Characterization of Liposomal Tacrolimus in Lung Surfactant-like Phospholipids and Evaluation of Its Immunosuppressive Activity

Abstract

Tacrolimus (FK506) is a hydrophobic immunosuppressive agent that rapidly penetrates the plasmatic membrane and inhibits the signal transduction cascade of T lymphocytes. The objective of this study was the characterization of liposomal FK506 with surfactant-like phospholipids to be administered intratracheally after lung transplantation or in inflammatory lung diseases. We evaluated the optimal incorporation of FK506 in dipalmitoylphosphatidylcholine (DPPC) and DPPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) monolayers and bilayers and the effects of FK506 on the physical properties of DPPC and DPPC/POPG (8:2 w/w) vesicles. In addition, we assessed the immunosuppressive effects of surfactant-like phospholipid vesicles containing different amounts of FK506 on T-cell proliferation and interleukin 2 production. From surface pressure measurements of FK506/DPPC and FK506/DPPC/POPG mixed monolayers, we determined that FK506 was embedded into these monolayers up to an FK506 concentration of about 0.4 mol %. Beyond this concentration, FK506 was not quantitatively incorporated into the monolayer, suggesting possible concentration-dependent aggregation of tacrolimus. The incorporation of FK506 into DPPC monolayers, at concentrations <or= 5 microM, occurred with a partition coefficient of (3.9 +/- 0.3) x 10(3) at the bilayer equivalence pressure. FK506 was incorporated in DPPC bilayers up to an FK506 concentration of about 0.7-1 mol %, which was about double that obtained via the monolayer technique. FK506 hardly affected the transition enthalpy, the T(m), and cooperativity of the phase transition of DPPC and DPPC/POPG vesicles as determined by differential scanning calorimetry and steady-state 1,6-diphenyl-1,3,5-hexatriene anisotropy. Finally, this study provides evidence that liposomal FK506 retains the immunosuppressive efficacy of the drug.

Physical properties and surface activity of surfactant‐like membranes containing the cationic and hydrophobic peptide KL4

“…Vesicles of DPPC ⁄ POPG (27 : 9, w ⁄ w), DPPC ⁄ POPG ⁄ PA (28 : 9.4 : 5.1, w ⁄ w ⁄ w), DPPC ⁄ POPC ⁄ PA (28 : 9.4 : 5.1, w ⁄ w ⁄ w) and DPPC ⁄ PA (28 : 5.1, w ⁄ w), with different amounts of KL 4 peptide, were prepared as previously reported [35,36]. The sample solutions were prepared by mixed stock solution of the lipid and the peptide, both prepared in chloroform ⁄ methanol, to achieve the desired lipid ⁄ peptide ratio.…”

“…The required amounts of KL 4 surfactant were mixed with DPH at a probe ⁄ phospholipid molar ratio of 1 : 200 (final phospholipid concentration of 1 mgAEmL )1 ), as previously described [35]. Excitation and emission wavelengths were set at 360 and 430 nm, respectively.…”

“…Calorimetric measurements were performed as previously reported [35] in a Microcal VP DSC (Microcal Inc., Northampton, MA, USA) at a heating rate of 0.5°CAEmin )1 .…”

Nanolayers for early diagnostics of proteins involved in degenerative amyloidosis

“…The dissociation constants, K d , for FK506 and Rfx were calculated according to literature and recent studies in our group [7,8] The ligands were embedded in a planar scaffold using a biomimetic approach: that is to say preparing Langmuir-Blodgett films of phospholipids. The optimal phospholipid composition for each ligand has been established from the analysis of previous literature [9] combined with our own experimental work.…”

“…Along the same lines, absorbance measurements of the depleted ligand solution after incubation confirmed that the overall uptake was much higher for Rfx than for FK506 with this procedure. Therefore, FK506 was co-spread at the water-air interface with a DPPC/POPG (8:2) mixture [9]. Spreading isotherms for monolayers at water-air interface obtained for co-spreading with 0.29% FK506 are reported in fig.…”