Juan M. Irache

Mannose-targeted systems for the delivery of therapeutics

Background: The specific targeting of nanomedicines to mannose receptors, highly expressed in cells of the immune system, performs a useful strategy for improving the efficacy of vaccines and chemotherapy. Objective: This review discusses the potential of mannose-targeted drug/antigen delivery systems for vaccination and treatment of diseases localized in macrophages and other antigen-presenting cells. Methods: The first part of the review describes the characteristics, localization and functions of mannose receptors. The following sections are devoted to the description of different methods used to deliver therapeutic agents, including mannose conjugates and mannosylated carriers or particulates (i.e., liposomes, nanoparticles and niosomes). Results/conclusions: A general overview of published reports confirms the effectiveness of mannosylation strategies, although the optimization and full exploitation of mannose-targeted drug delivery systems would require a deeper understanding of the structure–activity relationship. In the near future, these nanomedicines have the potential to treat a number of diseases (including cancer) and improve the quality of life of patients.

Ganciclovir-loaded albumin nanoparticles: characterization and in vitro release properties

Ganciclovir is one of the most widely used antiviral drug for the treatment of cytomegalovirus retinitis. Due to its short half-life in the vitreous, frequent administrations are necessary to maintain the therapeutic levels. In this context, the aim of this study was to characterise and in vitro evaluate the drug release properties of three different formulations of ganciclovir-loaded albumin nanoparticles. These carriers were prepared by a coacervation method and chemical cross-linking with glutaraldehyde. Depending on the step where the drug and/or cross-linking agent were added three different formulations were obtained, named models A, B and C. For model A nanoparticles, ganciclovir was incubated with the just-formed albumin nanoparticles. For the other two types of nanoparticulate formulations, the drug was added to a solution of albumin (model B) and glutaraldehyde (model C) prior to the formation of the carriers by coacervation. In all cases, the size of the different nanoparticulate formulations was comprised between 200 and 400 nm and the yield ranged from 50%, in model A, to 65% in model B. Concerning the ganciclovir loading, model B nanoparticles offered the higher capacity to carry this antiviral drug (around 30 microg ganciclovir/mg nanoparticle). On the contrary, the drug loading calculated for model A nanoparticles was only 14.6 microg/mg. The in vitro release profiles of the nanoparticles showed a biphasic pattern, with an initial and rapid release, followed by a slower step for up 5 days. This burst effect was especially relevant in model A (around 60% in 1 h), followed by model B (40%) and less important in model C (20%). The addition of trypsin to the release medium did not have a significant influence on the release characteristics. However, the release of the drug was increased in acidic or basic mediums, due to the disruption of the covalent binding between ganciclovir and the protein matrix via glutaraldehyde. This strong linkage was also confirmed by TLC experiences. In summary, a first step of incubation between the drug and the protein, prior to the preparation of nanoparticles, enabled us to obtain albumin carriers able to release ganciclovir in a sustained way.

In vitro phagocytosis and monocyte-macrophage activation with poly(lactide) and poly(lactide-co-glycolide) microspheres

Treatment of many intracellular infections in the mononuclear phagocytic system (MPS), requires targeting of antibiotics by a drug delivery system. The objective of this study was to examine whether the particular nature of microspheres, made of end-group capped and uncapped poly(lactide) [PLA] and poly(lactide-co-glycolide) [PLGA 50:50 and PLGA 75:25], affect the uptake into and also the activation of monocyte-macrophages. Placebo and gentamicin sulfate containing microspheres were incubated with J774 murine monocyte-macrophages and fresh human blood monocytes. Phagocytosis became more efficient with increasing polymer hydrophobicity, whereas opsonization of the particles in serum exerted inconsistent effects. Monocyte activation was determined by flow cytometry and measured as oxidative burst. The cellular oxidative burst induced by the particles was higher for end-group uncapped polymers. Opsonization increased significantly the oxidative activity of J774 monocytes, but affected inconsistently that of human blood monocytes. The results demonstrate that PLA and PLGA microspheres loaded with gentamicin sulfate were efficiently phagocytosed in vitro. The end-group uncapped polymer-type microspheres promoted significantly cell activation, which may be of importance for drug delivery and targeting to intracellular infections.

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.

Gentamicin encapsulation in PLA:PLGA microspheres in view of treating Brucella infections

In view of treating intracellular Brucella infections, microspheres made of poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) were developed as delivery system for the cationic and highly hydrophilic antibiotic gentamicin sulphate. Drug microencapsulation by spray drying yielded microspheres with regular morphology, an average particle size of approximately 3 micrometer and encapsulation efficiencies of up to 45%. Different copolymers of similar molecular weights gave varying encapsulation efficiencies and particle size distributions. The encapsulation efficiency generally increased with polymer hydrophilicity, except for the hydrophilic copolymer PLGA50:50H carrying carboxylic end groups. Encapsulation also depended on the pH value of the aqueous drug solution to be encapsulated. Moreover, increasing nominal gentamicin sulphate loading yielded lower efficiencies. For comparison, some formulations were also prepared by a (W(1)/O)W(2)-solvent evaporation method, which yielded lower encapsulation efficiencies, in the order of 13%. Finally, drug bioactivity was found to remain intact after microencapsulation, MS storage and MS incubation in aqueous medium. The results suggest that PLA/PLGA microspheres prepared by spray drying may be an appropriate delivery system for gentamicin sulphate to be used in the treatment of intracellular Brucella infections.

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.

Ocular disposition and tolerance of ganciclovir-loaded albumin nanoparticles after intravitreal injection in rats.

Cytomegalovirus (CMV) infection mainly affects endothelial cells of ocular vessels, optic nerve and the retina, resulting in direct or autoimmune damages, uveoretinitis and disturbed vision. The use of colloidal carriers for the intravitreal delivery of ganciclovir may prolong its residence in the eye, minimizing the opacification observed for macroscopic implants. The aim of this work was to evaluate the ocular toxicity induced by the prolonged presence of ganciclovir-loaded bovine serum albumin nanoparticles after their intravitreal injection. The intraocular disposition of these carriers was also studied by immunochemistry. Two weeks post-injection, a significant amount of nanoparticles remained in the vitreous cavity, mainly in a thin layer overlying the retina and in the area close to the blood aqueoUs barrier. Their prolonged residence in the eve seemed to be well tolerated and the histological evaluation of the retina, mainly the photoreceptor layer, and adjacent tissues revealed the absence of inflammatory reactions or alterations in the tissue architecture (i.e. cellular infiltrations or vascular inflammation). In addition, nanoparticles neither alter the expression and distribution of arrestin and rhodopsin autoantigens nor the mineralocorticoid receptor. In summary, the vision was not affected by autoimmune phenomena or alterations in the behavior of ophthalmic cells due to the intravitreal injection of these nanoparticles.

Combined hydroxypropyl-β-cyclodextrin and poly(anhydride) nanoparticles improve the oral permeability of paclitaxel

The aim of this work was to study the effect of the combination between 2-hydroxypropyl-beta-cyclodextrin (HPCD) and bioadhesive nanoparticles on the encapsulation and intestinal permeability of paclitaxel (PTX). In this context, a solid inclusion complex between PTX and HPCD was prepared by an evaporation method. Then, the complex was incorporated in poly(anhydride) nanoparticles by a solvent displacement method. The resulting nanoparticles, PTX-HPCD NP, displayed a size of about 300 nm and a drug loading of about 170 microg/mg (500-fold higher than in the absence of HPCD). The effect of these nanoparticles on the permeability of intestinal epithelium was investigated using the Ussing chamber technique. The apparent permeability (P(app)) of PTX was found to be 12-fold higher when formulated as PTX-HPCD NP than when formulated as Taxol (control). Furthermore, when interaction between nanoparticles and the mucosa was avoided, the permeability of PTX significantly decreased. In summary, the association between PTX-HPCD and poly(anhydride) nanoparticles would induce a positive effect over the intestinal permeability of paclitaxel, being the bioadhesion a mandatory condition in this phenomena.

Gliadin Nanoparticles as Carriers for the Oral Administration of Lipophilic Drugs. Relationships Between Bioadhesion and Pharmacokinetics

AbstractPurpose. The aim of this work was to evaluate the bioadhesive properties of non-hardened gliadin nanoparticles (NPs) and cross-linked gliadin nanoparticles (CL-NP) in the carbazole pharmacokinetic parameters obtained after the oral administration of these carriers. Methods. A deconvolution model was used to estimate the carbazole absorption when loaded in the different gliadin nanoparticles. In addition, the elimination rates of both adhered and non-adhered nanoparticulate fractions within the stomach were estimated. Results. Nanoparticles dramatically increased the carbazole oral bioavailability up to 49% and provided sustained release properties related to a decrease of the carbazole plasma elimination rate. The carbazole release rates from nanoparticles (NP and CL-NP), calculated by deconvolution, were found to be of the same order as the elimination rates of the adhered fractions of nanoparticles in the stomach mucosa. In addition, good correlation was found between the carbazole plasmatic levels, during the period of time in which the absorption process prevails, and the amount of adhered carriers to the stomach mucosa. Conclusion. Gliadin nanoparticles significantly increased the carbazole bioavailability, providing sustained plasma concentrations of this lipophilic molecule. These pharmacokinetic modifications were directly related to the bioadhesive capacity of these carriers with the stomach mucosa.

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.