F. Reig

Interaction of aminoglycosides and colistin with model membranes: Liposomes and monolayers

Abstract The interaction of gentamicin (G), kanamycin (K), spectinomycin (Sp), streptomycin (St) and a polymixin, colistin (C), with lipids was studied by using liposomes and monomolecular layers as membrane models. The lipids used were phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol disphosphate (PIP 2 ), gangliosides (Gan) and cholesterol (Chol). The results show that only colistin is able to induce the leakage of entrapped carboxyfluorescein (CF) from the liposomes after incubation. Moreover, this interaction is not clearly related to a single type of phospholipid, is dependent on the PL/colistin ratio and is slightly modified by the pH or the temperature of the incubation media. The interactions of these molecules with the polar heads of phospholipids were studied using 1-anilino-8-napthalenesulphonic acid (ANS) as fluorescent probe. The polarization values indicate that these antibiotics induce rigidification of the membrane. Monolayers having the same lipid composition of liposomes were spread on aqueous subphases and pressure increases, after injection of drug molecules, were recorded. In this set of experiments all molecules exhibit interaction, colistin and kanamycin being those with the maximum activity. Moreover, no specific differences could be assigned to the different lipids used except for kanamycin and gangliosides. The mathematical equations of the penetration kinetics for all the assays carried out were determined. The Linewe aver-Burk equation gave the best fit to the experimental values, the regression coefficient being higher than 0.98 in all cases. Compression isotherms of the same phospholipid mixtures were recorded, the antibiotic molecules being dissolved in the subphase. The compressibility and areaJmolecule were slightly affected by the presence of aminoglycosides in the aqueous phase. In contrast, the presence of colistin induced expansion of the monolayer especially at low pressures, thus indicating the existence of interactions. Comparing these results with those in the literature, it appears clear that the membrane model used exerts a strong influence on the results obtained.

A monolayer study on interactions of docetaxel with model lipid membranes.

Docetaxel (DCT) is an antineoplastic drug for the treatment of a wide spectrum of cancers. DCT surface properties as well as miscibility studies with l-alpha-dipalmitoyl phosphatidylcholine (DPPC), which constitutes the main component of biological membranes, are comprehensively described in this contribution. Penetration studies have revealed that when DCT is injected under DPPC monolayers compressed to different surface pressures, it penetrates into the lipid monolayer promoting an increase in the surface pressure. DCT is a surface active molecule able to decrease the surface tension of water and to form insoluble films when spread on aqueous subphases. The maximum surface pressure reached after compression of a DCT Langmuir film was 13 mN/m. Miscibility of DPPC and DCT in Langmuir films has been studied by means of thermodynamic properties as well as by Brewster angle microscopy (BAM) analysis of the mixed films at the air-water interface, concluding that DPPC and DCT are miscible and they form non-ideally mixed monolayers at the air-water interface. Helmholtz energies of mixing revealed that no phase separation occurs. In addition, Helmholtz energies of mixing become more negative with decreasing areas per molecule, which suggests that the stability of the mixed monolayers increases as the monolayers become more condensed. Compressibility values together with BAM images indicate that DCT has a fluidizing effect on DPPC monolayers.

Liposomes as vehicles for the presentation of a synthetic peptide containing an epitope of hepatitis A virus.

Previous work from our group showed that the entrapment of HAV-related synthetic peptides into multilamellar liposomes yielded satisfactory immunoresponses when administered to mice. In the present work we report investigative results for several liposome formulations of a 20-mer peptide related to VP3 capsid protein of HAV. In this sense, the recently introduced surface plasmon resonance technique has been applied to compare the different strategies of association between the synthetic peptide and phospholipid vesicles and to demonstrate that no significant alteration in antigenicity is produced when the peptide sequence is covalently coupled to the surface of small unilamellar vesicles. In addition, conformational data obtained by the circular dichroism technique have shown a decrease in the helical contribution of peptide-once linked to phospholipid; probably this change is due to the restriction introduced at the N-terminus of the sequence when coupled to the derivatized phospholipids at the surface of vesicle bilayers.

Evaluation of free and liposome-encapsulated gentamycin for intramuscular sustained release in rabbits

Gentamycin sulphate (GS) and gentamycin oleate (GO) were encapsulated in liposomes composed of phosphatidylcholine (HPC) and cholesterol (CHOL) (molar ratio 7:7:2 and 5:5:1, respectively), and were administered via intramuscular injection to rabbits, to evaluate their potential use as sustained release formulations. Five groups of five animals each were used for the pharmacokinetic study, and treatments were established as follows: 3 mg kg(-1) of GS i.v., 3 mg kg(-1) of GS i.m., 3 mg kg(-1) of liposome-containing gentamycin sulphate (LGS) i.m., 3 mg kg(-1) of GO i.m., and 3 mg kg(-1) of liposome-containing gentamycin oleate (LGO) i.m. Gentamycin plasma concentrations after i.m. administration of LGS were extremely low compared with those obtained after the i.m. administration of GS; the peak plasma concentration (Cmax) showed an eight-fold decrease with LGS, and the area under the concentration-time curve (AUC) was four-fold lower for the liposomal form. The apparent elimination half-life estimated after administration of LGS showed a three-fold increase compared with values calculated for free GS. After the administration of the same dose of LGO, Cmax obtained showed a 2.5-fold decrease in relation to peak concentrations of free GO, and the apparent beta-half life of encapsulated GO showed a three-fold increase compared with i.m. GO. Large-size liposomes containing gentamycin administered i.m. to rabbits gave sustained drug release from the injection site, providing prolonged plasma concentrations of the drug in the body.

Conformational behavior of the HAV-VP3(110–121) peptidic sequence and synthetic analogs in membrane environments studied by CD and computational methods

The present study was undertaken to examine the structural features that may be important to explain the immunogenicity of the (110-121) peptide sequence (FWRGDLVFDFQV) of VP3 capsid protein of hepatitis A virus. A conformational analysis of the preferred conformations by CD and molecular mechanics was carried out. Present results suggest that the interaction with liposomes as biomembrane model induces and stabilizes the amphipathic beta-structure of the peptide. To study the contribution of amino acid replacements at the RGD tripeptide as well as the influence of the peptide chain length on peptide conformation, solid-phase peptide synthesis of several peptide analogs was carried out and the peptide conformation was studied using CD spectroscopy. The results show that the RGD sequence is necessary to induce the beta-structure in the presence of liposomes.

Interaction of colistin with lipids in liposomes and monolayers

Abstract Colistin is an antibiotic member of the polymixin family, showing a high amphipathic character. Its mechanism of action has been related to its ability to disrupt phospholipid bilayers of the bacterial membrane. Due to its hydrophobic properties colistin can interact both with the polar heads and the alkyl chains of the phospholipids. Moreover, it has a polycationic structure, so its interactions with phospholipids should be highly dependent on the electric charge of the lipid and the ionisation state of polymixin molecule. In the present paper we report on physicochemical studies to define the type and characteristics of these interactions. The surface activity of colistin has been shown to be highly dependent on the pH of the medium, the less protonated forms being more stable at the air–water interface. Interaction with phospholipid monolayers shows the same tendency. Colistin is also able to form mixed monolayers with phospholipids with small deviations from ideality. Physicochemical studies carried out with fluorescent probes indicate the presence of pure colistin aggregates that can coexist with mixed micelles composed of colistin/phospholipids. In no case did the interaction cause significative changes in the transition temperature of phospholipids.

Anti‐Hepatitis A Virus Antibody Response Elicited in Mice by Different Forms of a Synthetic VP1 Peptide

Peptide VP1 (11‐25) of the capsid of hepatitis A virus was synthesized by the Fmoc‐polyamide solid phase method, and administered to mice in different forms: (1) free, (2) encapsulated in multilamellar liposomes, (3) coupled to keyhole limpet hemocyanin (KHL), and (4) incorporated into a tetrameric branched lysine core. The highest anti‐VP1 peptide responses were generated by synthetic peptides entrapped into liposomes and coupled to KLH. No anti‐HAV response was generated with the free peptide, while all the other forms induced both anti‐HAV and HAV‐neutralizing antibodies. Maximum neutralization indices were observed in ascites from mice treated with liposome‐entrapped and KLH peptides.

INTERACTION OF BRANCHED CHAIN POLYMERIC POLYPEPTIDES WITH PHOSPHOLIPID MODEL MEMBRANES

The surface properties at the air/water interface and the interaction of branched chain polymeric polypeptides with a general formula poly[Lys-(DL-Alam-X1)], where X = Π (AK), Ser (SAK), or Glu (EAK), with phospholipids were investigated. Polylysine derivatives with polycationic (SAK, AK) or amphoteric (EAK) were capable to spread and form stable monomolecular layers. The stability of monolayers at the air/water interface was dependent on the side-chain terminal amino acid residue of polymers and can be described by SAK < AK < EAK order. The area per amino acid residue values calculated from compression isotherms were in the same range as compared to those of linear poly-α-amino acids and proteins. Moreover, these polymers interact with phospholipid monomolecular layers composed of dipalmitoyl phosphatidyl choline (DPPC) or DPPC/PG (PG: phosphatidyl glycerol; 95/5, mol/mol). Data obtained from compression isotherms of phospholipids spread on aqueous polymer solutions at different initial surface pressure indicated that insertion into lipid monolayers for SAK or AK is more pronounced than for EAK. The interaction between branched polypeptides and phospholipid membranes was further investigated using lipid bilayers with DPPC/PG and fluorescent probes located either at the polar surface [1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) sodium anilino naphthalene sulfonate (ANS)] or within the hydrophobic core (DPH) of the liposome. Changes in fluorescence intensity and in polarization were observed when TMA-DPH or ANS, but not DPH were used. Comparative data also indicate that all three polymers interact only with the outer surface of the bilayer, but even the most marked penetration of polycationic polypeptide (SAK) did not result in alteration of the ordered state of the alkyl chains in the bilayer. Taken together, data obtained from mono- or bilayer experiments suggest that the interaction between branched polymers and phospholipids are highly dependent on the charge properties (Ser vs Glu) and on the identity (Ser vs Ala) of side-chain terminating amino acids. The binding of polymers to the model membranes could be mainly driven by electrostatic forces, but the significant role of hydrophilic properties in case of SAK cannot be excluded.

Mixed anhydrides in peptide synthesis. factors afffectlng urethane formation and racemization

Abstract A comparative study on the effectiveness of two mixed anhydrides procedures in the synthesis of a series of hydrophobic tripeptides by using three solvents and four different hydrophobic nucleophiles has been carried out. Dicyclohexylcarbodiimide (DCC) method has also been used as reference for comparative purposes. The results show that despite the system Dpp/DMF provides the best chemical yields with these specific couplings, when retention of configuration is concerned the coupling method of choice should be DCC/HOBt using either THF or DMF as solvent system.

Gentamicin Determination in Biological Fluids by HPLC, Using Tobramycin as Internal Standard

Abstract An improved procedure to quantify gentamicin in biological fluids is presented. The antibiotic is isolated from plasma and urine by using a silica column, and measured by reversed-phase chromatography. Pre-column derivatization with o-phthalaldehyde to form fluorescent products for detection is used. The method can accurately measure 0.3 mg of gentamicin per liter, and standard curves showed a linear response in plasma and urine at concentrations ranging from 0 to 10 mg/liter. The different processes described in the literature for aminoglycoside isolation are discussed and evaluated with reference to the present results. Moreover, the