LR12-peptide quantitation in whole blood by RP-HPLC and intrinsic fluorescence detection: Validation and pharmacokinetic study.

A simple, sensitive, selective and robust HPLC method based on intrinsic fluorescence detection was developed for the quantitation of a dodecapeptide (designated as LR12), inhibitor of Triggering Receptor Expressed on Myeloid cells-1, in rat whole blood. Sample treatment was optimized using protein precipitation and solid-phase extraction. Chromatographic separation was carried out in a gradient mode using a core-shell C18 column (150 × 4.6 mm, 3.6 µm) with mobile phases of acetonitrile and water containing trifluoroacetic acid at 1.0 mL/min. The method was validated using methodology described by the US Food and Drug Administration guidelines for bioanalytical methods. Linearity was demonstrated within the 50-500 ng/mL range and the lower limit of quantitation was 50 ng/mL. Finally, a preliminary pharmacokinetic study after intraperitoneal injection of LR12 in rats was conducted to evaluate both LR12 monomer and its corresponding disulfide dimer, the main product of degradation. Beyond the fact that this paper describes the first fully validated method for LR12 analysis in blood samples, the approach followed here to optimize pre-analytical steps could be beneficial to develop HPLC and/or MS methods for other pharmaceutical peptides.

Elaboration of Sterically Stabilized Liposomes for S-Nitrosoglutathione Targeting to Macrophages.

S-nitrosoglutathione (GSNO) is a potential therapeutic for infectious disease treatment because of its pivotal role in macrophage-mediated inflammatory responses and host defense in addition to direct antibacterial activities. In this study, sterically stabilized cationic liposomes (SSCL) and sterically stabilized anionic liposomes (SSAL) were developed as nanocarriers for macrophage targeting. Elaborated liposomes were characterized in terms of size, zeta potential, morphology, encapsulation efficiency, in vitro drug release behavior and cytotoxicity. Their versatility in targeting monocytes/macrophages was determined by confocal laser scanning microscopy and transmission electron microscopy. Flow cytometry revealed that cellular uptake of both SSCL and SSAL was governed by several endocytic clathrin- and caveolae-dependent mechanisms. Quantitative assessments of intracellular nitric oxide demonstrated highly efficient uptake of GSNO-loaded SSCL that was twenty-fold higher than that of GSNO-free molecules. GSNO-loaded SSCL displayed strong bacteriostatic effects on Staphylococcus aureus and Pseudomonas aeruginosa, which can be involved in pulmonary infectious diseases. These results reveal the potential of liposomal GSNO as an anti-infective therapeutic due to its macrophage targeting capacity and direct antibacterial effects.

Synthesis and in vitro release study of ibuprofen-loaded gelatin graft copolymer nanoparticles.

OBJECTIVE: This work deals with the preparation, characterization and in vitro release study of IBU-loaded gel graft copolymer nanoparticles. METHOD: Gelatin (Gel) graft copolymer nanoparticles were prepared using styrene (Sty) and/or 2-hydroxyethyl methacrylate (HEMA) monomers in the presence of potassium persulfate and glutaraldehyde as an initiator and cross-linker, respectively. The prepared nanoparticles as sustained release drug carriers were investigated using the nonsteriodal anti-inflammatory model drug, ibuprofen (IBU). RESULTS: The prepared nanoparticles as sustained release drug carriers were investigated using the nonsteriodal anti-inflammatory model drug, IBU. The prepared Gel/HEMA and Gel/Sty nanoparticles exhibited particles size ranging from 15 to 17 nm and from 0.42 to 5 mm, respectively. The dissolution of IBU in phosphate buffer, pH 7.4, at 37°C from the prepared nanoparticles was evaluated using UV spectroscopy. In addition, the prepared nanoparticles were characterized using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), transmitting electron microscope (TEM) and zeta potential/particle size analyzer. In vitro dissolution study showed that the dissolution rates of the crosslinked nanoparticles were retarded relative to the uncrosslinked ones. Moreover, the released amount constantly decreases with increasing gluteraldehyde content in the gel nanoparticles. CONCLUSION: Crosslinked gel-based graft copolymers exhibited slow IBU release within six hours. Furthermore, results from different characterization techniques such as TEM, particles size and zeta potential measurements confirmed the formation of pH-responsive gel-graft copolymer nanoparticles.

[Which future in cardiovascular therapy for nitric oxide and its derivatives?]. / Quel avenir en thérapeutique cardiovasculaire pour le monoxyde d'azote et ses dérivés?

Nitric oxide (NO) is involved in the regulation of several physiological processes such as vascular homeostasis. Exogenous NO supply offers major therapeutic interest, especially in the treatment of coronary artery disease, ischemic syndromes and other cardiovascular pathologies. Nevertheless, the administration of NO itself is limited by its short half-life. NO prodrugs have been marketed for decades, e.g. organic nitrates for angina pectoris. These prodrugs display undeniable advantages such as angina crisis relief and preconditioning effect. Nevertheless, they suffer from several drawbacks: toxicity, tolerance, endothelial dysfunction exacerbation. These negative effects are related to massive production of reactive species derived from oxygen or nitrogen, which trigger oxidative and nitrosative stress. New NO donors are under development to overcome those disadvantages, among which the S-nitrosothiols family seems especially promising.

Injectable PLA-based in situ forming implants for controlled release of Ivermectin a BCS Class II drug: solvent selection based on physico-chemical characterization.

In situ forming implants (ISI) prepared from biodegradable polymers such as poly(D,L-lactide) (PLA) and biocompatible solvents can be used to obtain sustained drug release after parenteral administration. The aim of this work was to study the effect of several biocompatible solvents with different physico-chemical properties on the release of ivermectin (IVM), an antiparasitic BCS II drug, from in situ forming PLA-based implants. The solvents evaluated were N-methyl-2-pyrrolidone (NMP), 2-pyrrolidone (2P), triacetine (TA) and benzyl benzoate (BB). Hansen's solubility parameters of solvents were used to explain polymer/solvent interactions leading to different rheological behaviours. The stability of the polymer and drug in the solvents were also evaluated by size exclusion and high performance liquid chromatography, respectively. The two major factors determining the rate of IVM release from ISI were miscibility of the solvent with water and the viscosity of the polymer solutions. In general, the release rate increased with increasing water miscibility of the solvent and decreasing viscosity in the following order NMP>2P>TA>BB. Scanning electron microscopy revealed a relationship between the rate of IVM release and the surface porosity of the implants, release being higher as implant porosity increased. Finally, drug and polymer stability in the solvents followed the same trends, increasing when polymer-solvent affinities and water content in solvents decreased. IVM degradation was accelerated by the acid environment generated by the degradation of the polymer but the drug did not affect PLA stability.

Impact of gold nanoparticle coating on redox homeostasis.

Gold nanoparticles (AuNP) hold great potential for biomedical applications. This study was aimed at examination of the effect of AuNP coating on the redox status of their environment. Two kinds of AuNP were tested, similar by shape and size, but with different surface coatings: either stabilized with citrate or functionalized with dihydrolipoic acid (Au@DHLA NP). Interestingly, whereas citrate-stabilized AuNP interact in vitro with reduced glutathione (GSH) and S-nitrosoglutathione, Au@DHLA NP do not interfere with both biomolecules. Albumin exhibits higher affinity toward citrate-stabilized AuNP than Au@DHLA NP, increasing their hydrodynamic diameter (8.0- and 1.3-fold, respectively). Furthermore, the AuNP coating affects also their internalization by macrophages (which was two fold higher for citrate-stabilized AuNP), following an exposure to a subtoxic NP concentration (10 nM, 80% viability). Citrate-stabilized AuNP were found to decrease the intracellular GSH level (ca. 20%), with no increase in reactive oxygen species production. Furthermore, these AuNP did not induce apoptosis (as shown by caspase-3 activity and nfkb2 transcription factor), and also did not activate gene expression related to oxidative stress (ncf1) and inflammatory response (tnfα). The present data highlight that the functionalization of AuNP with DHLA decreases their reactivity with biomolecules and cells, resulting in a promising medical platform.

Evaluation of subcutaneous forms in the improvement of pharmacokinetic profile of warfarin.

We attempted to prepare a subcutaneous pharmaceutical form of warfarin based on a suspension or poly(ε-caprolactone) microparticles to improve patient adherence. The warfarin suspension had a mean particle size of 20.0 µm and in vitro release close to 100% in 72 h. Microparticle size and encapsulation efficiencies ranged from 54.0 to 80.0 µm and 37.0 to 47.0%, respectively. After 72 h, warfarin microparticles exhibited in vitro drug release ranging from 62.0 to 80.0%. Warfarin subcutaneous dosage forms were administered to rabbits. Plasma concentration of warfarin was determined and biological activity was measured by prothrombin time monitoring. The observed relative bioavailabilities calculated from plasma concentrations and prothrombin times were 54.2 and 92.1%, and 61.8 and 61.4% for suspension and microparticles, respectively.

Low molecular weight heparin gels, based on nanoparticles, for topical delivery.

A commercial suspension of nanoparticles (Eudragit RS 30D) was used to manufacture a gel for topical application. Gels were prepared by mixing a polycationic polymer (Eudragit(®) RS 30D) and a low molecular weight heparin (LMWH), an antithrombotic agent. Gels formed spontaneously at a ratio of 1:1 as a result of electrostatic interactions between the polyanionic drug and the polycationic polymer. Different types of heparin were used: Bemiparin, Enoxaparin (Lovenox), Nadroparin (Fraxiparin) and Tinzaparin (Innohep). Several LMWH concentrations were tested. Rheological measurements were performed to investigate the gel behavior. Gel formation was confirmed by dynamic rheological measurements as the elastic modulus (G') was higher than the viscous one (G″). The amount of heparin incorporated into the gel matrix was determined. A maximum of incorporation (100%) was reached using a heparin solution of 600 IU/mL. The release kinetics of LMWH from the gel were also studied. Regardless of the LMWH used in the formulation, a biphasic release profile was observed. Accordingly, a burst effect was observed. Afterwards, the release rate became steady. The penetration of the LMWH through the dermal barrier was also investigated.

Drug delivery by polymeric nanoparticles induces autophagy in macrophages.

Drug delivery nanosystems are currently used in human therapy. In preliminary studies we have observed that Eudragit RS nanoparticles, prepared by nanoprecipitation or double emulsion techniques, are cytotoxic for NR8383 rat macrophages. In this study, we expand our previous analysis and suggest that unloaded Eudragit RS nanoparticles prepared by nanoprecipitation (NP/ERS) may induce important morphological and biochemical cellular modifications leading to cellular death. In NR8383 rat macrophages cell line exposed to doses varying from 15 to 100 µg/mL, NP/ERS nanoparticles are internalized inside the cells, reach the mitochondria and alter the structure of these organelles. In addition, the exposure to nanoparticles induces cellular autophagy as demonstrated by electron microscopy analysis, microchip array, qRT-PCR and Western blot assays. Although toxicity of nanoparticles has already been evidenced, it is the first time that results show clearly that the toxicity of polymeric nanovectors may be related to an activation of autophagy.

Production and characterization of testosterone undecanoate-loaded NLC for oral bioavailability enhancement.

PURPOSE: Nanostructured lipid carriers were loaded with testosterone undecanoate (TU), which has a low oral bioavailability. METHODS: Different NLC dispersions were produced using the hot high pressure homogenization method. Particles were characterized using dynamic and static light scattering techniques, differential scanning calorimetry and X-ray diffraction. And the bioavailability was compared to a marketed product. RESULTS: Nanostructured lipid carriers with up to 30% TU load and sizes of about 600 and 200 nm could be achieved, allowing a direct comparison of the size effect in in vivo bioavailability studies. The zeta potentials varied between - 20 and - 40 mV. The bioavailability of Andriol Testocaps® in the fed state was matched. CONCLUSIONS: This opens the perspective of administering a single dose of dose of TU in one oral dosage unit and simultaneously having a bioavailability less dependent on the fed state.

Photochemical internalization for pDNA transfection: evaluation of poly(d,l-lactide-co-glycolide) and poly(ethylenimine) nanoparticles.

The main objective of this study was to prepare two types of nanoparticles with poly(d,l-lactide-co-glycolide) (PLGA) and polyethylenimine (PEI) polymers. Plasmid DNA (pDNA) was adsorbed either on PLGA/PEI nanoparticles, or as PEI/DNA complex onto the surface of PLGA nanoparticles. Both types of nanoparticles were prepared by the double emulsion method. The nanoparticles were characterized by their size, zeta potential and pDNA or PEI/DNA complex adsorption. The PEI/DNA complex adsorption was confirmed with ethidium bromide assay. pDNA adsorption onto PLGA/PEI nanoparticles (PLGA/PEI-DNA) was studied by electrophoresis on agarose gel. Cytotoxicity and transfection efficiency of both types of nanoparticle and PEI/DNA complexes formulations were studied in head and neck squamous carcinoma cell line (FaDu). To improve endosomal release, photochemical internalization (PCI) was used. The zeta potential increased when the PEI/DNA complex adsorbed onto PLGA nanoparticles (PLGA-PEI/DNA). Optimal pDNA adsorption efficiency was achieved for nitrogen/phosphorous ratio≥20/1. In vitro transfection and cells viability on FaDu cells with or without PCI were found to be variable depending on the type and concentration of nanoparticles. The results showed that transfection efficiency for PLGA/PEI-DNA or PLGA-PEI/DNA nanoparticles ranged between 2 and 80%, respectively. PCI was found to slightly improve the transfection efficiency for all formulations.

Ivermectin-loaded microparticles for parenteral sustained release: in vitro characterization and effect of some formulation variables.

Ivermectin (IVM) is a BCS II drug with potent antiparasitic activity in veterinary applications. In this study, poly(lactide-co-glycolide) (PLGA) and poly(DL-lactide) (PLA) Ivermectin-loaded microparticles were prepared by the simple emulsion (O/W) solvent evaporation method in order to obtain sustained release formulations for parenteral applications. The effects of polymer end-groups (ester or free acid) and the addition of the hydrophilic polyvinylpyrrolidone polymer (PVP) in in vitro drug release profiles were also studied. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) analysis showed that IVM was present in an amorphous state or as a molecular dispersion within the polymers or theirs mixtures with PVP and that a PVP-drug complex was formed. Drug entrapment efficiency in the microparticles (>90%) was independent of the polymer composition, the end groups and the presence of PVP. However, microscopic (SEM) observations showed that the addition of PVP led to more porous microparticles accompanied by the increased rates of drug release.

Cytotoxicity assessment of heparin nanoparticles in NR8383 macrophages.

The bioavailability of low molecular weight heparin (LMWH) has been increased by encapsulation in nanoparticles. As a complement to these results, the cytotoxicity and apoptosis induced by LMWH nanoparticles prepared by two methods [nanoprecipitation (NP) and double emulsion (DE)] using Eudragit RS (RS) and poly-epsilon-caprolactone (PCL) have been analysed. Particle sizes varied from 54 to 400nm with zeta potential values between -65 and +63mV. Our results showed that the method of nanoparticle preparation affects their properties, especially in terms of drug incorporation and cell tolerance. Cell viability ranged from 6% to 100% depending on the preparation method and physicochemical properties of the particles and the type of toxicity assay. Particle diameter and zeta potential seemed to be the most valuable cytotoxicity markers when cell viability was measured by Trypan blue exclusion and MTT respectively. Nanoparticles prepared by DE were better tolerated than those of NP. LMWH encapsulation into the cationic nanoparticles reduces remarkably their toxicity. Apoptosis evaluation showed activated caspases in exposed cells. However, no nuclear fragmentation was detected in NR8383 cells whatever the tested nanoparticles. DE nanoparticles of RS and PCL can be proposed as a good LMWH delivery system due to their low toxicity (IC(50) approximately 2.33 and 0.96mg/mL, respectively).

Optimization of a new non-viral vector for transfection: Eudragit nanoparticles for the delivery of a DNA plasmid.

The development of new vectors to deliver DNA into cells for therapy of cancers or genetic diseases has been a major area of research for many years. However, the clinical application of this technology requires the development of efficient, reliable and sterile vectors enabling the transfer of genes in vivo. Non viral, polymer or lipid-based vectors offer a new impetus to gene therapy because they are less toxic than viral vectors (no endogenous recombination, fewer immunological reactions, easy production and delivery of large-sized plasmid). The aim of this study is to develop a new tool for DNA delivery composed of methacrylic polymeric (Eudragit RS and RL) nanoparticles. These nanoparticles were prepared by two methods: nanoprecipitation and double emulsion. The nanoparticles were characterized by their size, zeta potential and amount of DNA adsorption. Cytotoxicity tests based on mitochondrial activity (MTT test) revealed that the nanoparticles had limited cytotoxicity and that this depended on both the cell type and the nanoparticle concentration. Transgene expression was observed using the Green Fluorescence Protein gene as reporter gene, and was evaluated by flow cytometry in FaDu, MDA-MB 231 and MCF-7 cell lines. The results showed that transfection rates ranging between 4 and 7% were achieved in FaDu and MDA-MB 231 cells with nanoparticles prepared by the nanoprecipitation method. In MCF-7 cells transfected with nanoparticles prepared by either the double emulsion or the nanoprecipitation method, the transfection efficiency was between 2 and 4%. Nanoparticles prepared by nanoprecipitation were slightly more efficient than nanoparticles prepared from a double emulsion. Particle size was not an important factor for transfection, since no significant difference was observed with size between 50 and 350 nm. We showed that Eudragit RS and RL nanoparticles could introduce the transgene into different types of cells, but were generally less effective than the lipofectamine control.

Influence of polymers ratio on insulin-loaded nanoparticles based on poly-epsilon-caprolactone and Eudragit RS for oral administration.

Nanoparticles loaded with two different commercial insulins (Actrapid, Novorapid and based on different blends of a biodegradable polyester (poly-epsilon-caprolactone) and a polycationic non-biodegradable acrylic polymer (Eudragit RS) were characterized in vitro. The zeta potential was positive whenever Eudragit RS was part of the nanoparticles matrix. The encapsulation efficiency was ~ 96% except for Novorapid-loaded particles of poly-epsilon-caprolactone (only 35%). In vitro release studies revealed a burst release from nanoparticles, which may be of interest for oral delivery. Novorapid-loaded nanoparticles were orally administered to diabetic rats and allowed the glycemia to be decreased when compared with free nanoparticles.

Omega-3 fatty acids-loaded lipid nanoparticles for patient-convenient oral bioavailability enhancement.

Omega-3 fatty acids are commonly used as food supplements not only for their positive effects on the blood lipid profile but also for their cardioprotective properties. The majority of the commercially available products is made out of fish oil. Apart from the unpleasant side effects, up to 10 capsules per day have to be taken by the patients. This article describes the development and characterisation of an alternative lipid nanoparticle delivery system, which has the potential to reduce side effects and enhance bioavailability.

Composite microparticles with in vivo reduction of the burst release effect.

The aim of this study was to develop microparticles containing nanoparticles (composite microparticles) for prolonged drug delivery with reduced burst effect in vitro and in vivo. Such composite microparticles were prepared with hydrophobic and biodegradable polymers [poly(epsilon-caprolactone), poly(lactic-co-glycolic) acid]. Ibuprofen was chosen as the model drug, and microparticles were prepared by the extraction technique with ethyl acetate as the solvent. Nanoparticles and microparticles and an ibuprofen solution (Pedea) were administered subcutaneously at the dose of 1 mg of ibuprofen per kg to overnight-fasted rats (male Wistar). Composite microparticles showed prolonged ibuprofen release and less burst effect when compared to simple microparticles (without nanoparticles inside) or nanoparticles both in vitro (PBS buffer) and in vivo. Moreover, ibuprofen was still detected in the plasma after 96 h with composite microparticles. Consequently, it has been demonstrated that composite microparticles were able to reduce burst release and prolong the release of ibuprofen for a long period of time.

Stealth nanoparticles coated with heparin as peptide or protein carriers.

Nanoparticles (prepared from a mixture of polyester and a polycationic polymer) loaded with insulin were prepared by a double emulsion method followed by evaporation solvent. Low molecular weight heparin (LMWH) was bound by electrostatic interactions onto the surface of the particles to confer Stealth properties. These nanoparticles were characterized in vitro (mean diameter, zeta potential, encapsulation efficiency, and release kinetics) and compared with conventional (without LMWH) and unloaded nanoparticles. The pharmacokinetics of insulin were studied after intravenous injection into diabetic rats in the form of Stealth or conventional nanoparticles or as a solution. Stealth nanoparticles allowed an increase in the elimination half-life of insulin, showing that the hydrophilic layer of LMWH was able to limit recognition by the mononuclear phagocytosis system in vivo. However, complement activation studies (CH50) did not reveal significant difference between Stealth and conventional nanoparticles.

Encapsulation of low molecular weight heparins: influence on the anti-Xa/anti-IIa ratio.

Tinzaparin and nadroparin, two low molecular weight heparins (LMWH), were encapsulated within microparticles by the double emulsion method using ammonium methacrylate copolymer (Eudragit RS) alone or mixed with poly (d,l-lactic-co-glycolic acid) at different ratios. The resulting microparticles were characterized in vitro according to particle size, encapsulation rate and release profiles both by chemical and biological methods. The biological method was based on the measurement of the anti-Xa/anti-IIa ratio typical of each LMWH. This ratio also reflects the relative proportion between active chains below and above the critical chain length of 5000 Da (i.e. BCL and ACL chains), since LMWH are mixtures of chains with various lengths and activities. For both LMWH, high entrapment efficiencies, expressed as anti-Xa and anti-IIa activities, were obtained and amounted to anti-Xa/anti-IIa ratios close to the commercial ratio. During the in vitro release, whatever the formulation, more BCL chains were released than ACL chains: a higher (compared to commercial ratio) and stable anti-Xa/anti-IIa ratio was observed. This increase of the anti-Xa/anti-IIa ratio was influenced by the type of LMWH used and the composition of the Eudragit RS formulation. This type of microparticles could constitute a new pharmaceutical form of LMWH with a higher anti-Xa/anti-IIa ratio than commercial forms.

Granules in the improvement of oral heparin bioavailability.

Low molecular weight heparins (LMWHs), mainly used for the prevention of deep vein thrombosis, are so far only administered by parenteral route. Presumably, due to their large size and negative charge, LMWHs are not absorbed efficiently across the mucus of the gastrointestinal tract. Since parenteral administration requires medical assistance and is not the most convenient route of application, the development of an oral dosage form of heparin would improve patients' comfort and replace vitamin K antagonists. Thus, we developed granules of two LMWHs, enoxaparin and bemiparin, based on primary granules of microcrystalline cellulose and loaded with ethylcellulose (Aquacoat ECD) or a polycationic polymer (Eudragit RS30D (ERS)) or their mixtures. The highest maximal plasma anti-Xa activities and the highest bioavailabilities for enoxaparin granules (0.45+/-0.12IU/mL; 19.00+/-0.30%, respectively) and for bemiparin granules (0.54+/-0.08IU/mL; 29.02+/-4.12%, respectively) were found after oral administration of granules loaded with ERS alone at a dose of 600IU anti-Xa/kg to rabbits. These results confirm the oral absorption of LMWH from granules loaded with polycationic polymer and open up this technology for peptides and proteins that are very sensitive to organic solvents and have poor drug absorption from the gastrointestinal tract.