signal, increased 11-fold and 26-fold at 1 and 4 h of recovery, respectively, and then decreased to the control level after 24 h.

Attenuation Effect of misoprostol
  • -

    Pre-exposure of monolayers to the misoprostol (10 and 100 nM) for 30 min prior to 8.5 mM C10 addition for 60 min significantly attenuated C10′s capacity to reduce TEER and increase the [14C]-mannitol and FD-4K Papp.

  • -

    The protective effect of pre-incubation with misoprostol against C10 was detected by TEM.

  • -

    In a rat colonic loop instillation experiment, misoprostol reduced the mean AUC and Cmax of FD4K by 24% and 33%, respectively, compared with the results treatment with C10 alone.

  • -

    SC51322 (EP1 receptor antagonist) negated the effect of misoprostol in preventing the C10-induced changes in the intracellular Ca2+, mitochondrial membrane potential, and plasma membrane permeability.

 [104]
Caco-2 cell monolayer Membrane perturbation
(initial and fundamental mechanism)
TJ modulation
(by intracellular pathway arising from initial plasma membrane perturbation)		 Immunofluorescence of TJ proteins
  • -

    At 5 mM or higher C10, ZO-1 was internalized, and claudin-5 and occludin were also relocated and internalized.

Cytotoxicity assay
  • -

    LDH-Glo™, CellTox Green™, Neutral Red, and JC-1 assay results showed a decrease in the integrity of the plasma, nuclear, and mitochondrial membrane that was concentration-dependent (usually at 5 mM or higher) of C10. (This pattern was not observed in SNAC.)

 [105]
  • -

    CG-MD simulation

  • -

    US simulation

  • -

    TIRF microscopy

(FRAP analysis)		 Membrane perturbation
(insertion of C10 into membrane and transmembrane perturbation)		 CG-MD simulation
  • -

    When 100 mM C10 in the fluid composition of the fasting state was applied to the POPC membrane, it was inserted into the membrane at a level of 70–80% in 6 µs.

US simulation (PMF profile)
  • -

    In a POPC bilayer with a thickness of 4.05 nm, C10 had an energy minima at a distance of 1.46 nm from the membrane center. (Energy minima represent the maximum probability of finding the molecule.)

TIRF Microscopy (FRAP analysis)
  • -

    In the POPC-C10 mixed membrane composed of various concentrations of C10, the membrane diffusivity increased in a C10 concentration-dependent manner.

  • -

    When the pure POPC membrane was treated with 100 mM C10, diffusivity was increased, and it was observed that the generated bleached holes were recovered after 60 s when C10 was removed.

 [72]

TEM, transmission electron microscope; TJ, tight junction; PLC, phospholipase C; CaM, calmodulin; FD, FITC-dextran; TEER, transepithelial electrical resistance; ER, endoplasmic reticulum; Papp, apparent permeability; JAM, junctional adhesion molecule; YFP, yellow fluorescence protein; ZO-1, zonula occludens-1; EP1 receptor, prostaglandin E2 receptor 1; POPC, 1-Palmitoyl-2-oleoylphosphatidylcholine; CG-MD, coarse-grained molecular dynamics; US, umbrella sampling; TIRF, total internal reflection fluorescence; PMF, potential of mean force; FRAP, fluorescent recovery after photobleaching.