Francesca Reig

Coating liposomes with collagen (Mr 50,000) increases uptake into liver.

Collagen-coated small unilamellar liposomes were prepared by incubation of two hydrophobic derivatives of collagen (average Mr 50 000) with preformed vesicles. The introduction of hexyl and lauryl residues to the collagen molecule improved by 10-fold the ability of collagen to coat liposomes. In vitro stability of the different coated vesicles prepared, was studied by their ability to retain entrapped carboxyfluorescein as a function of the time. Coated vesicles were clearly more stable in vitro than control liposomes, except for those containing the lauryl derivative in a protein/phospholipid weight ratio higher than 10(-3). Vesicle clearance from circulation as well as tissue distribution were also determined. Pharmacokinetics (determined by both fluorescence and radioactive techniques) were highly dependent on the injected dose, phospholipids used and the content of collagen. Half-lives were maximum for liposomes composed of saturated phospholipids injected at a dose of 2 micromol phospholipid. Besides, blood elimination of collagen-containing vesicles was about 2-fold faster and liver uptake 1.5 to 2-fold higher than control liposomes.

A fluorescence and CD study on the interaction of synthetic lipophilic hepatitis B virus preS(120–145) peptide analogues with phospholipid vesicles

The interaction of the immunogenic peptide of human hepatitis B virus (HBV) preS(120-145), including B and T epitopes, with phospholipid vesicles has been studied by fluorescence techniques and CD. In addition, interaction of three lipopeptides derived from preS(120-145) containing stearoyl, cholanoyl, and tripalmitoyl-S-glyceryl-cysteine (Pam3C) SS moieties with dipalmitoylphosphatidylcholine (DPPC) has been investigated by polarization fluorescence spectroscopy. Fluorescence experiments showed an increase in fluorescence intensity and a blue shift of the maximum emission wavelength upon interaction of preS(120-145) with DPPC vesicles below the transition temperature (Tc), indicating that the tryptophan moiety enters a more hydrophobic environment. Moreover, fluorescence polarization experiments showed that the peptide decreased the membrane fluidity at the hydrophobic core, increasing the Tc of the lipid and decreasing the amplitude of the change of fluorescence polarization associated with the cooperative melting of 1,6-diphenyl-1,3,5-hexatriene labeled vesicles. The absence of leakage of vesicle-entrapped carboxyfluorescein indicates that the peptide did not promote vesicle lysis. Besides, the three lipopeptides derived from preS(120-145) showed a more pronounced rigidifying effect at the hydrophobic core of the bilayer, with a significative increase in the Tc. Stearoyl- and cholanoyl-preS(120-145) restricted the motion of lipids also at the polar surface, whereas Pam3CSS-preS(120-145) did not alter the polar head group order. Finally, CD studies in 2,2,2-trifluoroethanol or in presence of vesicles suggested that the bound peptide adopted amphiphilic alpha-helical and beta-sheet structures, with an important contribution of the beta-turn. It is concluded that preS(120-145) can interact with the lipid membrane through the formation of an amphipathic structure combination of beta-sheet and alpha-helix aligned parallel to the membrane surface, involving the N-terminal residues, and penetrating only a short distance into the hydrophobic core. The C-terminal part, with a combination of beta-turn and beta-sheet structure, remains at the outer part of the bilayer, being potentially accessible to immunocompetent cells. Furthermore, coupling of an hydrophobic moiety to the N-terminal part of the peptide favors anchoring to the membrane, probably facilitating interaction of the peptide with the immunoglobulin receptor. These results are in agreement with the induction of immune response by preS(120-145) and with the enhanced immunogenicity found in general for lipid-conjugated immunopeptides.

Kinetic study of α-chymotrypsin-catalyzed synthesis of kyotorphin

Abstract A kinetic analysis of reaction-rate data obtained during a series of optimization experiments of the α-chymotrypsin-catalyzed synthesis of kyotorphin has been performed. The kinetic data have been fitted to a model equation derived from a proposed sequential mechanism, which has been further simplified to a first-order equation as a function of the substrate consumption. Statistical tests performed validate the model, since the fitted constants were statistically significant. In addition, the activation energy of the process has been calculated and resulted to be 32.5 ± 2.3 kJ/mol which is within the range of other enzymatic reactions.

Interaction energies of mixing phosphatidylserine and phosphatidylcholine monolayers

Abstract The interactions between phosphatidylcholine and phosphatidylserine spread in monolayers of different molecular composition were studied. The influence in these mixtures of Na + and Ca 2+ ions was also determined. By applying the Goodrich, and Pagano and Gershfeld equations, the mixing energies and interactions at intermediate and collapse pressures were calculated. The maximum interaction occurs at higher pressures and in the presence of Ca 2+ ions.

Biodistribution study of doxorubicin encapsulated in liposomes: influence of peptide coating and lipid composition.

Abstract In this paper we describe the biodistribution of doxorubicin (DXR) encapsulated in three different types of liposomes. Common composition was hydrogenated phosphatidylcholine (HPC)/phosphatidylglycerol (PG) cholesterol (Chol)/X, X being either 10% N‐glutaryl phosphatidylethanolamine (NGPE), 10% NGPE + 6% distearoyl‐phosphatidylethanolamine‐polyethyleneglycol 2000 (DSPE‐PEG), or 10% NGPE + 6% DSPE‐PEG‐COOH. These series of vesicles were coated with an active or an inactive sequence of laminin (laminin receptors, integrins, are over‐expressed in tumor cells). Single doses of these preparations were injected, i.v., into healthy mice. For biodistribution experiments, mice were sacrificed at three different time‐points post‐treatment. Doxorubicin and doxorubicinol (DXOH) levels were determined in plasma, heart, lung, kidney, spleen, and liver using HPLC with daunorubicin (DNR) as internal standard. The results obtained indicate that compositions containing DSPE‐PEG have the longest half‐lives in plasma, as was to be expected according to the data in the literature. However, the presence of the peptides on the surface of liposomes reduces concentration values in this tissue. Distribution in other organs reveals high differences, among the liposomal samples studied, depending mainly on the presence of active or inactive peptide on the surface of vesicles. Liposomes coated with the laminin active sequence show lower accumulation in studied tissues than free DXR. This indicates that heart toxicity, associated to DXR treatments, could be diminished, and open promising perspectives for its future study in tumor‐bearing animals.

In vitro effects of SIKVAV retro and retro-enantio analogues on tumor metastatic events.

The SIKVAV peptide, located on the long arm of the laminin alpha1 chain, promotes cell adhesion, invasion and migration of tumor and endothelial cells, resulting in tumor growth, angiogenesis and metastasis. In this paper, we report the synthesis of the SIKVAV peptide and its retro (reverse l-amino acid order) and retro-enantio (reverse d-amino acid order) analogues and their effect on three critical steps in the metastatic process: cell-extracellular matrix protein (ECM) adhesion, cell migration and homotypic cell adhesion, using B16F10 melanoma cells. Results show that all peptides compete with laminin-1 cell attachment, but only SIKVAV induces peptide-cell adhesion. Retro analogue, but not retro-enantio, inhibits cell adhesion to SIKVAV, indicating that retro peptide recognizes the SIKVAV receptors while retro-enantio does not. Retro-enantio peptide is able to inhibit cell migration, by contrast of the SIKVAV chemoattractant activity. All three peptides reduce the homotypic cell adhesion in a dose-dependent manner, but retro-enantio sequence is the most effective reaching a 35% inhibition of controls at the higher concentration. These findings suggest that that both analogues of SIKVAV peptide, especially retro-enantio, may be considered as potential antimetastatic agents.

Design of a new formulation for sustained release of gentamicin: A Carbopol hydrogel

Two gentamicin-containing hydrogels of different water solubility have been prepared using Carbopol as a thickening agent. The most insoluble hydrogel (A) released its drug content extremely slowly, regardless of the absence or presence of plasma. In contrast, the rate of drug release from hydrogel B was just slightly slower than the free drug in saline, whereas in the presence of plasma components just 35% of the gentamicin contained in hydrogel B was eventually released from it. Pharmacokinetic studies showed a sustained release of gentamicin from the Carbopol hydrogel (B), with an elimination half-life in excess of 2.5 h.

Preparation and in vitro activity of liposome encapsulated opioids

Four opiate molecules: morphine, naloxone, meperidine and codeine have been encapsulated in liposomes. The encapsulation efficiency has been studied as a function of the following parameters: liposome preparation method, lipid composition and opioid molecule hydrophobicity. The most important parameter as far as the entrapment efficiency is concerned is the liposome preparation method. The opioid activity of these molecules in vitro (Guinea Pig Ileum preparation) has been determined. No differences in the IC50 values could be found between encapsulated and free drug molecules.

A Comparative Study of the Performance of Solid Supported and Soluble α-Chymotrypsin for the Enzymatic Synthesis of Kyotorphin

The performance of both free and solid-supported α-chymotrypsin for the synthesis of ZTyr-ArgNH2 (Z-kyotorphin amide) has been studied. The effect on the reaction yield of cosolvent/buffer proportions and reaction temperatures has been examined by a Box-Wilson method. Such factors influenced both preparations to a different extent. A kinetic analysis of reaction rate data has shown that the kinetic data are consistent with a proposed sequential mechanism. Model equations and numerically estimated kinetic parameters are presented. Statistical tests validate the model. In addition, the performance of the reaction has also been assessed by instantaneous and overall fractional yield calculations. Despite the high byproduct formation, the immobilized enzyme could be the choice for a technological process aiming at the large scale synthesis of such a peptide.

Solid phase synthesis of potential antigenic peptides and new lipopeptides of hepatitis B virus

Peptides belonging to the envelope protein of HBV [Tyr148]S(139–148) and preS(120–145) have been synthesized using the continuous-flow fluoren-9-ylmethoxycarbonyl (Fmoc)-polyamide solid phase methodology. Furthermore, the synthesis of a new series of hydrophobic derivatives involving Nα-acylation of both peptides with stearic and cholanic acid as well as the introduction of the synthetic B-cell and macrophage activator Pam3-Cys-Ser-Ser is also described. Benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) and benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate (BOP) proved to be convenient reagents to promote the coupling of these lipid moieties to peptides attached to Kieselguhr-supported polyacrylamide resins. Some synthetic aspects concerning reaction conditions and the use of different scavengers at the cleavage stage are discussed. Finally, a cyclic derivative of the S peptide was obtained through a disulphide bond formation.