An aquaporin 4 antisense oligonucleotide loaded, brain targeted nanoparticulate system design.

Aquaporins (AQPs), members of the water-channel protein family, are highly expressed in brain tissue especially in astrocytic end-feet. They are important players for water hemostasis during development of cytotoxic as well as vasogenic edema. Increased expression of AQPs is important in pathophysiology of neurological diseases such as neuroinflammation and ischemia. Unfortunately, there are a few pharmacological inhibitors of AQP4 with several side effects limiting their translation as a drug for use in clinical conditions. Another therapeutic approach is using antisense oligonucleotides (ASOs) to block AQP4 activity. These are short, synthetic, modified nucleic acids that bind RNA to modulate its function. However, they cannot pass the blood brain barrier (BBB). To overcome this obstacle we designed a nanoparticulate system made up of chitosan nanoparticles surface modified with PEG and conjugated with monoclonal anti transferrin receptor-1 antibody via streptavidin-biotin binding. The nanocarrier system could be targeted to the transferrin receptor-1 at the brain endothelial capillaries through monoclonal antibodies. It is hypothesized that the nanoparticles could pass the BBB via receptor mediated transcytosis and reach brain parenchyma. Particle size, zeta potential, loading capacity and release profiles of nanoparticles were investigated. It was observed that all types of chitosau (CS) nanoparticles had positive zeta potential values and nanoparticle particle size distribution varied between 100 and 800 nm. The association efficiency of ASOs into the nanoparticles was between 80-97% and the release profiles of the nanoparticles exhibited an initial burst effect followed by a controlled release. The results showed that the designed chitosan based nanocarriers could be a promising carrier system to transport nucleic acid based drugs to brain parenchyma.

Nanomedicine as a promising approach for the treatment and diagnosis of brain diseases: the example of Alzheimer's disease.

Targeting of the central nervous system (CNS) in order to treat disorders is actually challenging due to the necessity to cross the blood brain barrier (BBB). This review aims to show how nanomedicine can propose new approach for the treatment and the diagnosis of CNS diseases focusing on Alzheimer's disease (AD). AD is a neurodegenerative disorder prevalent in the senile population. It is characterized by severe neuronal loss and proliferation of plaques composed of ß-amyloid peptide (Aß) and Tau protein deposites. An imbalance between production and clearance leading to the aggregation of Aß peptides especially in neurotoxic forms, may be the initiating factor in AD. The absence of an effective therapeutic approach nowadays could be, in part, due to the bad knowledge of AD physiopathology and the lack of early diagnosis. Many drawbacks such as poor bioavailability or limited BBB arising of tested molecules in the current or new therapeutic strategies explain their failure but can be resolved by the use of nanotechnology. Examples of recently published works using nanoparticles for improving diagnosis and therapy of AD are presented. Ideal nanocarriers for this aim must be able to pass through the BBB and to interact with an AD marker as soluble extracellular Aß forms which are known as the most toxic ones. These first results, even if many ones were obtained in vitro, brought to light the potential of nanoparticles for this challenging issue.

(Poly-cyanoacrylate) nanomedicines for cancer and beyond: Lessons learned.

This « Magnum Opus ¼ emphasizes that serendipity is a corner stone in research. The paths of discovery and innovation often result from the interdisciplinarity of scientific areas that are a priori disconnected from each other. In the 1970s, fundamental discoveries in cell biology led to unexpected advances in galenic pharmacy with the emergence of nanotechnologies for the intracellular delivery of non diffusing molecules. As well, fluorescein-loaded polyacrylamide nanocapsules were shown to deliver this fluorescent agent precisely into cellular lysosomes which represented a seminal observation. However, due to the lack of biodegradability of this carrier polymer, this approach was still far from therapeutic application. The use of cyanoacrylates as surgical glue inspired us to use this material in the design of the first biodegradable nanoparticles for human use. Capable of transporting compounds with anti-tumor activity, these polyalkylcyanoacrylate nanoparticles demonstrated the unexpected property of overcoming multi-drug resistance. This discovery led to the development of a nanomedicine that has completed phase III clinical trials for the treatment of resistant hepatocarcinoma. Going beyond the state-of-the art, a step ahead in the nanomedicine field was the drug « squalenoylation ¼ technology, which represents a shift from the « physical ¼ to the « chemical ¼ encapsulation paradigm. The bioconjugation of anticancer and other drugs to squalene, a natural and biocompatible lipid, enabled a dramatic increase in drug payload, and eliminated the so-called « burst release ¼ of drug: Two major drawbacks commonly associated with drug nanoencapsulation. The drug « squalenoylation ¼ approach resulted in a generic nanomedicine platform with broad pharmacological applications.

Squalenoyl-gemcitabine/edelfosine nanoassemblies: Anticancer activity in pediatric cancer cells and pharmacokinetic profile in mice.

Despite the great advances accomplished in the treatment of pediatric cancers, recurrences and metastases still exacerbate prognosis in some aggressive solid tumors such as neuroblastoma and osteosarcoma. In view of the poor efficacy and toxicity of current chemotherapeutic treatments, we propose a single multitherapeutic nanotechnology-based strategy by co-assembling in the same nanodevice two amphiphilic antitumor agents: squalenoyl-gemcitabine and edelfosine. Homogeneous batches of nanoassemblies were easily formulated by the nanoprecipitation method. Their anticancer activity was tested in pediatric cancer cell lines and pharmacokinetic studies were performed in mice. In vitro assays revealed a synergistic effect when gemcitabine was co-administered with edelfosine. Squalenoyl-gemcitabine/edelfosine nanoassemblies were found to be capable of intracellular translocation in patient-derived metastatic pediatric osteosarcoma cells and showed a better antitumor profile than squalenoyl-gemcitabine nanoassemblies alone. The intravenous administration of this combinatorial nanomedicine in mice exhibited a controlled release behavior of gemcitabine and diminished edelfosine plasma peak concentrations. These findings make it a suitable pre-clinical candidate for childhood cancer therapy.

Vitamin C-squalene bioconjugate promotes epidermal thickening and collagen production in human skin.

Vitamin C (Vit C) benefits to human skin physiology notably by stimulating the biosynthesis of collagen. The main cutaneous collagens are types I and III, which are less synthesized with aging. Vit C is one of the main promotors of collagen formation but it poorly bypasses the epidermis stratum corneum barrier. To address this challenge, we developed a lipophilic version of Vit C for improving skin diffusion and delivery. Vit C was covalently conjugated to squalene (SQ), a natural lipid of the skin, forming a novel Vit C-SQ derivative suitable for cream formulation. Its biological activity was investigated on human whole skin explants in an ex vivo model, through histology and protein and gene expression analyses. Results were compared to Vit C coupled to the reference lipophilic compound palmitic acid, (Vit C-Palmitate). It was observed that Vit C-SQ significantly increased epidermal thickness and preferentially favored collagen III production in human skin after application for 10 days. It also promoted glycosaminoglycans production in a higher extent comparatively to Vit C-Palmitate and free Vit C. Microdissection of the explants to separate dermis and epidermis allowed to measure higher transcriptional effects either in epidermis or in dermis. Among the formulations studied, the strongest effects were observed with Vit C-SQ.

Preparation and evaluation of alpha-phenyl-n-tert-butyl nitrone (PBN)-encapsulated chitosan and PEGylated chitosan nanoparticles.

Alpha-phenyl-n-tert-butyl nitrone (PBN) shows its major effect by scavenging free radicals formed in the ischemia and it has the ability to penetrate through the blood brain barrier easily. The in vivo stability of PBN is very low and when administered systemically, it has a mean plasma half life of about three hours. Therefore, formulations which are able to prolong the plasma residence time of PBN are of major interest, because oxygen radicals are usually continuously formed under pathological conditions. In this study, PBN, a nitrone compound having neuroprotective properties, was encapsulated in chitosan (CS) and chitosan-poly(ethylene glycol) (CS-PEG) nanoparticles for treatment of diseases such as stroke, in which sustained free radical production is reported. The nanoparticles were characterized through particle size determination, zeta potential, encapsulation efficiency, surface morphology determinations and in vitro release studies. The surface morphologies were evaluated by transmission electron microscopy (TEM) and nanoparticles having spherical shapes were characterized. The particle size distribution was between approximately 97 nm and approximately 322 nm; and the zeta potentials varied between approximately 9 mV and approximately 33 mV. Size of the nanoparticle formulations was important for the release of PBN from nanoparticles. The quantitative determination of PBN has been evaluated by a validated analytical HPLC method. The presented chitosan-based nanotechnology opens new perspectives for testing antioxidant activity in vivo.

[Tick bite fever in West Africa: difficulties in diagnosis of an emerging disease]. / La fièvre récurrente à tique d'Afrique de l'Ouest: difficultés diagnostiques d'une maladie émergente.

West African tick bite fever is a prevalent emerging zoonosis from the coast of Senegal to Chad. It is characterized by recurrent fever in association with a deteriorating clinical state. It is now the second most common vector-borne disease in Senegal. The purpose of this report is to describe one case and to review the main clinical and epidemiological features of this disease.

A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine.

Among anticancer nanomedicines, squalenoyl nanocomposites have obtained encouraging outcomes in a great variety of tumors. The prodrug squalenoyl-gemcitabine has been chosen in this study to construct a novel multidrug nanosystem in combination with edelfosine, an alkyl-lysophopholipid with proven anticancer activity. Given their amphiphilic nature, it was hypothesized that both anticancer compounds, with complementary molecular targets, could lead to the formation of a new multitherapy nanomedicine. Nanoassemblies were formulated by the nanoprecipitation method and characterized by dynamic light scattering, transmission electron microscopy and X-ray photoelectron spectroscopy. Because free edelfosine is highly hemolytic, hemolysis experiments were performed using human blood erythrocytes and nanoassemblies efficacy was evaluated in a patient-derived metastatic pediatric osteosarcoma cell line. It was observed that these molecules spontaneously self-assembled as stable and monodisperse nanoassemblies of 51 ±â€¯1 nm in a surfactant/polymer free-aqueous suspension. Compared to squalenoyl-gemcitabine nanoassemblies, the combination of squalenoyl-gemcitabine with edelfosine resulted in smaller particle size and a new supramolecular conformation, with higher stability and drug content, and ameliorated antitumor profile.

Novel self-assembling nanogels: stability and lyophilisation studies.

The stability of new supramolecular nanoassemblies (nanogels), based on the association of a hydrophobically modified dextran (MD) and a beta-cyclodextrin polymer (pbetaCD), has been studied by two complementary methods: (i) size measurements and (ii) turbidity experiments using a Turbiscan optical analyser. Nanogels of about 120-150nm were obtained whatever the concentration of the two polymer solutions. At low concentrations, the suspensions presented little mean diameter variations upon storage. However, the concentrated ones tended to destabilize and their mean diameter increased upon time. Size measurements and Turbiscan investigations have demonstrated that destabilization in the MD-pbetaCD nanogel suspension was only due to particle aggregation and/or fusion, as no sedimentation or creaming occurred. The destabilization of MD-pbetaCD suspensions led to the formation of a highly viscous phase, as a final state. Moreover, the two methods have shown that aggregation and/or fusion phenomena were more pronounced in the concentrated MD-pbetaCD suspensions than in the diluted ones. The stability of MD-pbetaCD suspensions could be improved by their storage at 4 degrees C. Finally, freeze-drying was found to be a convenient method for the long-time storage of MD-pbetaCD nanoassemblies.

Encapsulation of mono- and oligo-nucleotides into aqueous-core nanocapsules in presence of various water-soluble polymers.

In a previous study, we have shown that cidofovir (CDV) and azidothymidine-triphosphate (AZT-TP) were poorly encapsulated in poly(iso-butylcyanoacrylate) (PIBCA) aqueous-core nanocapsules. This was attributed to the rapid leakage of these small and hydrophilic molecules through the thin polymer wall of the nanocapsules. In the present study, we have selected various water-soluble polymers as increasing Mw adjuvants and investigated their influence on the entrapment of mononucleotides (CDV, AZT-TP) as well as of oligonucleotides (ODN) into these PIBCA aqueous-core nanocapsules. We show here that the presence of cationic polymers (i.e. poly(ethyleneimine) (PEI) or chitosan) in the nanocapsule aqueous compartment allowed successful encapsulation of AZT-TP and ODN.

Liposomal formulation of a glycerolipidic prodrug for lymphatic delivery of didanosine via oral route.

Didanosine is a polar drug with poor membrane absorption and high hepatic first pass metabolism. This study aimed at developing a lipidic formulation of a glycerolipidic prodrug of didanosine in order to improve its bioavailability. In the course of a preformulation study, the glycerolipidic prodrug of didanosine was characterized by microscopy, DSC and XRDT. In anhydrous conditions, the prodrug displayed a polymorphic behaviour similar to that of triglycerides. Then, we evaluated three types of lipidic formulations (emulsions, mixed micelles and liposomes) in order to encapsulate the prodrug. Solubilities in water - even in the presence of taurocholate micelles - but also in some oils were very low (max 244 microg/mL) as the prodrug was found to be amphiphilic (log P=2). On the contrary, the prodrug was found to be perfectly incorporated in dipalmitoylphosphatidylcholine (DPPC) multilamellar liposomes up to a ratio of 1:5 (mol:mol) prodrug:DPPC as suggested by HPLC-UV and DSC experiments. Moreover, these liposomes could be freeze-dried whereas the chemical integrity of the prodrug was preserved. Then, the freeze-dried liposomal preparation could be formulated as gastro-resistant capsules to prevent didanosine from acidic degradation. Further experiments are on the way to evaluate in vitro the absorption of prodrug incorporated in liposomes by enterocytes.

Spontaneous association of hydrophobized dextran and poly-beta-cyclodextrin into nanoassemblies. Formation and interaction with a hydrophobic drug.

New nanoassemblies were instantaneously prepared by mixing two aqueous solutions, one containing a beta-cyclodextrin polymer (pbetaCD), and the other a hydrophobically modified by alkyl chains dextran (MD). The formation mechanism and the inner structure of these nanoassemblies were analysed using surface tension measurements and (1)H NMR spectroscopy. The effect of a hydrophobic guest molecule, such as benzophenone (BZ), on the formation and stability of the nanoassemblies was also evaluated. MD exhibited the typical behaviour of a soluble amphiphilic molecule and adsorbed at the air/water interface. Whereas the injection of native beta-CDs in the solution beneath the adsorbed MD monolayer did not produce any change in the surface tension, that of the pbetaCD resulted in an increase in the surface tension, indicating the desorption of the polymer from the interface. This result accounts for a cooperative effect of beta-CDs linked together in the pbetaCD polymer on dextran desorption. The presence of benzophenone in the system hindered the sequestration of dextran alkyl moieties by beta-CD in the polymer without impeding the formation of associative nanoassemblies of 100-200 nm. (1)H NMR investigations demonstrated that, in the BZ-loaded nanoassemblies, the hydrophobic molecule was mainly located into the cyclodextrin cavities.

Towards improved HIV-microbicide activity through the co-encapsulation of NRTI drugs in biocompatible metal organic framework nanocarriers.

The efficacy of the routinely used anti-HIV (Human Immunodeficiency Virus) therapy based on nucleoside reverse transcriptase inhibitors (NRTIs) is limited by the poor cellular uptake of the active triphosphorylated metabolites and the low efficiency of intracellular phosphorylation of their prodrugs. Nanoparticles of iron(iii) polycarboxylate Metal-Organic Frameworks (nanoMOFs) are promising drug nanocarriers. In this study, two active triphosphorylated NRTIs, azidothymidine triphosphate (AZT-Tp) and lamivudine triphosphate (3TC-Tp), were successfully co-encapsulated into the biocompatible mesoporous iron(iii) trimesate MIL-100(Fe) nanoMOF in order to improve anti-HIV therapies. The drug loaded nanoMOFs could be stored for up to 2-months and reconstituted after freeze drying, retaining similar physicochemical properties. Their antiretroviral activity was evidenced in vitro on monocyte-derived macrophages experimentally infected with HIV, making these co-encapsulated nanosystems excellent HIV-microbicide candidates.

Novel composite core-shell nanoparticles as busulfan carriers.

This study presents a method for the design of novel composite core-shell nanoparticles able to encapsulate busulfan, a crystalline drug. They were obtained by co-precipitation of mixtures of poly(isobutylcyanoacrylate) (PIBCA) and of a diblock copolymer, poly(epsilon-caprolactone)-poly(ethylene glycol) (PCL-PEG), in different mass ratios. The nanoparticle size, morphology and surface charge were assessed. The chemical composition of the top layers was determined by X-ray photo-electron spectroscopy (XPS). (3)H-labelled busulfan was used in order to determine the drug loading efficiency and the in vitro drug release by liquid scintillation counting. Physico-chemical techniques such as Zeta potential determination and XPS analysis provided evidence about a preferential surface distribution of the PCL-PEG polymer. Therefore, composite nanoparticles have a "core-shell"-type structure, where the "core" is essentially formed by the PIBCA polymer and the "shell" by the PCL-PEG copolymer. The use of PIBCA to form the core of the nanoparticles leads to a 2-4 fold drug loading increase, in comparison to the single PCL-PEG nanoparticles. In addition, the complement activation results showed a significant difference between the composite nanoparticles and the single PIBCA nanoparticles, thus demonstrating that PEG at the surface of the nanoparticles reduced the complement consumption. The PIBCA:PCL-PEG composite nanoparticles prepared using the new co-precipitation method here described represent an original approach for busulfan administration.

Innovative nanotechnologies for the delivery of oligonucleotides and siRNA.

One way to reach intracellular therapeutic targets in cells consists in the use of short nucleic acids which will bind specifically to on targets thanks to either Watson-Crick base pairing or protein nucleic acids recognition rules. Among these short nucleic acids an important class of therapeutic agents is antisense oligonucleotides and siRNAs. However, the major problem of nucleic acids is their poor stability in biological media. One method, among others, to solve the stability problem is the use of colloïdal carriers such as nanoparticles. Nanoparticles have already been applied with success to in vitro drug delivery to particular types of cells and in vivo in several experimental models. Many membrane and intracellular processes deal with nanosized structure (typically 100 nm) which are processed further through the recognition sites of receptors and enzymes. Thus non-viral nanoparticles are interesting candidates to present biochemical molecules such as nucleic acids and proteins to cells as well as to protect them in vivo during delivery. This review focuses on the recent developments in the design of nanotechnologies to improve the delivery of antisense oligonucleotides and siRNAs.

Encapsulation of antiviral nucleotide analogues azidothymidine-triphosphate and cidofovir in poly(iso-butylcyanoacrylate) nanocapsules.

Nucleoside analogues are widely used in the treatment of various viral infections. However, the poor in vivo conversion of the nucleoside analogues like azidothymidine (AZT) into their active triphosphate nucleotide counterpart limits their pharmacological efficacy. This could be overcome by the direct administration of azidothymidine triphosphate (AZT-TP), but it requires an appropriate drug delivery approach. Besides nucleoside analogues, nucleotide analogues like cidofovir (CDV) are also used in the treatment of viral infections. CDV has raised recent interest because of its promising activity against smallpox, but its use is limited by its poor bioavailability and nephrotoxicity. Here again, a proper drug delivery system should address these issues. In this study, we investigated the encapsulation of the nucleotide analogues AZT-TP and CDV into poly(iso-butylcyanoacrylate) aqueous core nanocapsules, known to efficiently entrap oligonucleotides. We show here that the encapsulation of these mono-nucleotides is less efficient than with oligonucleotides and that a rapid release of AZT-TP from the nanocapsules occurred in vitro. This highlights the importance of the molecular weight of the entrapped molecules which, if they are too small, are diffusing through the thin polymer membrane of the nanocapsules. On the other hand, a good protection of the encapsulated AZT-TP was observed.

Antineoplastic busulfan encapsulated in a metal organic framework nanocarrier: first in vivo results.

Nanoparticles of a mesoporous iron(iii) trimesate MIL-100 nanocarrier encapsulating high amounts of the challenging antineoplastic busulfan were administered to rats and compared with the commercial Busilvex®. Large differences in serum concentration of both busulfan and trimesate revealed the great impact of drug encapsulation both on the drug and on nanoparticle pharmacokinetics during the first 24 h of administration.

In vivo behavior of MIL-100 nanoparticles at early times after intravenous administration.

Metal-organic frameworks have shown interesting features for biomedical applications, such as drug delivery and imaging agents. The benchmarked mesoporous iron(III) trimesate MIL-100 MOF nanocarrier combines progressive release of high drug cargoes with absence of visible in vivo toxicity. Although in a previous study pharmacokinetics and biodistribution of MIL-100 nanoparticles were evaluated in the long term (from 24h to 1 month), the crucial times for drug targeting and delivery applications are shorter (up to 24h). Thus, this work aims to study the blood circulating profile and organ accumulation of MIL-100 nanocarrier at early times after administration. For this purpose, after intravenous administration to rats, both constitutive components of MIL-100 (trimesate and iron) were quantified by high performance liquid chromatography and a spectrophotometric method, respectively. The pharmacokinetic profile suggested that the nanoparticles act as a depot in the blood stream during the first hours before being cleared. Accumulation took mainly place in the liver and, in some extent, in the spleen. Nevertheless, histological studies demonstrated the absence of morphological alterations due to the presence of the particles in these organs. Liver function was however slightly altered as reflected by the increased plasma aspartate aminotransferase concentrations. Finally trimesate was progressively eliminated in urine.

Retrovirus budding may constitute a port of entry for drug carriers.

This paper investigates the relation between viral infection and cell uptake of liposomes and nanoparticles. A defective virus was used to infect two types of cells: cells allowing virus budding (psi2neo cells) and cells bereft of a virus exit process (NIH 3T3 cells). This study has revealed that cell uptake of pH-sensitive-liposomes is highly dependent on the virus exit process, since it ensued only when virus budding occurred. This preferential uptake of pH-sensitive liposomes by infected cells was not carrier-specific because similar uptake was observed with non-biodegradable fluorescent nanoparticles using confocal microscopy. Also, inhibition of neo gene expression by oligonucleotide pH-sensitive-liposomes was only observed in the cell system (psi2neo) endowed with a virus exit process. Finally, increased membrane fluidity was noted in the infected cells, possibly reflecting membrane perturbation due to virus budding. We suggest that this membrane perturbation may be the key to the uptake of the different colloidal carriers. Infected cells could, thus, constitute a natural target for particulate drug carriers.

pH-sensitive liposomes as a carrier for oligonucleotides: a physico-chemical study of the interaction between DOPE and a 15-mer oligonucleotide in quasi-anhydrous samples.

pH-sensitive liposomes made of dioleoylphosphatidylethanolamine (DOPE)/oleic acid (OA)/cholesterol (CHOL) mixtures were shown to be very promising carriers for oligonucleotides (ON). However, it appeared necessary to clarify the structural consequence of the interactions of ON with the liposome, and especially on DOPE, the lipid responsible for the pH sensitivity. The present study was carried out by differential scanning calorimetry and X-ray diffraction, at low hydration. In such a case, DOPE generally adopt a hexagonal phase. It could be shown that ON increased DOPE transition temperature and increased v/al, as a result of electrostatic interactions between ON and DOPE headgroups. OA was found to have exactly opposite effects, its presence between DOPE molecules inhibiting the formation of hydrogen bonds. The presence of both ON and OA allowed the system to organize in a lamellar phase below the solid/liquid transition, whereas above this temperature ON preferably interacted with DOPE in a hexagonal phase and led OA to separate.