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.

Increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes.

A novel lipidic vector composed of DOTAP/Chol liposomes, asialofetuin (AF), protamine sulfate and DNA has been developed. The resulting protamine-AF-lipoplexes improved significantly the levels of gene expression in cultured cells and in the liver upon i.v. administration. Lipoplexes containing the optimal amount of AF (1 microg/microg DNA) showed a 16-fold higher transfection activity in HepG2 cells than non-targeted (plain) complexes. The uptake by cells having asialoglycoprotein receptors (ASGPr) on their plasma membrane was decreased by the addition of free AF, indicating that AF-lipoplexes were taken up specifically by cells via ASGPr-mediated endocytosis. Results from transfections performed in cells defective in ASGPr, ie HeLa cells, confirmed this mechanism. By addition of the condensing peptide, protamine sulfate, smaller complexes were obtained, which enhanced even more the uptake of AF-complexes in HepG2 cells and in the liver. The optimal amount of protamine was 0.4 microg/mcirog DNA, and gene expression was about 5-fold over that obtained with AF-lipoplexes in the absence of the peptide, and 75-fold higher than that with plain conventional lipoplexes. Protamine-AF-lipoplexes increased by a factor of 12 luciferase gene expression in the liver of mice administered systemically via the tail vein, compared to plain complexes. In summary, our findings extend the scope of previous studies where AF-lipoplexes were used to introduce DNA into hepatocytes. The combination of targeting and protamine condensation obviated the need for partial hepatectomy, commonly required to obtain efficient gene delivery in this organ. Since protamine sulfate has been proven to be non-toxic in humans, the novel liver-specific vector described here may be useful for the delivery of clinically important genes to this organ.

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.

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.

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.

Enhanced gene delivery in vitro and in vivo by improved transferrin-lipoplexes.

Cationic liposomes and the complexes they form with DNA (lipoplexes) constitute the most promising alternative to the use of viral vectors for gene therapy. One of the limitations to their application in vivo, however, is the inhibition of gene delivery by serum. In a previous study, we demonstrated that transferrin (Tf)-lipoplexes were superior to plain lipoplexes in transfecting HeLa cells in the presence of high concentrations of serum. With the goal of obtaining efficient gene expression in vivo, we evaluated the efficacy of Tf-lipoplexes (containing DOTAP and cholesterol) in transfecting primary hepatocytes and adipocytes in the presence of high serum concentrations. The association of transferrin with cationic liposomes increased luciferase expression compared to plain lipoplexes in primary cells as well as in HepG2 and 3T3-L1 differentiated adipocytes. The complexes were not cytotoxic and were highly effective in protecting DNA from attack by DNase I. An efficient and reliable method was developed to prepare lipoplexes containing both Tf and protamine sulfate, where the latter was mixed with transferrin, followed by the addition of cationic liposomes and DNA. The resulting protamine-Tf-lipoplexes increased significantly the levels of gene expression in cultured cells and in various tissues in mice following i.v. administration.

Development of a chromatographic method for the determination of saquinavir in plasma samples of HIV patients.

A simple, sensitive and reproducible high-performance liquid chromatographic method for detecting and quantifying saquinavir in human plasma is described. Verapamil was used as internal standard. The method employes a single liquid-liquid extraction step with tert-butil methyl ether followed by chromatography on a Lichrospher 60 Select B C8 reversed-phase column. Ultraviolet detection was used to identify the compounds of interest. The quantitation limit of saquinavir was 1 ng/mL and only 0.5 mL of plasma sample was required for the determination. The average saquinavir recoveries over a concentration range of 2.5-500 ng/mL ranged from 86 to 95%. Precision and accuracy did not exceed 5%.

Improved selectivity in detection of polar basic drugs by liquid chromatography-electrospray ionization mass spectrometry. Illustration using an assay method for the determination of famotidine in human plasma.

It is well to assume that bioanalytical chromatographic methods for the determination of polar basic drugs are developed and optimised according to a standardised procedure which involves two alternatives: (a) modifications in the sample preparation procedures, and (b) changes in the stationary phase of the chromatographic system. In this paper, a simple and rapid chromatographic procedure using a specific analytical detection method (ESI tandem mass spectrophotometric detection) in combination with a fast and efficient sample work-up procedure, protein precipitation, is presented. A demonstration of the entire chromatographic procedure is given for an HPLC method for the determination of famotidine in human plasma, a basic polar drug with poor solubility in organic solvents. In order to optimize the mass detection of famotidine, several parameters such as ionization mode, fragmentor voltage, m/z ratios of ions monitored, type of organic modifier and eluent additive, were investigated. Each analysis required 5 min. The calibration curve of famotidine in the range 1-200 ng/ml was linear with a correlation coefficient of 0.9992 (n = 6), and a detection limit a signal-to-noise ratio of 3 was approximately 0.2 ng/ml. The within- and between-day variations in the famotidine analysis were 5.2 (n = 6) and 6.7% (n = 18), respectively. The applicability of this method was also demonstrated for the analysis of plasma samples in a Phase-I human pharmacokinetic study.

Gliadin nanoparticles as carriers for the oral administration of lipophilic drugs. Relationships between bioadhesion and pharmacokinetics.

PURPOSE: 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.

Bioadhesive potential of gliadin nanoparticulate systems.

The objective of this work was to prepare, characterise and evaluate the adhesive potential of gliadin nanoparticulate carriers. Firstly, lectin-nanoparticle conjugates were obtained by the carbodiimide (CDI) covalent binding of Dolichos biflorus lectin (DBA) to the surface of gliadin nanoparticles (NP) containing carbazole (as a model lipophilic drug). The DBA binding efficiency was favoured in mild acidic conditions. Similarly, a CDI concentration of about 0.63 mg/mg nanoparticles, acting during at least 1 h, provided binding efficiencies of about 50% bulk lectin. Under optimised experimental conditions, the DBA conjugates showed a size of around 500 nm and the amount of loaded carbazole and the DBA content were calculated to be around 15 and 23.5 microg/mg, respectively. The bioadhesive activity of NP and DBA conjugates was determined in samples of small and large rat intestinal mucosa. The amount of adsorbed NP was calculated to be around 8 and 4 g/m(2) in the small and large intestine, respectively. This high capacity to interact with the mucosa may be explained by gliadin composition. In fact, gliadin is rich in neutral and lipophilic residues. Neutral amino acids can promote hydrogen bonding interactions with the mucosa, while the lipophilic components can interact with the biological tissue by hydrophobic interactions. The bioadhesive activity of DBA conjugates was calculated to be about 2 g/m(2) in the small intestine and greater than 4 g/m(2) in the caecum and distal colon. These degrees of interaction were always significantly higher than those obtained with controls. Finally, DBA did not provide the specificity for interaction with Peyer's patches. In summary, gliadin nanoparticles show a high capacity of non-specific interaction with the intestine, whereas DBA binding to the surface of these carriers provided a greater specificity for colonic mucosa.

Preparation of Ulex europaeus lectin-gliadin nanoparticle conjugates and their interaction with gastrointestinal mucus.

One approach to improve the bioavailability and efficiency of drugs consists of the association of a ligand (i.e. lectins), showing affinity for biological structures located on the mucosa surfaces, to nanoparticulate drug delivery systems. In this context, Ulex europaeus lectin-gliadin nanoparticle conjugates (UE-GNP) were prepared with the aim of evaluating their in vitro bioadhesive properties. The lectin was fixed by a covalent procedure to gliadin nanoparticles by a two-stage carbodiimide method. Typically, the amount of bound lectin was calculated to be approximately 15 microg lectin/mg nanoparticle, which represented a coupling efficiency of approximately 16% of the initial lectin concentration. In addition, the activity of these conjugates was tested with bovine submaxillary gland mucin (BSM) and the level of binding to this mucin was always much greater with UE-GNP than with controls (gliadin nanoparticles). However, the presence of 50 micromol fucose, which is the reported specific sugar for U. europaeus lectin, specifically inhibited the activity of these conjugates and, therefore, the UE-GNP binding to BSM was attenuated by 70%. These results clearly showed that the activity and specificity of U. europaeus lectin was preserved after covalent coupling to these biodegradable carriers.