In this work, a peptide for ocular delivery (POD) and human immunodeficiency virus transactivator... more In this work, a peptide for ocular delivery (POD) and human immunodeficiency virus transactivator were conjugated with biodegradable poly(lactic-co-glycolic acid) (PGLA)polyethylene glycol (PEG)-nanoparticles (NPs) in an attempt to improve ocular drug bioavailability. The NPs were prepared by the solvent displacement method following two different pathways. One involved preparation of PLGA NPs followed by PEG and peptide conjugation (PLGA-NPs-PEG-peptide); the other involved self-assembly of PLGA-PEG and the PLGA-PEGpeptide copolymer followed by NP formulation. The conjugation of the PEG and the peptide was confirmed by a colorimetric test and proton nuclear magnetic resonance spectroscopy. Flurbiprofen was used as an example of an anti-inflammatory drug. The physicochemical properties of the resulting NPs (morphology, in vitro release, cell viability, and ocular tolerance) were studied. In vivo anti-inflammatory efficacy was assessed in rabbit eyes after topical instillation of sodium arachidonate. Of the formulations developed, the PLGA-PEG-POD NPs were the smaller particles and exhibited greater entrapment efficiency and more sustained release. The positive charge on the surface of these NPs, due to the conjugation with the positively charged peptide, facilitated penetration into the corneal epithelium, resulting in more effective prevention of ocular inflammation. The in vitro toxicity of the NPs developed was very low; no ocular irritation in vitro (hen's egg test-chorioallantoic membrane assay) or in vivo (Draize test) was detected. Taken together, these data demonstrate that PLGA-PEG-POD NPs are promising vehicles for ocular drug delivery.
Introduction Evidence suggests that citrullinated fibrin(ogen) may be a potential in vivo target ... more Introduction Evidence suggests that citrullinated fibrin(ogen) may be a potential in vivo target of anticitrullinated protein/ peptide antibodies (ACPA) in rheumatoid arthritis (RA). We compared the diagnostic yield of three enzyme-linked immunosorbent assay (ELISA) tests by using chimeric fibrin/ filaggrin citrullinated synthetic peptides (CFFCP1, CFFCP2, CFFCP3) with a commercial CCP2-based test in RA and analyzed their prognostic values in early RA.
Introduction: Comparative data on synovial cell infiltrate and cytokine levels in anti citrullina... more Introduction: Comparative data on synovial cell infiltrate and cytokine levels in anti citrullinated peptide/protein antibody (ACPA)-positive and ACPA negative rheumatoid arthritis (RA) patients are scarce. Our aim was to analyze synovial cell infiltrate and synovial fluid (SF) levels of cytokines in patients with RA according to the presence or absence of ACPA in serum. Methods: A cross-sectional study in a single center including consecutive RA patients was performed. Patients were defined as 'ACPA negative' if serum was negative to two different ACPAs [second generation commercial anti-cyclic citrullinated peptide antibodies (CCP2) and chimeric fibrin/filaggrin citrullinated antibodies]. Parallel synovial tissue (ST) biopsies and SF were obtained by knee arthroscopy. Synovial cell infiltrate and endothelial cells were analyzed by immunohistochemistry and SF levels of Th1, Th2, Th17 and pro-inflammatory cytokines by Quantibody(R) Human Array. Results: A total of 83 patients underwent arthroscopy, with a mean age of 55.9 ± 12 years, and mean disease duration of 45 months (interquartile range, IQR 10.8 to 122). 62% were female and 77% were ACPA positive. No significant differences were found in clinical variables, acute phase reactants, synovial cell infiltrate or lymphoid neogenesis (LN) between ACPA positive and negative patients. However ACPA positive patients had significantly higher levels of IL-1β, IL-10, IL-17 F and CC chemokine ligand 20 (CCL-20) than ACPA negative patients. Conclusions: In our cohort of patients with RA no significant differences were found in synovial cell infiltrate or synovial LN according to ACPA status. However, ACPA positive patients had higher levels of T-cell derived and pro-inflammatory cytokines than ACPA negative patients. As systemic and local inflammation was similar in the two groups, these findings support a distinct synovial physiopathology.
To analyze the effects of cigarette smoking on disease activity and radiographic damage in patien... more To analyze the effects of cigarette smoking on disease activity and radiographic damage in patients with early rheumatoid arthritis (RA). Study subjects were 156 patients with early RA (< 2 yrs). Disease activity, therapeutic response, and radiographic progression were compared in smokers and nonsmokers at 24 months. At baseline, ever-smokers had earlier disease onset and a closer association with the shared epitope (SE), but not more seropositive disease. No significant differences were observed in disease activity and European League Against Rheumatism therapeutic responses between smokers and nonsmokers. Multivariate analysis showed that baseline Larsen score, the HLA-DRB*04 genotype, being female, and current smoking were associated with radiographic progression. In patients with early RA, smoking was associated with earlier disease onset and the SE. Smoking was an independent factor of radiographic progression.
Following the report of beneficial effects of co-infection by GB virus C (GBV-C) for HIV-infected... more Following the report of beneficial effects of co-infection by GB virus C (GBV-C) for HIV-infected patients, we have studied synthetic GBV-C peptides and their relationship with HIV type-1. This paper reports the design and synthesis of new forms of presentation of two peptide inhibitors corresponding to the envelope proteins E1 and E2 of GBV-C, together with a study of their anti-HIV-1 activity. Homogeneous and heterogeneous multiple antigenic peptides (MAPs), lipophilic derivatizations, cyclization and peptide-gold conjugations are the chemical design strategies adopted. Our aim is to enhance the anti-viral potency of the GBV-C peptide domains. Of all the GBV-C peptide derivatives studied, peptide-gold complexes derived from the (22-39) sequence of the GBV-C E1 protein were the most active entry inhibitors. These results support the putative modulation of HIV-1 infection by the GBV-C E1 protein and open new perspectives for the development of novel peptide-derived HIV-1 entry inhib...
The synthesis of three cyclic peptides related to an active sequence of Laminin is described. The... more The synthesis of three cyclic peptides related to an active sequence of Laminin is described. These molecules have no, one, or two stearoyl residues linked to the amino terminal end of the sequence. The physicochemical properties of these peptides as well as their interactions with dipalmitoylphosphatidylcholine molecules ordered in mono- and bilayers are described. Properties based on aqueous solutions of peptides show maximum activity for the analogue with one stearoyl residue. But properties measured from organic solutions of peptides (compression isotherms), as well as those in water but at high temperatures, gave as the most surface active the distearoyl derivative. This behavior is interpreted as a strong tendency of this highly hydrophobic molecule to form aggregates (probably micelles), very stable in aqueous media. Thus, the monostearoyl-substituted peptide seems to be the most adequate for efficient insertion into mono- and bilayers.
The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural a... more The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (πe) were clearly lower than the biological membrane pressure (24-30mN m(-1)) and the HIV-1 FP (35mN m(-1)). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (G(E)). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored ...
The synthesis of a peptide belonging to the VP3 capsid protein of Hepatitis A virus has been acco... more The synthesis of a peptide belonging to the VP3 capsid protein of Hepatitis A virus has been accomplished by the continuous flow Fmoc-polyamide solid phase method. The use of methoxytrimethylbenzenesulphonyl (Mtr) and pentamethylchromansulphonyl (Pmc) as arginine side-chain protecting groups in the presence of tryptophan without lateral protection or protected with t-Boc is discussed. The synthetic VP3 peptide has been administered to mice in different forms: (i) free, (ii) coupled to keyhole limpet hemocyanin, (iii) encapsulated in multilamellar (MLV) liposomes, and (iv) incorporated to a tetrameric branched lysine core. The immune response induced by these preparation is reported.
between an opioid antagonist, naloxone, and several lipids and lipid mixtures was studied using m... more between an opioid antagonist, naloxone, and several lipids and lipid mixtures was studied using monomolecular layers as a membrane model. No specific interactions could be detected. The main factor affecting the penetration of naloxone in monolayers seems to be the initial ordered state of the molecules instead of the chemical composition of the monolayer. A new membrane model was assayed based on the retention times in high-pressure liquid chromatography working with hydrophobically modified columns.
An improved procedure to quantify gentamicin in biological fluids is presented. The antibiotic is... more 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
ABSTRACT The interaction of gentamicin (G), kanamycin (K), spectinomycin (Sp), streptomycin (St) ... more 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 (PIP2), 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.
Previous work from our group showed that the entrapment of HAV-related synthetic peptides into mu... more 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 dierent strategies of association between the synthetic peptide and phospholipid vesicles and to demonstrate that no signi®cant 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. #
The physicochemical characterization of the peptide sequences E2 (39 Á/53) and E2 (32 Á/59) corre... more The physicochemical characterization of the peptide sequences E2 (39 Á/53) and E2 (32 Á/59) corresponding to the structural protein E2 of the GB virus C was done by studying their interaction with model membranes. The peptides showed surface activity concentration dependent when injected beneath a buffered solution. This tendency to accumulate into the air/water interface suggested a potential ability of these peptides to interact with bilayers. For that reason, Small Unilamellar Liposomes (SUVs) of 1,2-dimyiristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2dimyiristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG) were chosen as a mimetic membranes. A series of fluorescence experiments based on tryptophan peptide fluorescence or with fluorescence labeled SUVs, were done to cover different aspects of peptide interaction with bilayers. Steady state fluorescence anisotropy studies with N -(7-nitro-2-1,3-benzoxadiazol-4-yl) dioleoylphosphatidylethanolamine (NBD-PE) or 1-[4-(trimethylammonium) phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) labeled SUVs indicated that only the long peptide was able to change the lipid microenvironment of DMPG vesicles by slightly increasing the rigidity of the bilayer both above and under the lipid main transition temperature. These results were concordant with the slight blue shift of the maximum tryptophan wavelength emission after E2 (32 Á/53) peptide incubation with DMPG vesicles. Our data provide useful information for the design of synthetic immunopeptides that can be incorporated into a liposomal system with a potential to promote a direct delivery of the membrane-incorporated immunogen to the immunocompetent cells, thus increasing the immuno response from the host. #
Three synthetic peptide sequences of 18 amino acid each, corresponding to different fragments of ... more Three synthetic peptide sequences of 18 amino acid each, corresponding to different fragments of the E2 capsid protein of GB virus C (GBV-C): SDRDTVVELSEWGVPCAT (P45), GSVRFPFHRCGAGPKLTK (P58) and RFPFHRCGAGPKLTKDLE (P59) have been characterized in order to find a relationship between their physicochemical properties and the results obtained in cellular models. Experiments were performed in presence and absence of the HIV fusion peptide (FP-HIV) due to the evidences that GBV-C inhibits AIDS progression. P45 peptide showed lower surface activity and less extent of penetration into 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) (3:2, mol/mol) lipid monolayers than P58 and P59. However, P45 peptide presented higher capacity to inhibit FP-HIV induced cell-cell fusion than the other two sequences. These results were supported by fluorescence anisotropy measurements which indicated that P45 had a significant effect on the inhibition of FP-HIV perturbation of liposomes of the same lipid composition. Finally, atomic force microscopy (AFM) studies have evidenced the modification of the changes induced by the FP-HIV in the morphology of lipid bilayers when P45 was present in the medium.
Merocyanine 540 (MC540) has been used as external probe to determine the interaction of the pepti... more Merocyanine 540 (MC540) has been used as external probe to determine the interaction of the peptide sequence 125 Á/ 139 corresponding to the E2 protein of Hepatitis G virus, with lipid bilayers. The probe was incorporated into large unilamellar vesicles (LUVs) or small unilamellar vesicles (SUVs) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). When incorporated into bilayers, MC540 shows two absorption maxima corresponding to the monomer (570 nm) and dimer (530 nm) form of the probe. Changes in the probe microenvironment are reflected by a modification in the position and/or intensity of these maxima. Addition of increasing amounts of peptide resulted in a slight decrease of the ratio A570/A530 thus indicating a change in MC540 partition into the membrane, going from a hydrophobic to a more hydrophilic environment. This effect was concomitant with an increase in dimer formation as stated from the values of the apparent dimerization constant (K app ) obtained. Fluorescence spectra as well as steady state anisotropy measurements were in agreement with the above results indicating that the peptide was able to relocate the probe and displacing MC540 from its initial location into the bilayer. Results with SUVs or LUVs were similar for what curvature does not seem to play any role on peptide activity. These results reflect the ability of peptide to interact with biomimetic membranes in the lipid head group region. #
European journal of medicinal chemistry, Jan 30, 2014
The development of peptide fusion inhibitors based on short synthetic peptides represents a promi... more The development of peptide fusion inhibitors based on short synthetic peptides represents a promising option in the fight against HIV-1 infection, especially in individuals infected with multiresistant HIV-1 strains. GBV-C has the beneficial effect of retarding the progression of AIDS in people who are co-infected with both the GBV-C and HIV viruses. In previous works, the E1(22-39) GBV-C sequence (E1P8lin) was found to be capable of inhibiting the interaction of HIV-1 FP with bilayers and its cyclic analogue (E1P8cyc) showed a higher anti-HIV-1 activity. In the present work, in an attempt to gain a better understanding of the interaction of E1P8 peptides with HIV-1 FP, we analyzed direct interactions between peptides at the molecular level. Our results support that E1P8cyc might be more potent at blocking HIV-1 entry than E1P8lin as a consequence of the structure induced in the complex formed with HIV-1 FP, which is able to modify the conformation adopted by this functional domain ...
In this work, a peptide for ocular delivery (POD) and human immunodeficiency virus transactivator... more In this work, a peptide for ocular delivery (POD) and human immunodeficiency virus transactivator were conjugated with biodegradable poly(lactic-co-glycolic acid) (PGLA)polyethylene glycol (PEG)-nanoparticles (NPs) in an attempt to improve ocular drug bioavailability. The NPs were prepared by the solvent displacement method following two different pathways. One involved preparation of PLGA NPs followed by PEG and peptide conjugation (PLGA-NPs-PEG-peptide); the other involved self-assembly of PLGA-PEG and the PLGA-PEGpeptide copolymer followed by NP formulation. The conjugation of the PEG and the peptide was confirmed by a colorimetric test and proton nuclear magnetic resonance spectroscopy. Flurbiprofen was used as an example of an anti-inflammatory drug. The physicochemical properties of the resulting NPs (morphology, in vitro release, cell viability, and ocular tolerance) were studied. In vivo anti-inflammatory efficacy was assessed in rabbit eyes after topical instillation of sodium arachidonate. Of the formulations developed, the PLGA-PEG-POD NPs were the smaller particles and exhibited greater entrapment efficiency and more sustained release. The positive charge on the surface of these NPs, due to the conjugation with the positively charged peptide, facilitated penetration into the corneal epithelium, resulting in more effective prevention of ocular inflammation. The in vitro toxicity of the NPs developed was very low; no ocular irritation in vitro (hen's egg test-chorioallantoic membrane assay) or in vivo (Draize test) was detected. Taken together, these data demonstrate that PLGA-PEG-POD NPs are promising vehicles for ocular drug delivery.
Introduction Evidence suggests that citrullinated fibrin(ogen) may be a potential in vivo target ... more Introduction Evidence suggests that citrullinated fibrin(ogen) may be a potential in vivo target of anticitrullinated protein/ peptide antibodies (ACPA) in rheumatoid arthritis (RA). We compared the diagnostic yield of three enzyme-linked immunosorbent assay (ELISA) tests by using chimeric fibrin/ filaggrin citrullinated synthetic peptides (CFFCP1, CFFCP2, CFFCP3) with a commercial CCP2-based test in RA and analyzed their prognostic values in early RA.
Introduction: Comparative data on synovial cell infiltrate and cytokine levels in anti citrullina... more Introduction: Comparative data on synovial cell infiltrate and cytokine levels in anti citrullinated peptide/protein antibody (ACPA)-positive and ACPA negative rheumatoid arthritis (RA) patients are scarce. Our aim was to analyze synovial cell infiltrate and synovial fluid (SF) levels of cytokines in patients with RA according to the presence or absence of ACPA in serum. Methods: A cross-sectional study in a single center including consecutive RA patients was performed. Patients were defined as 'ACPA negative' if serum was negative to two different ACPAs [second generation commercial anti-cyclic citrullinated peptide antibodies (CCP2) and chimeric fibrin/filaggrin citrullinated antibodies]. Parallel synovial tissue (ST) biopsies and SF were obtained by knee arthroscopy. Synovial cell infiltrate and endothelial cells were analyzed by immunohistochemistry and SF levels of Th1, Th2, Th17 and pro-inflammatory cytokines by Quantibody(R) Human Array. Results: A total of 83 patients underwent arthroscopy, with a mean age of 55.9 ± 12 years, and mean disease duration of 45 months (interquartile range, IQR 10.8 to 122). 62% were female and 77% were ACPA positive. No significant differences were found in clinical variables, acute phase reactants, synovial cell infiltrate or lymphoid neogenesis (LN) between ACPA positive and negative patients. However ACPA positive patients had significantly higher levels of IL-1β, IL-10, IL-17 F and CC chemokine ligand 20 (CCL-20) than ACPA negative patients. Conclusions: In our cohort of patients with RA no significant differences were found in synovial cell infiltrate or synovial LN according to ACPA status. However, ACPA positive patients had higher levels of T-cell derived and pro-inflammatory cytokines than ACPA negative patients. As systemic and local inflammation was similar in the two groups, these findings support a distinct synovial physiopathology.
To analyze the effects of cigarette smoking on disease activity and radiographic damage in patien... more To analyze the effects of cigarette smoking on disease activity and radiographic damage in patients with early rheumatoid arthritis (RA). Study subjects were 156 patients with early RA (< 2 yrs). Disease activity, therapeutic response, and radiographic progression were compared in smokers and nonsmokers at 24 months. At baseline, ever-smokers had earlier disease onset and a closer association with the shared epitope (SE), but not more seropositive disease. No significant differences were observed in disease activity and European League Against Rheumatism therapeutic responses between smokers and nonsmokers. Multivariate analysis showed that baseline Larsen score, the HLA-DRB*04 genotype, being female, and current smoking were associated with radiographic progression. In patients with early RA, smoking was associated with earlier disease onset and the SE. Smoking was an independent factor of radiographic progression.
Following the report of beneficial effects of co-infection by GB virus C (GBV-C) for HIV-infected... more Following the report of beneficial effects of co-infection by GB virus C (GBV-C) for HIV-infected patients, we have studied synthetic GBV-C peptides and their relationship with HIV type-1. This paper reports the design and synthesis of new forms of presentation of two peptide inhibitors corresponding to the envelope proteins E1 and E2 of GBV-C, together with a study of their anti-HIV-1 activity. Homogeneous and heterogeneous multiple antigenic peptides (MAPs), lipophilic derivatizations, cyclization and peptide-gold conjugations are the chemical design strategies adopted. Our aim is to enhance the anti-viral potency of the GBV-C peptide domains. Of all the GBV-C peptide derivatives studied, peptide-gold complexes derived from the (22-39) sequence of the GBV-C E1 protein were the most active entry inhibitors. These results support the putative modulation of HIV-1 infection by the GBV-C E1 protein and open new perspectives for the development of novel peptide-derived HIV-1 entry inhib...
The synthesis of three cyclic peptides related to an active sequence of Laminin is described. The... more The synthesis of three cyclic peptides related to an active sequence of Laminin is described. These molecules have no, one, or two stearoyl residues linked to the amino terminal end of the sequence. The physicochemical properties of these peptides as well as their interactions with dipalmitoylphosphatidylcholine molecules ordered in mono- and bilayers are described. Properties based on aqueous solutions of peptides show maximum activity for the analogue with one stearoyl residue. But properties measured from organic solutions of peptides (compression isotherms), as well as those in water but at high temperatures, gave as the most surface active the distearoyl derivative. This behavior is interpreted as a strong tendency of this highly hydrophobic molecule to form aggregates (probably micelles), very stable in aqueous media. Thus, the monostearoyl-substituted peptide seems to be the most adequate for efficient insertion into mono- and bilayers.
The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural a... more The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (πe) were clearly lower than the biological membrane pressure (24-30mN m(-1)) and the HIV-1 FP (35mN m(-1)). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (G(E)). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored ...
The synthesis of a peptide belonging to the VP3 capsid protein of Hepatitis A virus has been acco... more The synthesis of a peptide belonging to the VP3 capsid protein of Hepatitis A virus has been accomplished by the continuous flow Fmoc-polyamide solid phase method. The use of methoxytrimethylbenzenesulphonyl (Mtr) and pentamethylchromansulphonyl (Pmc) as arginine side-chain protecting groups in the presence of tryptophan without lateral protection or protected with t-Boc is discussed. The synthetic VP3 peptide has been administered to mice in different forms: (i) free, (ii) coupled to keyhole limpet hemocyanin, (iii) encapsulated in multilamellar (MLV) liposomes, and (iv) incorporated to a tetrameric branched lysine core. The immune response induced by these preparation is reported.
between an opioid antagonist, naloxone, and several lipids and lipid mixtures was studied using m... more between an opioid antagonist, naloxone, and several lipids and lipid mixtures was studied using monomolecular layers as a membrane model. No specific interactions could be detected. The main factor affecting the penetration of naloxone in monolayers seems to be the initial ordered state of the molecules instead of the chemical composition of the monolayer. A new membrane model was assayed based on the retention times in high-pressure liquid chromatography working with hydrophobically modified columns.
An improved procedure to quantify gentamicin in biological fluids is presented. The antibiotic is... more 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
ABSTRACT The interaction of gentamicin (G), kanamycin (K), spectinomycin (Sp), streptomycin (St) ... more 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 (PIP2), 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.
Previous work from our group showed that the entrapment of HAV-related synthetic peptides into mu... more 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 dierent strategies of association between the synthetic peptide and phospholipid vesicles and to demonstrate that no signi®cant 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. #
The physicochemical characterization of the peptide sequences E2 (39 Á/53) and E2 (32 Á/59) corre... more The physicochemical characterization of the peptide sequences E2 (39 Á/53) and E2 (32 Á/59) corresponding to the structural protein E2 of the GB virus C was done by studying their interaction with model membranes. The peptides showed surface activity concentration dependent when injected beneath a buffered solution. This tendency to accumulate into the air/water interface suggested a potential ability of these peptides to interact with bilayers. For that reason, Small Unilamellar Liposomes (SUVs) of 1,2-dimyiristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2dimyiristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG) were chosen as a mimetic membranes. A series of fluorescence experiments based on tryptophan peptide fluorescence or with fluorescence labeled SUVs, were done to cover different aspects of peptide interaction with bilayers. Steady state fluorescence anisotropy studies with N -(7-nitro-2-1,3-benzoxadiazol-4-yl) dioleoylphosphatidylethanolamine (NBD-PE) or 1-[4-(trimethylammonium) phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) labeled SUVs indicated that only the long peptide was able to change the lipid microenvironment of DMPG vesicles by slightly increasing the rigidity of the bilayer both above and under the lipid main transition temperature. These results were concordant with the slight blue shift of the maximum tryptophan wavelength emission after E2 (32 Á/53) peptide incubation with DMPG vesicles. Our data provide useful information for the design of synthetic immunopeptides that can be incorporated into a liposomal system with a potential to promote a direct delivery of the membrane-incorporated immunogen to the immunocompetent cells, thus increasing the immuno response from the host. #
Three synthetic peptide sequences of 18 amino acid each, corresponding to different fragments of ... more Three synthetic peptide sequences of 18 amino acid each, corresponding to different fragments of the E2 capsid protein of GB virus C (GBV-C): SDRDTVVELSEWGVPCAT (P45), GSVRFPFHRCGAGPKLTK (P58) and RFPFHRCGAGPKLTKDLE (P59) have been characterized in order to find a relationship between their physicochemical properties and the results obtained in cellular models. Experiments were performed in presence and absence of the HIV fusion peptide (FP-HIV) due to the evidences that GBV-C inhibits AIDS progression. P45 peptide showed lower surface activity and less extent of penetration into 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) (3:2, mol/mol) lipid monolayers than P58 and P59. However, P45 peptide presented higher capacity to inhibit FP-HIV induced cell-cell fusion than the other two sequences. These results were supported by fluorescence anisotropy measurements which indicated that P45 had a significant effect on the inhibition of FP-HIV perturbation of liposomes of the same lipid composition. Finally, atomic force microscopy (AFM) studies have evidenced the modification of the changes induced by the FP-HIV in the morphology of lipid bilayers when P45 was present in the medium.
Merocyanine 540 (MC540) has been used as external probe to determine the interaction of the pepti... more Merocyanine 540 (MC540) has been used as external probe to determine the interaction of the peptide sequence 125 Á/ 139 corresponding to the E2 protein of Hepatitis G virus, with lipid bilayers. The probe was incorporated into large unilamellar vesicles (LUVs) or small unilamellar vesicles (SUVs) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). When incorporated into bilayers, MC540 shows two absorption maxima corresponding to the monomer (570 nm) and dimer (530 nm) form of the probe. Changes in the probe microenvironment are reflected by a modification in the position and/or intensity of these maxima. Addition of increasing amounts of peptide resulted in a slight decrease of the ratio A570/A530 thus indicating a change in MC540 partition into the membrane, going from a hydrophobic to a more hydrophilic environment. This effect was concomitant with an increase in dimer formation as stated from the values of the apparent dimerization constant (K app ) obtained. Fluorescence spectra as well as steady state anisotropy measurements were in agreement with the above results indicating that the peptide was able to relocate the probe and displacing MC540 from its initial location into the bilayer. Results with SUVs or LUVs were similar for what curvature does not seem to play any role on peptide activity. These results reflect the ability of peptide to interact with biomimetic membranes in the lipid head group region. #
European journal of medicinal chemistry, Jan 30, 2014
The development of peptide fusion inhibitors based on short synthetic peptides represents a promi... more The development of peptide fusion inhibitors based on short synthetic peptides represents a promising option in the fight against HIV-1 infection, especially in individuals infected with multiresistant HIV-1 strains. GBV-C has the beneficial effect of retarding the progression of AIDS in people who are co-infected with both the GBV-C and HIV viruses. In previous works, the E1(22-39) GBV-C sequence (E1P8lin) was found to be capable of inhibiting the interaction of HIV-1 FP with bilayers and its cyclic analogue (E1P8cyc) showed a higher anti-HIV-1 activity. In the present work, in an attempt to gain a better understanding of the interaction of E1P8 peptides with HIV-1 FP, we analyzed direct interactions between peptides at the molecular level. Our results support that E1P8cyc might be more potent at blocking HIV-1 entry than E1P8lin as a consequence of the structure induced in the complex formed with HIV-1 FP, which is able to modify the conformation adopted by this functional domain ...
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Papers by Isabel Haro