Biomedical subjects

J M Irache

Publications and source records attributed to J M Irache.

At least 19 recordsLinked to original sources

PubMed2007

Gamma interferon loaded onto albumin nanoparticles: in vitro and in vivo activities against Brucella abortus.

The aim of this study was to evaluate the activity of gamma interferon (IFN-gamma) when it was either adsorbed onto or loaded into albumin nanoparticles. Brucella abortus-infected macrophages and infected BALB/c mice were selected as the models for testing of the therapeutic potentials of these cytokine delivery systems, in view of the well-established role of IFN-gamma-activated macrophages for the control of Brucella sp. infections. Whereas the encapsulation of IFN-gamma inside the matrix of nanoparticles completely abrogated its activity, adsorbed IFN-gamma increased by 0.75 log unit the bactericidal effect induced by RAW macrophages activated with free IFN-gamma, along with a higher level of production of nitric oxide. In infected BALB/c-mice, IFN-gamma adsorbed onto nanoparticles was also more active than free cytokine in reducing the number of bacteria in the spleens, and the effect was mediated by an increased ratio of IFN-gamma-secreting (Th1) to interleukin-4-secreting (Th2) cells. Overall, albumin nanoparticles would be suitable as carriers that target IFN-gamma to macrophages and, thus, potentiate their therapeutic activity.

Albumins↗

PubMed2006

Evaluation of bioadhesive potential and intestinal transport of pegylated poly(anhydride) nanoparticles.

Nanoparticles based on the poly(methyl vinyl ether-co-maleic anhydride) were pegylated with different types of PEGs, namely, two hydroxyl-functionalized PEGs (PEG and mPEG) and two amino-PEGs (DAE-PEG and DAP-PEG). The resulted nanoparticles demonstrated reduction of the negative surface charge compared to the non-modified particles. Further, in vivo experiments showed that all types of pegylated particles possessed higher affinity to adhere to intestinal rather than to the stomach mucosa. Higher bioadhesive potential was observed in the case of PEG-NP and DAE-PEG-NP which was attributed to the flexibility and specific properties of the surface "brush" layer of these particles. The lower bioadhesive potential of mPEG-NP was due to the low presence of coating "brush" layer, whereas for DAP-PEG-NP to the fact that the double end coupled chains ("loop"-conformation) were not available for intensive interactions with the mucosa. The observations made by optic microscopy illustrated an intracellular transport of PEG-NP in vivo with preferable location in the apical area of enterocytes.

Adhesiveness↗

PubMed2005

Albumin nanoparticles for the intravitreal delivery of anticytomegaloviral drugs.

Albumin nanoparticles (NP) were proved to be effective and safe carriers for delivering anticytomegaloviral compounds in the vitreous. NP improved the antiviral activity of both ganciclovir and the phosphodiester oligonucleotide analog to formivirsen. NP appeared to be fusogenic carriers able to target the nucleus of cells. In addition, these drug carriers were well tolerated when administered by the intravitreal route and did not induce autoimmune reactions.

Albumins↗

PubMed2004

Influence of the co-encapsulation of different excipients on the properties of polyester microparticle-based vaccine against brucellosis.

This work evaluates the influence of different pharmaceutical auxiliaries (Pluronic F68, polyvinylpyrrolidone [PVP] or Tween 20), when mixed with an antigenic extract from Brucella ovis (hot saline; HS), on the characteristics of the resulting poly(epsilon-caprolactone) (PEC) and poly(lactide-co-glycolide) (PLGA) microparticles. In all cases, PEC microparticles were smaller than PLGA ones. Concerning the HS loading, PLGA microparticles were highly dependent on the type of the excipient used, whereas all the PEC formulations displayed similar encapsulation efficiencies. For both types of microparticles, the presence of PVP induced a burst release effect. On the contrary, the use of Tween 20 or Pluronic F68 dramatically modified this profile. For PLGA-Tween 20 and PEC-Pluronic F68 microparticles, the HS was released in a pulsatil way during the first 7 days followed by a continuous release for at least 3 weeks. The antigenicity of the HS components was kept in all cases. Phagocytosis by murine monocytes showed a clear difference based just on the hydrophobicity of the polymer, being PEC microparticles better engulfed. Cell activation quantified by the release of H2O2 did not showed major differences between batches, however, microparticles of PEC and Pluronic F68 induced the highest nitric oxide production. Together, these results confirm the advantageous qualities of the "HS-PEC-Pluronic F68 microparticles" as favorable candidate for vaccine purposes against brucellosis.

Animals↗

PubMed2004

Albumin nanoparticles improved the stability, nuclear accumulation and anticytomegaloviral activity of a phosphodiester oligonucleotide.

The goal of this study was to evaluate the potential of albumin nanoparticles as a delivery system for antisense oligonucleotides. Nanoparticles were prepared by a coacervation process and cross-linkage with glutaraldehyde. Phosphodiester (PO) and phosphorotioate (PS) oligonucleotides were either adsorbed on the surface of nanoparticles (PO-NPA and PS-NPA) or incorporated in the nanoparticle matrix (PO-NPB and PS-NPB). When PO-loaded nanoparticles were incubated with phosphodiesterase, only NPB was able to keep the oligonucleotide hybridization capability for at least 60 min. The antiviral activity was evaluated in MRC-5 fibroblasts infected with human cytomegalovirus at a MOI of 0.0035. Both PO nanoparticle formulations significantly increased the antiviral activity of free PO (P<0.001) and NPB showed slightly higher efficacies than NPA (P<0.05). On the other hand, PS exhibited significant higher activity than free PO (P<0.001), however, no significant differences were found between PS-nanoparticle and PO-nanoparticle formulations. These findings were well correlated with the intracellular distribution observed for fluorescent oligonucleotide-loaded albumin nanoparticles. Even these carriers delayed and decreased the uptake of PO by MRC-5 cells, they finally induced a diffused cytoplasmic distribution and major nuclear accumulation. In summary, albumin nanoparticles partially protected a PO against enzymatic degradation and improved their presence in the nucleus and thus, increased its efficiency.

Albumins↗

PubMed2004

Fluconazole encapsulation in PLGA microspheres by spray-drying.

Fluconazole-loaded PLGA microspheres were prepared by the spray-drying process. The influence of some process parameters on the physical characteristics of the microspheres was evaluated. Neither type nor polymer concentration influenced significantly the mean diameter of the microspheres, their size distribution and encapsulation efficiency of the drug. However, the drug loading greatly affected their size and the physical state in which fluconazole can exist in the matrix of the carriers, and, thus, affected the release rate of the drug. Results obtained by differential thermal analysis and X-ray powder diffraction revealed that at low nominal drug loading, fluconazole was incorporated in an amorphous state or in a molecular dispersion in the matrix of the microspheres and at high nominal drug loading part of the drug was in a crystalline form. Release profiles of fluconazole from the microspheres displayed a biphasic shape. The duration and extent of each phase were affected mainly by polymer nature, drug loading and physical state in which fluconazole existed in the polymeric matrix.

Antifungal Agents↗

PubMed2003

Polymeric carriers for amphotericin B: in vitro activity, toxicity and therapeutic efficacy against systemic candidiasis in neutropenic mice.

OBJECTIVE: To study the toxicity and activity of two new amphotericin B formulations: poly(epsilon-caprolactone) nanospheres coated with poloxamer 188 (AmB-NP) and mixed micelles with the same surfactant (AmB-MM). MATERIALS AND METHODS: The toxicity of these formulations was evaluated in erythrocytes, J774.2 macrophages and LLCPK1 renal cells, as well as in mice. Activity was determined in clinical isolates and in neutropenic mice. Mice were made neutropenic with 5-fluorouracil, infected with Candida albicans and treated with the antifungal formulations for three consecutive days. AmB association in cells and accumulation in kidneys and liver of animals was quantified by HPLC. RESULTS: Both formulations decreased between 8- and 10-fold the MIC of the polyene against clinical isolates of C. albicans. However, their activity was lower than or equal to that of AmB-deoxycholate when it was assessed against C. albicans-infected macrophages. When given as a single intravenous dose in mice, AmB-MM and AmB-NP had an LD50 of 9.8 and 18.6 mg/kg, respectively, compared with 4 mg/kg for AmB-deoxycholate. Comparison of residual infection burdens in the liver and kidneys showed that AmB-deoxycholate (0.5 mg/kg) was more effective and faster in eradicating yeast cells than polymeric formulations. This fact can be related to a lower AmB accumulation inside macrophages and in liver and kidneys (about 1.5 mg drug/g tissue) of mice, compared with those detected for AmB-deoxycholate (4 mg drug/g). Overall, the efficacy of these formulations at 2 mg/kg was equal to that of AmB-deoxycholate at 0.5 mg/kg. CONCLUSIONS: AmB-MM and AmB-NP decreased the in vivo antifungal activity of AmB, and higher concentrations were therefore necessary to obtain a similar therapeutic effect. However, these higher concentrations were achievable owing to the reduced toxicity of these formulations.

Amphotericin B↗

PubMed2003

Influence of the surface characteristics of PVM/MA nanoparticles on their bioadhesive properties.

The aim of this work was to investigate the influence of the cross-linkage of poly(methylvinylether-co-maleic anhydride) (PVM/MA) nanoparticles with increasing amounts of 1,3-diaminopropane (DP) and, eventually, bovine serum albumin (BSA) on their gastrointestinal transit and bioadhesive properties. The fluorescently-labelled formulations were orally administered to rats and, at different times, the amount of nanoparticles in both the lumen content and adhered to the gut mucosa were quantified. The gut transit was evaluated by calculating the gastric (k(ge)) and intestinal (k(ie)) emptying rates. The adhered fraction of nanoparticles in the whole gut was plotted versus time and, from these curves, the intensity, capacity and extent of the adhesive interactions were estimated. The bioadhesive potential of PVM/MA was much higher when formulated as nanoparticles (NP) than in the solubilised form in water. However, k(ge) and k(ie) increased by increasing the extent of cross-linkage of nanoparticles with DP, while the capacity to develop adhesive interactions and the intensity of the adhesive phenomenon were significantly higher for non-hardened than for DP-cross-linked carriers. In contrast, the BSA-coating of cross-linked nanoparticles significantly decreased k(ge) and k(gi), whereas the intensity of the bioadhesive phenomenon was significantly higher than for NP. In summary, the adhesivity of the nanoparticles appears to modulate their gastrointestinal transit profile.

Adhesiveness↗

PubMed2003

Loading of plasmid DNA into PLGA microparticles using TROMS (Total Recirculation One-Machine System): evaluation of its integrity and controlled release properties.

Loading plasmid DNA into poly(ester) microparticles usually involves the formation of a multiple emulsion, using homogenisation techniques such as sonication or Ultra-Turrax. These procedures may negatively affect the integrity of the macromolecule and consequently its activity. The aim of this study was to prepare and evaluate DNA-loaded microparticles by TROMS (Total Recirculation One-Machine System), a new procedure that is based on the formation of a multiple emulsion by the injection of the phases under a turbulent regime. Microparticles were prepared with either Resomer) RG 502 (MP 502) or RG 756 (MP 756) and DNA loading was quantified fluorimetrically. DNA loading in MP 756 was almost twice as high as in MP 502 (510 vs. 285 ng/mg, respectively). Under both formulations, the loaded plasmid was released while maintaining its integrity for at least 24 days (MP 502) and 40 days (MP 756). Finally, the transfection efficiency was studied after injection of the microparticles (MP 502) into rat skeletal muscle and compared with naked DNA injection. Injection of naked DNA (150 microg DNA per muscle) achieved higher but variable expression levels that decreased after 3 weeks. In contrast, the muscles injected with microparticles (6.8 microg DNA per muscle) showed lower but homogeneous expression values, which were maintained for at least 3 weeks.

Animals↗

PubMed2003

Bovine serum albumin modified the intracellular distribution and improved the antiviral activity of an oligonucleotide.

In the present work, we describe the ability of bovine serum albumin (BSA) to improve the cytoplasmatic delivery of a phosphodiester oligonucleotide (PO), whose phosphorotioate analogue is ISIS 2922 (Vitravene). The changes in intensity and lambda(max) in fluorescence spectroscopy and the increase in fluorescence anisotropy upon complex formation between the oligonucleotide and the protein (PO-BSA) were used to determine the dissociation constant, Km=6.3 x 10(-8) M. The stoichiometry of the complex is mainly 1:1, although 2 PO per BSA have been detected at high PO/BSA ratios. The complexation did not protect PO against enzymatic degradation by snake venom phosphodiesterase (0.1 mg/mL). Fluorescence microscopy revealed that PO-BSA complexes showed a decreased uptake and modified pattern of intracellular distribution with a rapid nuclear accumulation (rather than vesicular localization in the cytoplasm observed for free oligonucleotide). The effect was only observed over a certain threshold of BSA concentration (higher than found in media supplemented with 10% serum). By this way, the interaction with albumin increased the antiviral activity of the oligonucleotide, tested by a plaque reduction assay in MRC-5 fibroblasts infected with human cytomegalovirus (strain RC 256) at a MOI of 0.0035. At 10 microM PO concentration, free PO decreased virus plaques to 85% of the control, while PO-BSA complexes reduced to 60%, in the same order than ISIS 2922. The phosphorotioate analogue complexed by BSA (ISIS 2922-BSA) showed the highest activity with 45% of the control plaques.

Animals↗

PubMed2002

Modulation of the cellular immune response after oral or subcutaneous immunization with microparticles containing Brucella ovis antigens.

An antigenic extract (HS) from Brucella ovis was encapsulated in either poly-epsilon-caprolactone (PEC) or poly-lactic-co-glycolic acid 75:25 (PLGA) microparticles containing beta-cyclodextrin and Pluronic F-68 as stabilising agents. The resulting microparticles displayed sub-5 microm sizes. Antigen loading was 5.2 and 3.8 microg/mg for HS-PEC and HS-PLGA microparticles, respectively. Specific HS cytokine profiles were determined after subcutaneous and oral immunisation of BALB/c mice. Gut distribution studies of the formulations after oral administration showed that HS-PEC microparticles interacted more strongly with mucosa and Peyer's patches than HS-PLGA. Accordingly, oral immunisation with HS-PLGA induced a negligible immune response, whereas HS-PEC elicited a Th1 response although of low intensity. Subcutaneous immunisation with HS-PEC induced high IFN-gamma and IL-2 release; in contrast, HS-PLGA particles induced a Th2 profile characterized by significant levels of IL-4. Splenic cells from free-HS immunised mice released IFN-gamma and IL-2 but not IL-4. A less intense Th1 pattern was also found from HS stimulated nai;ve splenic cells. These results suggest that the HS itself possesses Th1 immunopotentiating properties, required to control brucellosis, that can be specifically increased by encapsulation in PEC microparticles. In contrast, PLGA microparticles modulate the response toward a Th2 pathway.

Administration, Oral↗

PubMed2002

Gantrez AN as a new polymer for the preparation of ligand-nanoparticle conjugates.

The aim of this study was to evaluate the feasibility and in vitro activity of ligand-conjugates based on the use of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA or Gantrez AN). Fluorescently labelled PVM/MA nanoparticles were prepared by desolvation and cross-linkage with 1,3-diaminopropane (DP). Conjugates were obtained by incubation between the carriers and Sambucus nigra agglutinin (SNA) for 1 h in an aqueous medium. The lectin binding to the surface of nanoparticles was increased by both increasing the bulk ligand concentration and decreasing the amount of cross-linker. However, a concentration of about 0.3-0.4 mg DP per mg polymer was necessary to obtain maximum agglutination activity. Under optimal conditions, the amount of fixed ligand was 46 microg/mg nanoparticle (binding efficiency of 86%); although the activity of SNA conjugates was 13.3 microg/mg particle. The activity of nanoparticles, measured by the association to Caco-2 monolayers, was higher when SNA was covalently bound onto their surface. The lectin-conjugate interaction was 6-fold higher than conventional nanoparticles. Moreover, energy-dependent mechanisms were only observed in SNA-PVM/MA particles. Finally, the decrease in association in the presence of lactose demonstrates that both SNA- and SNA-conjugate-binding was due to a true lectin-sugar interaction.

Animals↗

PubMed2002

Albumin nanoparticles as carriers for a phosphodiester oligonucleotide.

The goal of this study was the evaluation of albumin nanoparticles as drug delivery systems for antisense oligonucleotides. Bovine serum albumin (BSA) nanoparticles were prepared by a coacervation process. A phosphodiester oligonucleotide was either incorporated into the matrix of the particles by incubation with the albumin prior the coacervation process or adsorbed onto the pre-formed nanoparticles. Incorporated and/or adsorbed oligonucleotide was estimated by capillary electrophoresis and fluorescence spectroscopy. The adsorbed amount of oligonucleotide was dramatically dependent on the pH of the medium. Desorption of the oligonucleotide was also affected by the pH and ionic strength of the medium. This indicated that electrostatic forces play a major role in the interaction between the oligonucleotide and the nanoparticles. When the oligonucleotide was incubated with the albumin prior to nanoparticle formation, the profile of release confirmed that a fraction was incorporated into the matrix and its release was controlled by the albumin degradation. The hybridisation capability of the oligonucleotide in both nanoparticle formulations was retained. However, only the oligonucleotide incorporated into the nanoparticle matrix was protected against enzymatic degradation.

Albumins↗

PubMed2002

Quantification of the bioadhesive properties of protein-coated PVM/MA nanoparticles.

This work describes the bioadhesive properties of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) nanoparticles fluorescently-labelled with rhodamine B isothiocyanate, and coated with either Sambucus nigra lectin (SNA-NP) or bovine serum albumin (BSA-NP). The different formulations (10 mg) were administered to animals by the oral route and the fraction of adhered particles to the mucosa was estimated by measuring the fluorescent marker after the digestion of the tissue. Plotting the amount of adhered particles in the whole gut versus time enabled us to determine the affinity of the formulation for the biological support (expressed as Q(max)), the intensity and relative duration of the bioadhesive phenomenon (AUC(adh) and MRT(adh), respectively), and the elimination rate of the adhered particles (k(adh)). SNA-NP displayed a similar adhesive affinity and adhesive intensity for the gut mucosa than the control particles; although, its maximum of interaction with the mucosa was observed 1 h post-administration, whereas control and BSA-NP took place only 30 min post-administration. On the other hand, the coating of nanoparticles with SNA significantly reduced the k(adh) (P<0.01) and, thus, MRT(adh) was 35 min longer for the lectin-conjugate than for the control. BSA-NP displayed a highest initial affinity for the gut mucosa and AUC(adh) was calculated to be 1.5 fold higher than for the control or SNA-NP. However, BSA-NP were eliminated more rapidly from the mucosa than SNA-NP and, thus, the MRT(adh) was only 27 min longer than control. In summary, the parameters describing the bioadhesive profile of a given formulation may be useful to quantify the potential of colloidal particulates to interact with a mucosa and to evaluate the influence of different ligands on the bioadhesive properties of the resulting drug carriers.

Adhesives↗

PubMed2002

Development of microparticles prepared by spray-drying as a vaccine delivery system against brucellosis.

The antigenic extract Hot Saline from Brucella ovis was microencapsulated by the spray-drying technique with different polyesters (poly-lactide-co-glycolide RG502H [PLGA], and blends with poly- epsilon -caprolactone [PEC]) in order to obtain microparticles smaller than 5 microm. Microparticles were tested for encapsulation efficiency, release studies, acidification of the in vitro release medium, and in vitro J744-macrophage experiments (phagocytosis and toxicity of the preparations) to determine the optimal formulation for vaccination purposes. Formulation containing no PCL showed the highest encapsulation efficiency, although the differences were not significant. The in vitro release kinetics were characterized by a high burst effect after 1 h of incubation, followed by a slow and continuous release. For the formulation based on PLGA, the pH of the medium during release dropped from 7.4 to 3.5 while the presence of PEC attenuated the pH drop. All formulations showed light toxicity by the MTT assay, but differences were observed in terms of phagocytosis, as particles prepared with PEC showed the higher uptake by J744-macrophages and cell respiratory burst, determined by oxygen peroxide release. All these characteristics suggest that the microparticulated antigenic formulation containing the higher ratio of PEC is susceptible to be used in animal vaccination studies.

Bacterial Vaccines↗

PubMed2002

In vitro antileishmanial activity of amphotericin B loaded in poly(epsilon-caprolactone) nanospheres.

The activity of formulations for amphotericin B (AmB) associated with poly(epsilon-caprolactone) nanospheres and coated with variable amounts of a non ionic surfactant poloxamer 188, was evaluated against AmB-susceptible (WT) and AmB-resistant (AmB(r)) strains of Leishmania donovani amastigotes in thioglycolate-elicited peritoneal macrophages. AmB-nanospheres were more actives than free AmB only against amastigotes of wild strain. The activity was not influenced by the concentration of poloxamer 188 used to stabilize the nanospheres in spite of this surfactant was previously reported to synergy with AmB on the membrane of the resistant parasite. Similarly, this improvement was not mediated through macrophage activation. In fact, these nanoparticle formulations appeared to inhibit both NO and TNF-alpha production induced by the free drug. Therefore, we suggest that the association of AmB with nanospheres may improve the capability of the drug to interact with ergosterol. This hypothesis appears to be supported by the fact that nanospheres did not show any improvement of the AmB activity against the resistant strain (characterized by the absence of ergosterol).

Amphotericin B↗

PubMed2002

Adaptive immune responses of legumin nanoparticles.

Legumin is one of the main storage proteins in the pea seeds (Pisum sativum L.) and the molecules of this protein have the capacity of binding together to form nanoparticles after aggregation and chemical cross-linkage with glutaraldehyde. The aim of this work was to study the adaptive immune response of legumin nanoparticles in rats. Following intradermal immunisation with the native protein legumin and legumin nanoparticles of about 250 nm, the humoral and cell-mediated immune responses were analysed in rats. The humoral responses against legumin and legumin nanoparticles were examined by western blot and ELISA analysis. Both techniques clearly showed that sera from rats immunised with legumin strongly expressed antibodies against this protein. On the contrary, serum samples from rats inoculated with legumin nanoparticles did not contain detectable amounts of antibodies. These results may be explained by a reduction on the antigenic epitopes of the protein induced by the glutaraldehyde used during the cross-linking step. Concerning the cell-mediated response, neither legumin nor legumin nanoparticles stimulated an immunogenic response. This absence of response of spleen lymphocytes for legumin and legumin nanoparticles may be explained by a cytostatic effect of legumin which was corroborated by the evaluation of the middle phase of cell apoptose. In fact, both legumin and legumin nanoparticles are potent inductors of a cytostatic phenomenon and showed a significant increase of the chromatin condensation (p < 0.05) as compared with control.

Animals↗

PubMed2002

Polyester microparticles as a vaccine delivery system for brucellosis: influence of the polymer on release, phagocytosis and toxicity.

Microparticles, containing an antigenic complex from Brucella ovis (HS), were evaluated for vaccine purposes against brucellosis. They were prepared by the double emulsion solvent evaporation method using two different polyesters, poly-lactide-co-glycolide acid (75:25; RG 756) and poly-epsilon-caprolactone. The encapsulation efficiency and release of HS from the microparticles, their capacity to be phagocytosed and also their toxicity on murine monocytes J774.2 were investigated. Both polymers lead to smooth and spherical sub-5 microm particles, with approximately 30% of the antigen initial dose encapsulated. SDS-PAGE and immunoblot of extracted antigens confirmed that the apparent molecular weight and antigenicity remained unaltered after the encapsulation procedure. However, the in vitro release of the antigens differed among them. The release profile for PLGA microparticles was continuous, whereas PEC ones released the antigens in a triphasic release pattern. Phagocytosis was clearly influenced by the hydrophobicity of the polymer, increasing in the case of PEC microparticles. Toxicity assay showed that both types of microparticles induced similar levels of mitochondrial damage. In conclusion, HS-PEC microparticles could be used as an effective vaccine against brucellosis, as the antigen is released in boosters and they are greatly phagocytosed by macrophages.

Animals↗