Polyisobutylcyanoacrylate nanocapsules containing an aqueous core for the delivery of oligonucleotides.

Antisense oligonucleotides (ODNs) with base sequences complementary to a specific RNA can, after binding to intracellular mRNA, selectively modulate the expression of a gene. However, these molecules are poorly stable in biological fluids and are characterized by a low intracellular penetration. In view of using ODNs as active molecules, the development of nanocapsules containing ODNs in their aqueous core was considered. Nanocapsules were prepared by interfacial polymerization of isobutylcyanoacrylate (IBCA) in a W/O emulsion. After ultracentrifugation and re-suspension in water, the nanocapsules displayed a size of 350+/-100 nm. Oligonucleotide loading did not significantly influence the zeta potential, suggesting that they were located within the core of the nanocapsules. Fluorescence quenching assays confirmed this localization. When encapsulated in the nanocapsules and incubated in the presence of serum, the ODNs were efficiently protected from degradation by nucleases, whereas ODNs adsorbed onto nanospheres were less efficiently protected. This paper describes, for the first time, a nanotechnology able to encapsulate ODNs, rather than adsorbing them at the surface of a solid support. Such a formulation has great potential for oligonucleotide delivery.

EWS fli-1 antisense nanocapsules inhibits ewing sarcoma-related tumor in mice.

EWS Fli-1, a fusion gene resulting from a t(11;22) translocation is found in 90% of both Ewing's sarcoma and primitive neuroectodermal tumor (PNET). In the present study, we show that recently developed polyisobutylcyanoacrylate nanocapsules with an aqueous core were able to encapsulate efficiently high amounts of phosphorothioate oligonucleotides (ODN) directed against EWS Fli-1 chimeric RNA. Release of these ODN in serum medium was shown to be biphasic which was explained by the presence of two types of nanocapsules able to release ODN with different kinetics. In addition, nanocapsules were found to provide protection of these oligonucleotides from the degradation in serum. These ODN nanocapsules permitted to obtain inhibition of Ewing sarcoma-related tumor in mice after intratumoral injection of a cumulative dose as low as 14.4 nanomoles. This new type of non viral vector shows great potential for in vivo administration of oligonucleotides.

Polyisobutylcyanoacrylate nanocapsules containing an aqueous core as a novel colloidal carrier for the delivery of oligonucleotides.

PURPOSE: The goal of the present paper was to encapsulate oligonucleotides in a new particulate carrier in order to protect them from enzymatic degradation. METHODS: Nanocapsules with an aqueous core containing oligonucleotides were prepared by interfacial polymerization of isobutylcyanoacrylate in a W/O emulsion. Ultracentrifugation and re-suspension in water yielded a dispersion of these containing an aqueous core nanocapsules. Zeta potential measurements and quenching of fluorescence of fluorescein-bounded oligonucleotides were used to study the localization of the oligonucleotides. Oligonucleotide degradation studies were carried out in fetal calf serum. RESULTS: Polydisperse nanocapsules of size ranging from 20 to 400 nm were obtained. Oligonucleotide loading did not significantly influence the zeta potential, suggesting they were located within the core of the nanocapsules. Fluorescence quenching assays confirmed this localization. When encapsulated in the nanocapsules and incubated in the presence of serum, the oligonucleotides were efficiently protected from degradation by nucleases, whereas oligonucleotides adsorbed onto nanospheres were protected less efficiently. CONCLUSIONS: This paper describes, for the first time, a nanotechnology able to encapsulate oligonucleotides rather than adsorbing them at the surface of a solid support. Such a formulation has great potential for oligonucleotide delivery.

Poly(D,L-lactide) nanocapsules prepared by a solvent displacement process: influence of the composition on physicochemical and structural properties.

Nanocapsules (NC) were prepared by interfacial deposition of preformed biodegradable polymer (PLA(50)) after a solvent displacement process. The influence of the composition used for the preparation of NC was evaluated in terms of particle size, polydispersity, zeta potential, homogeneity, and structural characteristics of the systems. The nature of the oil phase, polymer molecular weight, type and concentration of different surfactants were investigated to optimize the formulation to obtain NC suitable for intravenous administration. The influence of the physicochemical properties of the different oils used in NC preparation on the NC size was evaluated. The interfacial tension between the oil and water phases seems to have a greater effect on NC size than the oil viscosity. Miglyol 810 and ethyl oleate lead to the formation of smaller NC, probably because of the reduced interfacial tension. The polymer molecular weight plays only a small role in NC surface charge in the presence of lecithin, whereas NC surface charge, size, polydispersity, and short-term stability were highly influenced by lecithin purity. It appears that the absence of poloxamer 188 leads to smaller polydispersity, less contamination with nanospheres, and reduced formation of structures other than NC. Furthermore, electron microscopy and density gradient density techniques were used to examine the structure of the particles formed and their homogeneity. NC formation was evidenced by the bands with intermediate density between nanoemulsion and nanospheres; however, other bands of low intensity were observed. The presence of liposomes and multilayers in NC preparation was confirmed by electron microscopy. The percentage of carboxyfluorescein entrapped in different NC formulations allowed us to estimate the contamination by liposomes. It has been show that, under our experimental conditions, an excess of lecithin is an essential prerequisite for a stable preparation of PLA NC.

Targeted delivery of antibiotics using liposomes and nanoparticles: research and applications.

This review examines current technologies for increasing the bioavailability of antibiotics by means of liposomes or nanoparticles. The main focus is on liposomes. These carriers were preferentially developed because their composition is compatible with biological constituents. Biodegradable polymers in the form of colloidal particles have also been used and show promise for future applications in antimicrobial chemotherapy. The in vivo behaviour of both types of carriers and consequently their therapeutic potential, are determined by their route of administration. Conventional carrier strategies permit the mononuclear phagocyte system to be targeted by intravenous injection of antibiotics. Stealthy strategies avoid major uptake by these cells and extend the systemic presence of these carriers. The purpose of this review is to provide background information in antibiotic targeting gathered from papers published over the last twenty years. It seems clear that such drug carriers (liposomes, nanoparticles) allow increased drug concentration at infected sites but reduce drug toxicity.

Visualization of in vitro protein-rejecting properties of PEGylated stealth polycyanoacrylate nanoparticles.

The in vitro protein-rejecting properties of PEG-coated polyalkylcyanoacrylate (PACA) nanoparticles were for the first time visualized after freeze-fracture of the nanoparticles pre-incubated with fibrinogen as a model blood protein. The reduced protein association to the nanoparticles was evidenced also by two-dimensional PAGE after incubation of the nanoparticles with human plasma. In vivo experiments showed the 'stealth' long-circulating properties of the PEGylated nanoparticles after intravenous administration to mice. Thus, the images obtained after nanoparticle-protein incubation were predictive of the behavior observed in vivo. In conclusion, freeze-fracture analysis represents a novel and original qualitative approach to investigate the interactions between proteins and particulate systems.

Optimization of the encapsulation and release of beta-lactoglobulin entrapped poly(DL-lactide-co-glycolide) microspheres.

The goal of the present paper was to optimize the encapsulation of beta-lactoglobulin (BLG) within poly(lactide-co-glycolide) (PLGA) microparticles prepared by the multiple emulsion solvent evaporation method. The role of the pH of the external phase and the introduction of the surfactant Tween 20, in the modulation of the entrapment and release of BLG from microparticles, was studied. Reducing the solubility of BLG by decreasing the pH of the external phase to a value close to the pI of BLG resulted in a better encapsulation with, however, a larger burst release effect. By contrast, Tween 20 was shown to increase the encapsulation efficiency of BLG and reduce considerably the burst release effect. In fact, Tween 20 was shown to be responsible for removing the BLG molecules that were adsorbed on the particle surface. In addition, Tween 20 reduced the number of aqueous channels between the internal aqueous droplets as well as those communicating with the external medium. Thus, the more dense structure of BLG microspheres could explain the decrease in the burst release. These results constitute a step ahead in the improvement of an existing technology in controlling protein encapsulation and delivery from microspheres prepared by the multiple emulsion solvent evaporation method.

A polysorbate-based non-ionic surfactant can modulate loading and release of beta-lactoglobulin entrapped in multiphase poly(DL-lactide-co-glycolide) microspheres.

PURPOSE: The goal of the present paper was to investigate the role of a surfactant, Tween 20, in the modulation of the entrapment and release of beta-lactoglobulin (BLG) from poly (DL-lactide-co-glycolide) microspheres. METHODS: Poly(DL-lactide-co-glycolide) microspheres containing BLG were prepared by a water-in-oil-in-water emulsion solvent procedure. Tween 20 was used as a surfactant in the internal aqueous phase of the primary emulsion. BLG entrapment efficiency and burst release were determined. Displacement of BLG from microsphere surface was followed by confocal microscopy observations and zeta potential measurements, whereas morphological changes were observed by freeze-fracture electron microscopy. RESULTS: Tween 20 was shown to increase 2.8 fold the encapsulation efficiency of BLG without any modification of the stability of the first emulsion and the viscosity of the internal aqueous phase. In fact, Tween 20 was shown to be responsible for removing the BLG molecules that were adsorbed on the particle surface or very close to the surface as shown by confocal microscopy and zeta potential measurements. Tween 20 reduced the number of aqueous channels between the internal aqueous droplets as well as those communications with the external medium. Thus, the more dense structure of BLG microspheres could explain the decrease of the burst release. CONCLUSIONS: These results constitute a step forward in the improvement of existing technology in controlling protein encapsulation and delivery from microspheres prepared by the multiple emulsion solvent evaporation method.

Development of ciprofloxacin-loaded nanoparticles: physicochemical study of the drug carrier.

This paper describes the optimization of the preparation of ciprofloxacin-loaded polyethylbutylcyanoacrylate (PEBCA) nanoparticles. The association of ciprofloxacin with nanoparticles was performed by emulsion polymerization, but successful entrapment was only obtained in the presence of acetone in the polymerization medium. This preparation process led to a stable ciprofloxacin nanoparticle suspension, with a mean size value twice as high as that obtained in the absence of drug, and an association efficiency of 82%. Moreover, the molecular weight value of ciprofloxacin nanoparticles was shown to be reduced as compared with unloaded nanoparticles. Drug release from the colloidal carrier in medium containing esterase was found to be very slow (a maximum of 51.5% after 48 h), suggesting that this release resulted from bioerosion of the polymer matrix. Interestingly, it was observed that 30.5% of the initial amount of ciprofloxacin was not detectable by HPLC analysis after nanoparticle preparation and corresponded either to ciprofloxacin covalently bound to PEBCA or to ciprofloxacin chemically degraded during the polymerization process. 19F-NMR analysis demonstrated that ciprofloxacin entrapped into nanoparticles was only in its neutral form. The measurements of molecular weight suggest the participation of the antibiotic as an anionic polymerization initiator, leading to the formation of a chemical bond between some of the drug and the polymer. These data allowed us to propose a model describing the association of ciprofloxacin with PEBCA nanoparticles obtained by emulsion polymerization.

Drug targeting by polyalkylcyanoacrylate nanoparticles is not efficient against persistent Salmonella.

PURPOSE: We have investigated the efficacy of colistin and ciprofloxacin, free or bound to polyalkylcyanoacrylate nanoparticles, for the targeting and eradication of Salmonella persisting in the organs of the mononuclear phagocyte system. METHODS: A model of persistent S. typhimurium infection was developed in C57BL/6 mice using i.v. inoculation of the plasmid-cured strain C53. RESULTS: In vivo and ex vivo experiments showed that the persisting bacteria seem to evolve to a nongrowing state during experimental salmonellosis. In vivo treatment with free or nanoparticle-bound colistin did not significantly reduce the number of viable Salmonella C53, either in the liver or the spleen of infected mice. In contrast, in vivo treatment with ciprofloxacin led to a significant decrease of bacterial counts in the liver whatever the stage of infection and the form used. However, none of the treatments were able to sterilize the spleen or the liver. In ex vivo experiments, colistin was only active against bacteria recovered during the early phase of infection, whereas ciprofloxacin exerted its activity at all times postinfection. CONCLUSIONS: We suggest that the micro-environment in which the bacterial cells persist in vivo probably causes dramatic changes in their susceptibility to antimicrobial agents.

Intracellular distribution of ampicillin in murine macrophages infected with Salmonella typhimurium and treated with (3H)ampicillin-loaded nanoparticles.

The intracellular distribution of (3H)ampicillin-loaded polyisohexylcyanoacrylate nanoparticles was studied in murine macrophages (peritoneal cells and the J774 cell line) infected by Salmonella typhimurium C5, using ultrastructural autoradiography. Ampicillin penetration and retention into the cells obviously increased by means of nanoparticles. After short-term (2-4 h) treatment with the nanoparticle formulation, numerous intracellular bacteria were seen to be in the process of destruction. The tritium labelling was located in the cell cytoplasm and inside vacuoles in which bacteria undergoing degradation were often present. After long-term (12 h) treatment, numerous spherical bodies (d: 100 nm to 500 nm) and larger forms (2 microns) were seen in the vacuoles. Radioactivity was mainly found to be localized on the spherical bodies, indicating marked damaging action of the ampicillin on the bacterial walls. The targeting of ampicillin therefore allowed its penetration into the macrophages and vacuoles infected with S. typhimurium.

A new method to isolate polyalkylcyanoacrylate nanoparticle preparations.

A simple method for the separation of polyalkylcyanoacrylate nanoparticles was developed using polyisohexylcyanoacrylate (PIHCA) as a model. Fluorescein isothiocyanate dextran 70 was used to label the nanoparticles. Ultracentrifugation onto a performed sucrose gradient allowed the easy elimination of the dextran and of the free molecules remaining in the upper phase. After such treatment, the physicochemical characteristics of the PIHCA nanoparticles were not modified. This method could be usefully extended to other types of nanoparticles.

The uptake of ampicillin-loaded nanoparticles by murine macrophages infected with Salmonella typhimurium.

The purpose of this study was to investigate the in-vitro interaction between [3H]ampicillin-loaded polyisohexylcyanoacrylate nanoparticles and murine macrophages (peritoneal and J774) infected with Salmonella typhimurium. The multiplicity of infection was ten bacteria to each macrophage and the mean (+/- S.D.) diameter of the nanoparticles was 220 (+/- 20 nm), corresponding to an ampicillin concentration of 2 g/L. The uptake of nanoparticle-bound [3H]ampicillin by non-infected J774 and peritoneal macrophages was six- and 24-fold greater respectively than that of free [3H]ampicillin. For infected cells, uptake by J774 and peritoneal macrophages was nine- and 20-fold greater respectively. However, there was no difference between nanoparticle-bound ampicillin and free ampicillin in terms of bactericidal activity against intracellular S. typhimurium. This unexpected observation might be accounted for by bacterium-induced inhibition of phagosome-lyosome fusion within the macrophages, thereby preventing contact between the bacteria in the phagosomes and the nanoparticles in the secondary lysosomes.

Intracellular visualization of ampicillin-loaded nanoparticles in peritoneal macrophages infected in vitro with Salmonella typhimurium.

Intracellular targeting of ampicillin by means of polyisohexylcyanoacrylate (PIHCA) nanoparticles was studied in murine peritoneal macrophages infected with Salmonella typhimurium. The intracellular distribution of actively endocytosed nanoparticles was visualized by transmission electron microscopy and confocal microscopy. Nanoparticles were either isolated or closely associated with bacteria within phagosomes or phagolysosomes. Thus the potential of ampicillin-loaded nanoparticles in targeting of intracellular bacteria is demonstrated. Consequently, ampicillin, which usually penetrates into cells at a low level, is directly carried in, when loaded on nanoparticles, and brought into contact with intracellular bacteria.

Emergence of aminoglycoside resistance genes aadA and aadE in the genus Campylobacter.

Resistance to streptomycin or spectinomycin or both in five Campylobacter coli strains, two Campylobacter jejuni strains, and a Campylobacter-like strain was studied by enzymatic assays and dot blot hybridization. Resistance was due to 6- or 3",9-aminoglycoside adenylyltransferases and to new types of phospho- and adenylyltransferases.