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The aim of the present study was to evaluate the inhibitory effect on protein kinase C alpha (PKC alpha) neosynthesis of antisense oligonucleotides delivered by two types of carriers. First, PKC alpha antisense oligonucleotides were associated with polyisobutylcyanoacrylate (PIBCA) nanoparticles pre-coated with cetyltrimethyl ammonium bromide (CTAB), a hydrophobic cation. Adsorption of oligonucleotides onto PIBCA nanoparticles was shown to be a saturating process. From these studies, it was possible to identify two types of particles: positively and negatively charged. Secondly, Lipofectin was used as another carrier system. These systems were incubated with HepG2 cells. Toxicity was evaluated by the MTT assay, and PKC alpha neosynthesis was determined by Western blots in conditions where nanoparticles and Lipofectin were not inducing cytotoxicity. It was observed that both mismatch and antisense oligonucleotides induced an inhibition of PKC alpha neosynthesis when loaded onto cationic or anionic nanoparticles as well as when complexed to cationic liposomes (Lipofectin). This non-specific effect was only observed in the phase of PKC alpha neosynthesis when the cells were first depleted in PKC alpha by phorbol 12-myristate beta-acetate (12-PMA) and in the absence of serum. These results strongly suggest that delivery systems, PIBCA nanoparticles or Lipofectin, containing a positively charged component (CTAB or cationic lipids), are able to induce a perturbation in the intracellular metabolic activity. In conclusion, it was shown that the commonly used strategy of oligonucleotides targeting with cationic non-viral vectors may display non-specific effects which can lead to artifactual results.

The distribution of a model 16-mer oligothymidylate (pdT16) in several ocular tissues (cornea, conjunctiva, sclera, iris, lens, aqueous and vitreous humors) was determined after instillation in the eye of various dosage forms in a rabbit model. Radiolabelled pdT16 was applied as a simple solution, a 27% poloxamer 407 gel, a suspension of liposomes or liposomes dispersed within a 27% poloxamer 407 gel. pdT16 concentrations were measured in the tissues and fluids by radioactivity counting at time intervals of 10 min, 2 h and 24 h. When the pdT 16 solution was used, the highest concentrations were observed in the conjunctiva and the cornea, while a substantial amount of drug was also present in the sclera. Low concentrations were measured in the iris. Using the same treatment protocol, the two liposomal formulations (liposomes suspension or liposomes dispersed within the poloxamer gel) delivered low amounts of pdT16 to all ocular tissues, and particularly to the conjunctiva and the cornea. The poloxamer gel provided higher tissue concentrations of pdT16 than liposomes but lower than those observed with the solution except 10 min after administration in the iris where the amounts of pdT16 were higher when administered under the gel form. These findings indicate that liposomal forms may not be considered useful delivery systems for topical administration of oligonucleotides in superficial ocular diseases.

International audience; The aim of this study was to design a new antisense oligonucleotide (ON) carrier system based on alginate nanoparticles and to investigate its ability to protect ON from degradation in the presence of serum. Pharmacokinetics and tissue distribution of ON-loaded nanoparticles have been determined after intravenous administration. An original and dynamic process for ON loading into polymeric nanoparticles has been applied. It is based on the diffusion of ON or ON/polylysine complex into the nanoparticle or the alginate gel, respectively. Indeed, the single coincubation of ON with nanoparticles led, within a few days, to an extremely efficient association. The diffusion kinetic of ON was shown to be dependent on several parameters, incubation temperature, ON concentration, presence or absence of polylysine, polylysine molecular weight, and nanoparticle preparation procedure. This new alginate-based system was found to be able to protect [33P]-radiolabeled ON from degradation in bovine serum medium and to modify their biodistribution, as an important accumulation of radioactivity was observed in the lungs, in the liver, and in the spleen after intravenous administration into mice. ON may be associated efficiently with calcium alginate in a colloidal state. Such nanosponges are promising carriers for specific delivery of ON to lungs, liver, and spleen.

The aim of the work was to develop small microspheres made from a biodegradable polymer, poly(lactide-co-glycolide), in order to entrap small peptides. Microspheres prepared by a water-in-oil-in-water emulsion solvent evaporation technique displayed a mean diameter below than 10 microns and showed high encapsulation efficiency of a 33 amino acid peptide (V3 BRU). In vitro release kinetics studies showed that such microparticles could be employed for both oral immunization and controlled release. The encapsulation of a seven aminoacid peptide in the same conditions, led to a very low encapsulation efficiency. In order to increase the entrapment efficiency, two strategies were adopted: taking into account the solubility of pBC 264 at different pH, a pH gradient was created to prevent the leakage of the encapsulated peptide into the outer aqueous phase. The inner aqueous phase was maintained at basic pH where the peptide was soluble, while the external aqueous phase was acidic: ovalbumin was added during preparation to stabilize the inner emulsion. These two strategies allowed to increase significantly the encapsulation rate of pBC 264. Nevertheless, the in vitro release kinetics of the peptide were strongly influenced by the presence of ovalbumin which seems to form pores in the microsphere structure (80% of the total peptide content was released after 30 minutes). By contrast, when ovalbumin was replaced by Pluronic F 68 microspheres did not have pores, thus the release profile and the extent of the burst were much smaller. When microspheres were stereotactically implanted in the rat brain, in vivo release profiles were in good agreement with the release observed in vitro. In conclusion, these microspheres are well suited for the slow delivery of neuropeptides in the brain, a feature expected to facilitate the study of long term effects of these compounds.

Polymeric injectable carriers, able to deliver drugs or other compounds to specific sites of action for a prolonged time, represent a potential therapeutic approach for several diseases. However, therapeutic activity is compromised by particle recognition by the macrophages of the mononuclear phagocyte system (MPS) and their elimination within seconds, or minutes after intravenous injection. Blood proteins (opsonins) adsorb onto the particles surface, making them recognizable to the macrophages of the MPS.

So far gene transfer to the muscle has mainly been based on viral vectors, given their efficiency to transfer DNA in vivo. However, virus-derived vectors have numbers of limitations, such as insert size, tissue specificity or immunogenicity, therefore restricting the possibilities of repeated administrations. After intramuscular injection, major hurdles remain tissue diffusion and intracellular entry.

Blanca Tobar-Grande,1 Ricardo Godoy,1 Paulina Bustos,2 Carlos von Plessing,1 Elias Fattal,3,4 Nicolas Tsapis,3,4 Claudia Olave,1 Carolina Gómez-Gaete11Departamento de Farmacia, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile; 2Departamento de Bioquímica Clínica e Inmunología, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile; 3Univ Paris-Sud, Institut Galien Paris-Sud, Faculté de Pharmacie, Châtenay-Malabry, France; 4CNRS, UMR 8612, Faculté de Pharmacie, Châtenay-Malabry, FranceAbstract: In this work, microparticles were prepared by spray-drying using albumin, chondroitin sulfate, and hyaluronic acid as excipients to create a controlled-release methylprednisolone system for use in inflammatory disorders such as arthritis. Scanning electron microscopy demonstrated that these microparticles were almost spherical, with development of surface wrinkling as the methylprednisolone load in the formulation was increased. The methylprednisolone load also had a direct influence on the mean diameter and zeta potential of the microparticles. Interactions between formulation excipients and the active drug were evaluated by x-ray diffraction, differential scanning calorimetry, and thermal gravimetric analysis, showing limited amounts of methylprednisolone in a crystalline state in the loaded microparticles. The encapsulation efficiency of methylprednisolone was approximately 89% in all formulations. The rate of methylprednisolone release from the microparticles depended on the initial drug load in the formulation. In vitro cytotoxic evaluation using THP-1 cells showed that none of the formulations prepared triggered an inflammatory response on release of interleukin-1ß, nor did they affect cellular viability, except for the 9.1% methylprednisolone formulation, which was the maximum test concentration used. The microparticles developed in this study have characteristics amenable to a therapeutic role in inflammatory pathology, such as arthritis.Keywords: spray-drying, microparticles, methylprednisolone, chondroitin sulfate, hyaluronic acid, cytotoxic assays

The present work investigated the magnitude of microsphere-induced acute myotoxicity and determined whether this myotoxicity is related to microsphere size and/or reconstitution solvent. Using a high molecular weight poly(dl-lactide-co-glycolide) copolymer, the myotoxicity of two different size microsphere formulations (3.6 microns and 19 microns) in normal saline or distilled water was quantified using a previously validated isolated rat muscle system. Overall, microspheres were found to be relatively nontoxic compared to known myotoxic agents (e.g., phenytoin) and control muscles. The smaller microspheres were found to be significantly more myotoxic than larger microspheres. Furthermore, the myotoxicity was lower in large microspheres reconstituted with normal saline or normal saline with 0.5% (w/v) carboxymethylcellulose (to prevent aggregation) compared to those reconstituted with distilled water. Smaller microspheres were found to be extremely difficult to inject, due to aggregation, which could not be prevented by the addition of carboxymethylcellulose. This study suggests that larger microspheres are less myotoxic than smaller microspheres.

A quantitative polyacrylamide gel electrophoresis (PAGE) analysis using a multichannel radioactivity counter was designed for the evaluation of 33P-labeled antisense oligonucleotide associated with polymeric drug carrier (nanoparticles). The proposed analytical method was first validated. The criteria of specificity, linearity, reliability, detection and quantification limits, and resolution power were determined. Results were compared to those obtained using liquid scintillation counting of crude samples or after solubilization of gel slices. The proposed method gave a better linearity and reliability than liquid scintillation counting of solubilized gel slices. In comparison with the liquid scintillation counting of crude samples, the method presented the advantage of being able to directly separate oligonucleotides differing by only one nucleotide in length. This method was applied for the separation of free oligonucleotides and oligonucleotides bound onto nanoparticles, allowing quantification of the amount of free and bound oligonucleotides without any further separation steps. Thus, because it is easy and rapid, the quantitative PAGE analysis using a multichannel radioactivity counter offers interesting possibilities for the characterization of oligonucleotide nanoparticles.

The understanding of the interrelationships among dynamics, structure, and function of membrane-interacting peptide segments has been intensely studied during the last decade.

To introduce optical imaging among methods available to follow nanoparticle biodistribution, we have evaluated the concept of covalently labeling poly(lactide-co-glycolide) (PLGA) with a near-infrared (NIR) dye to obtain stable NIR fluorescent nanoparticles. PLGA was coupled with the NIR dye (DY-700, Dyomics) by an amide bond with 38% efficiency. Incorporating 1% of this conjugate into PLGA nanoparticles stabilised by polyvinyl alcohol (PVA) leads to stable nanoparticles (NPs) without affecting their colloidal characteristics (average diameter, polydispersity and zeta potential). In addition, nanoparticles remain strongly fluorescent and display good storage stability for 4 weeks at 4 °C or over one week at 37 °C. Nanoparticle cytotoxicity evaluated using HUVEC, NIH/3T3 and J774.A1 cell lines was similar for unlabeled or labeled NPs. Fluorescent nanoparticles and free dye were injected intravenously into mice and their biodistribution was followed for 24 h by NIR imaging, in vivo and ex vivo. Nanoparticles were found mainly in the liver whereas the free dye was not accumulating preferentially in this organ. The DY-700 NIR conjugate incorporated into PLGA NPs shows good performance both in vitro and in vivo, thus paving the way to finely traceable PLGA nanosystems for in vivo administration.

Adenosine triphosphate (ATP) was proposed in various medical applications, as a possible bioenergetic substrate. Unfortunately, ATP is very difficult to use at a therapeutic level because of its high sensitivity to enzymatic hydrolysis making this molecule unstable in biological fluids. ATP is also a highly hydrophilic molecule that is unable to cross biological membranes. To try to develop a system able to protect ATP against degradation and to efficiently deliver this bioenergetic substrate, its liposomal encapsulation in multilamellar vesicles was carried out. One of the studies described in this paper deals with the efficiency of liposomal ATP in the treatment of cerebral ischemia. Our results show that encapsulation was able to protect ATP from its degradation by ectonucleotidases and that liposomal ATP was active against experimental brain ischemia. The other study deals with the effect of ATP on the motility and the acrosomal reaction of human spermatozoa. The results show that co-incubating ATP-loaded liposomes with sperm cells was able to induce the process of capacitation in vitro and might therefore be a useful tool in the procedure of in vitro fertilization.

This paper describes the conditions of preparation of poly(lactide-coglycolide) microspheres with a mean diameter lower than 10 μm obtained by a (w1/o)w2 emulsion solvent evaporation method. Different parameters influencing respectively the size of the inner emulsion and the diameter of the microspheres were determined. V3 BRU, which is a specific immunogenic fraction from GP120 of HIV, was encapsulated in those microspheres. The entrapment efficiency was shown to be superior to that of microspheres prepared according to the single emulsion solvent evaporation method. Electron microscopy observations demonstrated the presence within the microspheres of globules corresponding to the w / o initial inner emulsion in which the peptide was dissolved in the aqueous phase. Analysis of the release kinetics was carried out in phosphate buffer (PBS), in artificial gastric and intestinal medium. V3 BRU release in PBS was slow, reaching a plateau at 24 h corresponding to 25% of drug release. In addition, V3 BRU was not released in gastric medium within 4 h whereas under the same time conditions, 60% of the drug was released in the presence of intestinal medium. These results open up interesting prospects for the use of these microspheres as an oral adjuvant for HIV vaccination.

Simona Mura1,2, Herve Hillaireau1,2, Julien Nicolas1,2, Benjamin Le Droumaguet1,2, Claire Gueutin1,2, Sandrine Zanna3, Nicolas Tsapis1,2, Elias Fattal1,2 1Univ Paris-Sud, UMR 8612, Châtenay Malabry, F-92296; 2CNRS, Châtenay Malabry, F-92296; 3Laboratoire de Physico-Chimie des Surfaces, CNRS-ENSCP (UMR 7045), Ecole Nationale Superiore de Chimie de Paris, France Background: Because of the described hazards related to inhalation of manufactured nanoparticles, we investigated the lung toxicity of biodegradable poly (lactide-co-glycolide) (PLGA) nanoparticles displaying various surface properties on human bronchial Calu-3 cells. Methods: Positively and negatively charged as well as neutral nanoparticles were tailored by coating their surface with chitosan, Poloxamer, or poly (vinyl alcohol), respectively. Nanoparticles were characterized in terms of size, zeta potential, and surface chemical composition, confirming modifications provided by hydrophilic polymers. Results: Although nanoparticle internalization by lung cells was clearly demonstrated, the cytotoxicity of the nanoparticles was very limited, with an absence of inflammatory response, regardless of the surface properties of the PLGA nanoparticles. Conclusion: These in vitro results highlight the safety of biodegradable PLGA nanoparticles in the bronchial epithelium and provide initial data on their potential effects and the risks associated with their use as nanomedicines. Keywords: nanoparticles, PLGA, surface properties, Calu-3, toxicity, inflammation

Chitosan, a natural polysaccharide obtained from chitin deacetylation, complexes with metal ions by coordination with the free electron pairs of amine groups. Based on this complexation mechanism, cross-linked chitosan-metal microparticles were prepared by spray drying using iron (II or III) or zinc ions and characterized in terms of size distribution and capacity to specifically adsorb ciprofloxacin. Chitosan-Zn(II) and chitosan-Fe(III) microparticles appear to adsorb more ciprofloxacin than plain chitosan or chitosan-Fe(II) microparticles. Adsorption isotherms for CH and CH–Fe(II) microparticles can be fitted by a single logarithm model (slope 1) with one ciprofloxacin per adsorption site, whereas for CH–Fe(II) and CH–Zn(II) microparticles, isotherms are bilogarithmic with an initial slope of 2, suggesting that a single adsorption site can bind two molecules of ciprofloxacin. In addition, the pseudo second order kinetic model fits well experimental data, proving that adsorption is mediated by a chemical reaction. CH–Fe(II) and CH–Zn(II) appear very promising for drug elimination, either from hospital waste water or from the gastrointestinal tract to prevent the emergence of antibiotic resistance. Chitosan, a natural polysaccharide obtained from chitin deacetylation, complexes with metal ions by coordination with the free electron pairs of amine groups. Based on this complexation mechanism, cross-linked chitosan-metal microparticles were prepared by spray drying using iron (II or III) or zinc ions and characterized in terms of size distribution and capacity to specifically adsorb ciprofloxacin. Chitosan-Zn(II) and chitosan-Fe(III) microparticles appear to adsorb more ciprofloxacin than plain chitosan or chitosan-Fe(II) microparticles. Adsorption isotherms for CH and CH–Fe(II) microparticles can be fitted by a single logarithm model (slope 1) with one ciprofloxacin per adsorption site, whereas for CH–Fe(II) and CH–Zn(II) microparticles, isotherms are bilogarithmic with an initial slope of 2, suggesting that a single adsorption site can bind two molecules of ciprofloxacin. In addition, the pseudo second order kinetic model fits well experimental data, proving that adsorption is mediated by a chemical reaction. CH–Fe(II) and CH–Zn(II) appear very promising for drug elimination, either from hospital waste water or from the gastrointestinal tract to prevent the emergence of antibiotic resistance.

We modulate precisely the surface morphology of polymer microcapsules containing perfluorooctyl bromide prepared by the solvent-evaporation technique by varying the percentage of PLGA-PEG copolymer in the formulation. As copolymer percentage increases, protrusions start to appear, become more numerous and finally fuse to yield sponge-like capsules. It has been postulated that the shell morphology arises from an interfacial tension instability. We show here that if an interfacial tension instability develops, it happens during the final step of polymer desolvation, when the interface is no longer fluid and interfacial tension changes cannot be monitored. The formation of protrusions occurs in order to accommodate all the PEG chains of the copolymer in a hydrophilic environment, since PEG and PLGA moieties are not miscible in the solid state.

In this paper, we report the physico-chemical properties of negatively charged liposomes and of polyisohexylcyanoacrylate nanoparticles loaded with ampicillin. Although the carriers were of the same size (200 nm), drug-loading capacity was 20 times higher for nanoparticles than for liposomes. After freeze-drying or storage at +4 degrees C, no drug escaped from polymeric nanoparticles. On the other hand, in the same conditions, ampicillin leaked rapidly from liposomes. Drug release in foetal calf serum was gradual (of zero order) with nanoparticles, whereas it was rapid with liposomes. Finally, the antimicrobial activity of ampicillin-entrapped liposomes or nanoparticles was studied in vitro.

The APV (German “International Association for Pharmaceutical Technology”) and the APGI (French “Association for Industrial Pharmaceutical Technology”) jointly sponsored the 6th World Meeting on Pharmaceutics, Biopharmaceutics, and Pharmaceutical Technology (PBP Meeting) in Barcelona, Spain on April 7–10, 2008. Sixteen other pharmaceutical and engineering societies participated in this event. Although this meeting focuses on the fields of Pharmaceutics, Biopharmaceutics, and Pharmaceutical Technology, the biannual World Meeting has become the most important pharmaceutical sciences congress in Europe. Nearly 1,400 participants from more than 50 countries met to discuss the latest developments. The slogan of this World Meeting was “Industry Meets Academia and Authorities: Shared Research for Scientific Progress.” The scientific program consisted of five plenary lectures, five symposia with invited speakers, more than 60 short oral presentations, and 900 poster presentations. Value added programming for participants consisted of an industrial exhibition and social events. The opening lecture, “Generics—making more for less” by Dr. Andreas Rummelt, CEO of Sandoz Pharmaceuticals, provided an overview of the challenges and possibilities for generic drug products from an industrial perspective. Specifically, Dr. Rummelt explained how generic products profit from advanced dosage forms, and how the relevance of generic products differs from country to country. This excellent lecture also detailed the future perspectives of generic products for small and large molecules, and set the stage for many symposia such as this, on biosimilars. The prestigious Maurice Janot Award Lecture by Professor Patrick Couvreur, University de Paris-Sud, France provided an overview of his research, aimed at intelligent design of nanomedicines for treatment of complex diseases. Professor Chris Porter, of Monash University, Melbourne, Australia, gave a plenary lecture on the role of lipid transport pathways in drug absorption and emerging concepts in this area. Biopharmaceutical aspects of increasing oral bioavailability were the focus of a lecture by Professor Jennifer B. Dressman of Goethe-University, Frankfurt, Germany. She reported on progress in the development of biorelevant dissolution media to provide a better correlation between in vitro and in vivo dissolution behavior of oral dosage forms. Professor Daan Crommelin, of Utrecht University, The Netherlands, gave a stimulating overview on the most recent developments in the area of Biotechnology and future trends. He critically summarized the current status and achievements of the field, noting the perspectives are manifold. He put the developments into the context of future public health. Invited speaker symposia topics were “Quality by Design and PAT,” “Formulation of Poorly soluble Drugs,” “Biosimilars,” “Pre-formulation and New Excipients,” and “Polymorphism and Amorphous Systems”. Many oral and poster presentations of extreme high quality demonstrated the impressive need to bring the expertise of industry, academia, and regulatory authorities together in order to meet the challenges of modern medicine design, production, and application. This created an atmosphere of truly international scientific collaboration and exchange during the meeting. The 7th World Meeting on Pharmaceutics, Biopharmaceutics, and Pharmaceutical Technology will be held in Malta on March 8–11, 2010. In continuation of the successful Barcelona meeting, emphasis will be placed on industrially-relevant topics in order to help bridge the gap between fundamental academic research and industrial applications. For more detailed information, visit the meeting’s Web site at http://www.worldmeeting.org, or email info@worldmeeting.org.

PURPOSE: To reestablish the immunosuppressive microenvironment of the eye, disrupted by ocular inflammation during endotoxin-induced uveitis (EIU), by means of intravitreal injection of vasoactive intestinal peptide (VIP) in saline or encapsulated in liposomes, to increase its bioavailability and efficiency. METHODS: EIU was induced in Lewis rats by subcutaneous injection of lipopolysaccharide (LPS). Simultaneously, animals were intravitreally injected with saline, saline/VIP, VIP-loaded liposomes (VIP-Lip), or unloaded liposomes. EIU severity and cellular infiltration were assessed by clinical examination and specific immunostaining. VIP concentration was determined in ocular fluids by ELISA. Ocular expression of inflammatory cytokine and chemokine mRNAs was detected by semiquantitative RT-PCR. Biodistribution of rhodamine-conjugated liposomes (Rh-Lip) was analyzed by immunohistochemistry in eyes and regional cervical lymph nodes (LNs). RESULTS: Twenty-four hours after intravitreal injection of VIP-Lip, VIP concentration in ocular fluids was 15 times higher than after saline/VIP injection. At that time, EIU clinical severity, ocular infiltrating polymorphonuclear leukocytes (PMNs), and, to a lesser extent, ED1(+) macrophages, as well as inflammatory cytokine and chemokine mRNA expression, were significantly reduced in VIP-Lip-injected rats compared with rats injected with saline/VIP, unloaded liposomes, or saline. Rh-Lip was distributed in vitreous, ciliary body, conjunctiva, retina, and sclera. It was internalized by macrophages and PMNs, and VIP colocalized with liposomes at least up to 14 days after injection. In cervical LNs, resident macrophages internalized VIP-Rh-Lip, and some adjacent lymphocytes showed VIP expression. CONCLUSIONS: VIP was efficient at reducing EIU only when formulated in liposomes, which enhanced its immunosuppressive effect and controlled its delivery to all tissues affected by or involved in ocular inflammation.

Background: Concerning myopathies, the treatment of myocardial impairment raises specific issues due to the structure and function of the myocardium. Thus, original routes of administration can be considered as direct injection into the myocardium, coronary infusion or intrapericardial injection. Gene therapy is mainly based on two different vector systems: viral or non-viral formulation of the DNA. Considering that repeated administrations of plasmid DNA (pDNA) would be mandatory to achieve the therapeutic goals and that viral transfection's main issue remains immunogenicity, non-viral vectors seem to be the best suited. Several lipidic or polymeric systems have already been tested, and in vivo, one of the most efficient appears to be polyethyleneimine (PEI). Our study aims at designing a new non-viral system for myocardial gene delivery, displaying low toxicity and the best efficiency. This system is based on condensation of plasmid DNA with PEI. These polyplexes are then included in a thermosensitive gel in order to improve the residence time of the system and administered via the intrapericardial route.

Summary ATP entrapped into liposomes was administered intracarotidally to rats submitted to brain ischemics episodes by clamping of the carotid arteries and lowering of the systemic blood pressure. It was observed that when entrapped into liposomes, ATP greatly increased the number of ischemic episodes tolerated before brain electrical silence and death appeared. These results added to very similar previous data obtained by i.c.v. treatment excluding the prominent role of cardiovascular effects, could open new possibilities in brain antihypoxic protection. Here and now it cannot be stated if ATP provides direct energetic supply.

Liposomally entrapped adenosine triphosphate (ATP) was administered intracerebroventricularly and intracarotidally to rats subjected to brain ischaemic episodes by clamping of the carotid arteries and lowering of the systemic blood pressure. It was observed that, when entrapped in liposomes, ATP greatly increased the number of ischaemic episodes before brain electrical silence and death. The results open new perspectives in brain ATP supply, which will potentially be useful in human resuscitation from deep brain hypoergic states.

ATP was entrapped inside negatively charged liposomes composed of sulfatide, in order to improve its penetration into the brain and to reduce its degradation into other tissues. These liposomes were prepared according to an original method allowing a satisfying stability of the formulation. Liposomally entrapped ATP were administered intracerebroventricularly to rats submitted to brain ischemic episodes by both carotid arteries clamping and systemic blood pressure lowering (during 3 minutes every 15 minutes). Such treatment importantly increases the number of ischemic episodes before brain silence appeared. So, this paper allows new perspectives in the administration of drugs into the brain. Several authors (1,2) have already proposed administration of ATP for the treatment of severe hypoergic syndromes. However, it is generally accepted that strongly charged anions cannot enter into the cells through the plasma membrane (2,3). For this reason, improvement in the treatment of hemorhagic shock after systemic administration of ATP was mostly attributed to peripheral effects, taking place mainly in the liver and the kidneys(1). However, such results were not confirmed by Schloerb et al. (4) who showed that ATP-MgCl2 i.v. neither increased survival rate, nor enhanced tissue levels of ATP in shocked animals. Furthermore, they showed that the administered ATP was rapidly and extensively degraded in various tissue. These results could be explained by the fact that the central nervous system and especially the neo-cortex is characterized by a very tight blood brain barrier, suggesting that ATP does not enter it. On the other hand, liposomes were proposed for introduction of hydrophilic molecules (i.e. enzymes, cytostatics) into the brain (5,6). Furthermore, liposomes composed of sulfatide, in addition to phospholipids, were found to be the optimal system for brain targeting (7). Owing to these considerations, we looked at the possibility of entrapping ATP inside negatively charged liposomes composed of sulfatide. This paper describes preparation and characterization of liposomes loaded with ATP, as well as their efficiency in experimental brain ischemia in the rat.

The effectiveness of nanoparticle-bound ampicillin was tested in the treatment of experimental Listeria monocytogenes infection in congenitally athymic nude mice. Nanoparticles of polyisohexylcyanoacrylate (PIHCA) 187 +/- 13 nm in diameter were bound to ampicillin at an ampicillin/PIHCA ratio of 0.2:1. The proportion of ampicillin bound was 90% +/- 3%. After adsorption onto nanoparticles, the therapeutic activity of ampicillin increased dramatically over that in the free state. Thus, 2.4 mg of nanoparticle-bound ampicillin (three doses of 0.8 mg each) had a greater therapeutic effect than 48 mg of free ampicillin (three doses of 16 mg each). These results might provide an incentive for further development of intracellular targeting of antibiotics on biodegradable polymeric carriers.

We tested the effectiveness of ampicillin bound to nanoparticles of polyisohexylcyanoacrylate (PIHCA) in treating C57BL/6 mice experimentally infected with Salmonella typhimurium C5. The diameter of the nanoparticles was 187 +/- 13 nm, and the ampicillin/PIHCA ratio was 0.2/1. The proportion of ampicillin bound was 90 +/- 3%. All control mice and all those treated with nonloaded nanoparticles died within 10 days of infection. By contrast, all mice treated with a single injection of 0.8 mg of nanoparticle-bound ampicillin survived. With free ampicillin, a similar curative effect required three doses of 32 mg each. Lower doses delayed but did not reduce mortality. The sharp increase in the therapeutic index of ampicillin after linkage to PIHCA nanoparticles was explained by studies of the distribution of ampicillin, which showed that when bound to nanoparticles, the ampicillin was concentrated mainly in the liver and spleen, the primary foci of infection in the experimental model that we used. These findings warrant further development of intracellular targeting of antibiotics on biodegradable polymeric carriers such as PIHCA.

The milk model protein, beta lactoglobulin (BLG), was encapsulated into microspheres prepared by a multiple emulsion/solvent evaporation method. The effect of the pH of the outer aqueous phase on protein encapsulation and release as well as on microsphere morphology has been investigated. At all tested pH values, the encapsulation efficiency was shown to decrease with increasing the initial amount of BLG. This was correlated with the reduced stability of the primary emulsion as the initial BLG increased. In addition, reducing the solubility of BLG in the external aqueous phase by decreasing the pH to the isoelectric point of BLG (pI 5.2) resulted in an improved protein encapsulation. Moreover, it was shown that combining pH modification and optimal stability of the first emulsion yielded microspheres with a high encapsulation efficiency. However, release kinetic studies revealed that a significant burst release was observed with microspheres loaded with large amounts of BLG, especially when prepared in a medium at pH 5.2. This burst effect was attributed to morphology changes in the microsphere surface which was characterized by the presence of pores or channels able to accelerate the release of BLG. These pores were assumed to result from the presence of large amounts of protein molecules on the microsphere surface, that aggregate during microsphere formation at pH 5.2. Indeed, single adsorption experiments have shown that BLG had a higher affinity for the particle surface when the pH was close to the pI. Thus, reducing the solubility of a protein in the external aqueous phase allows the product of microspheres with a better encapsulation efficiency, although this benefit is provided by a strong adsorption of the protein on microsphere surface.

We study the influence of ultrasound on paclitaxel-loaded nanocapsules in vitro and in vivo. These nanocapsules possess a shell of poly(dl-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) and a liquid core of perfluorooctyl bromide (PFOB). In vitro experiments show that mechanical effects such as cavitation are negligible for nanocapsules due to their small size and thick and rigid shell. As the mechanical effects were unable to increase paclitaxel delivery, we focused on the thermal effects of ultrasound in the in vivo studies. A focused ultrasound sequence was therefore optimized in vivo under magnetic resonance imaging guidance to obtain localized mild hyperthermia with high acoustic pressure. Ultrasound-induced mild hyperthermia (41-43°C) was then tested in vivo in a subcutaneous CT-26 colon cancer murine model. As hyperthermia is applied, an inhibition of tumor growth for both paclitaxel-loaded nanocapsules and the commercial formulation of paclitaxel, namely Taxol® have been observed (p

Encapsulation of vaccines in biodegradable microspheres provides excellent mucosal immunogens with a high potential for immunization against bacterial infections. We tested the protective immunity elicited by intragastric vaccination with phosphorylcholine (PC) encapsulated in poly(DL-lactide-co-glycolide) (DL-PLG) microspheres against Salmonella typhimurium in a mouse model of invasive intestinal infection. We chose PC as the antigen because it was found to elicit an immune response after intestinal exposure of mice to PC-bearing S. typhimurium and because anti-PC immunity protects mice against Streptococcus pneumoniae, another PC-bearing microorganism. Mice were primed intragastrically on days 1, 2, and 3 and boosted on days 28, 29, and 30 with PC (280 microg) coupled to porcine thyroglobulin (PC-thyr) encapsulated in DL-PLG microspheres, free PC-thyr, or blank microspheres. A significant rise in anti-PC immunoglobulin A (IgA) titers, as measured by an enzyme-linked immunosorbent assay, was observed in the intestinal secretions after immunization with PC-loaded microspheres, compared to the titers of mice immunized with free PC-thyr or blank microspheres. This antibody response peaked 14 days after the last boost and correlated with a highly significant resistance to oral challenge by S. typhimurium C5 (P < 10(-3)). Control mice were primed intraperitoneally on day 1 with 15 microg of PC in complete Freund's adjuvant and boosted on days 10, 14, and 20 with the same dose without adjuvant but via the same route. In these mice, the levels of anti-PC IgA in intestinal secretions were equivalent to those of the mice intragastrically immunized with PC-loaded microspheres, but protection was significantly weaker, suggesting that either the IgAs were not functional or that other immune mechanisms are important in protection. Taken together, our results highlight the potential of antigen encapsulation in DL-PLG microspheres for eliciting protective immunity against invasive intestinal bacterial diseases and suggest that a similar strategy could be used against diseases caused by other PC-bearing microorganisms.

Neuropeptides have been shown to play a critical role in adaptational processes, probably by long-term modulation of neuronal pathways. It could therefore be interesting to study behavioral changes induced by chronic local stimulation of neuropeptide receptors. With this aim poly(lactide-co-glycolide) microspheres loaded with a highly potent, peptidase-resistant, cholecystokinin (CCK)-B-selective CCK peptidomimetic agonist (pBC 264) were prepared by a water in oil in water emulsion solvent evaporation method and stereotaxically implanted into the anterior part of the rat nucleus accumbens. Two different kinds of loaded polymeric microspheres differing only by the stabilizing agent [ovalbumin (OVA) or Pluronic F 68] added to the inner emulsion were used. The histological and behavioral studies done 24 h and 8 days after implantation of nonloaded microspheres in the nucleus accumbens indicated that the microspheres were well tolerated. The in vivo release of the selective CCK-B agonist pBC 264 (associated with a tracer dose of [3H]pBC 264) from microspheres prepared with OVA was very fast (92% after 6 h), whereas only 26% (88 pmol) of pBC 264 was released from the formulation with Pluronic F 68 after 24 h. Eight days after implantation 36% of pBC 264 had diffused from the microspheres, and 8% (approximately 30 pmol) was still present in the brain concentrated around the site of administration. In all cases the released material was found to correspond to intact pBC 264, thus demonstrating the possibility of obtaining a slow controlled release of peptide in vivo. This method opens up interesting perspectives to study the long-term effects of neuropeptides.

Salcaprozate sodium (SNAC) and sodium caprate (C10) are two of the most advanced intestinal permeation enhancers (PEs) that have been tested in clinical trials for oral delivery of macromolecules. Their effects on intestinal epithelia were studied for over 30 years, yet there is still debate over their mechanisms of action. C10 acts via openings of epithelial tight junctions and/or membrane perturbation, while for decades SNAC was thought to increase passive transcellular permeation across small intestinal epithelia based on increased lipophilicity arising from non-covalent macromolecule complexation. More recently, an additional mechanism for SNAC associated with a pH-elevating, monomer-inducing, and pepsin-inhibiting effect in the stomach for oral delivery of semaglutide was advocated. Comparing the two surfactants, we found equivocal evidence for discrete mechanisms at the level of epithelial interactions in the small intestine, especially at the high doses used in vivo. Evidence that one agent is more efficacious compared to the other is not convincing, with tablets containing these PEs inducing single-digit highly variable increases in oral bioavailability of payloads in human trials, although this may be adequate for potent macromolecules. Regarding safety, SNAC has generally regarded as safe (GRAS) status and is Food and Drug Administration (FDA)-approved as a medical food (Eligen®-Vitamin B12, Emisphere, Roseland, NJ, USA), whereas C10 has a long history of use in man, and has food additive status. Evidence for co-absorption of microorganisms in the presence of either SNAC or C10 has not emerged from clinical trials to date, and long-term effects from repeat dosing beyond six months have yet to be assessed. Since there are no obvious scientific reasons to prefer SNAC over C10 in orally delivering a poorly permeable macromolecule, then formulation, manufacturing, and commercial considerations are the key drivers in decision-making. European Commission Horizon 2020 Science Foundation Ireland SANOFI-AVENTIS Recherche & Development French National Agency of Research and Technology CÚRAM Center for Medical Devices