Quantification of surface GalNAc ligands decorating nanostructured lipid carriers by UPLC-ELSD

Nanoparticles have been extensively studied for drug delivery and targeting to specific organs. The functionalization of the nanoparticle surface by site-specific ligands (antibodies, peptides, saccharides) can ensure efficient recognition and binding with relevant biological targets. One of the main challenges in the development of these decorated nanocarriers is the accurate quantification of the amount of ligands on the nanoparticle surface. In this study, nanostructured lipid carriers (NLC) were functionalized with N-acetyl-D-galactosamine (GalNAc) units, known to target the asialoglycoprotein receptor (ASGPR). Different molar percentages of GalNAc-functionalized surfactant (0%, 2%, 5%, and 14%) were used in the formulation. Based on ultra-high-performance liquid chromatography separation and evaporative light-scattering detection (UPLC-ELSD), an analytical method was developed to specifically quantify the amount of GalNAc units present at the NLC surface. This method allowed the accurate quantification of GalNAc surfactant and therefore gave some insights into the structural parameters of these multivalent ligand systems. Our data show that the GalNAc decorated NLC possess large numbers of ligands at their surface and suitable distances between them for efficient multivalent interaction with the ASGPR, and therefore promising liver-targeting efficiency

ASSOCIATION DU POLY(L-LYSINE CITRAMIDE) AVEC UNE ANTIPROTEASE DU VIH ET EVALUATION DE L'ACTIVITE ANTIVIRALE SUR CELLULES INFECTEES

CETTE THESE VISAIT A VECTORISER UN INHIBITEUR DE LA PROTEASE DU VIH VIA UNE ASSOCIATION AVEC LE POLY(L-LYSINE CITRAMIDE), VECTEUR MACROMOLECULAIRE HYDROSOLUBLE, BIOCOMPATIBLE ET BIORESORBABLE AFIN DE FAVORISER LA PENETRATION INTRACELLULAIRE DU PRINCIPE ACTIF A FAIBLE BIODISPONIBILITE. LE MEMOIRE DEBUTE PAR UN RAPPEL BIBLIOGRAPHIQUE DES CONNAISSANCES SUR LA PROTEASE DU VIH ET SUR LES SYSTEMES MACROMOLECULAIRES VECTEURS POTENTIELS D'INHIBITEURS DE LA PROTEASE. LE CHAPITRE II EST CONSACRE A UNE NOUVELLE VOIE DE SYNTHESE DU POLY(L-LYSINE CITRAMIDE) EN PHASE HOMOGENE, ET A LA CARACTERISATION DU VECTEUR OBTENU. PAR COMPARAISON AVEC LA POLYCONDENSATION INTERFACIALE, IL EST MONTRE QUE LES POLYMERES PREPARES PAR LES DEUX VOIES SONT DE MEME NATURE CHIMIQUE MAIS DIFFERENT PAR LA PROPORTION DES UNITES DE TYPE IMIDE ET OXOLACTONE. LE CHAPITRE III RAPPORTE LA SYNTHESE ET LA CARACTERISATION DE CONJUGUES POLYMERE PLCA/INHIBITEUR (PSI) OU NON INHIBITEUR (PNI) DE LA PROTEASE DU VIH VIA L'AGENT DE COUPLAGE BOP. UNE ETUDE PHYSICO-CHIMIQUE DE CES CONJUGUES RENSEIGNE SUR LEUR COMPORTEMENT EN MILIEU AQUEUX. AFIN DE DISTINGUER L'INFLUENCE DE LA NATURE MACROMOLECULAIRE DES CONJUGUES AU COURS DES TESTS CELLULAIRES, DES COMPOSES MODELES DES UNITES CONSTITUTIVES DE LA PRODROGUE MACROMOLECULAIRE SONT SYNTHETISES ET ETUDIES COMPARATIVEMENT (CIPSI ET LYPSI). LE CHAPITRE IV TRAITE DE L'EVALUATION IN VITRO DE L'ACTIVITE ANTIVIRALE DES CONJUGUES ET DE LEUR COMPORTEMENT AU CONTACT DE CELLULES INFECTEES PAR LE VIH. L'INTERACTION VECTEURS SYNTHETIQUES/CELLULES INFECTEES EST ETUDIEE. AFIN D'ENVISAGER UNE EVENTUELLE EXPLOITATION THERAPEUTIQUE, L'ABSENCE DE TOXICITE D'UN CONJUGUE IN VIVO EST VERIFIEE. IL EST MONTRE QUE L'ACTIVITE ANTIVIRALE EST EN FAIT LIEE A LA NATURE MACROMOLECULAIRE DES CONJUGUES ET NON A UN COMPORTEMENT DE PRODROGUES.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Development and validation of a novel UPLC-ELSD method for the assessment of lipid composition of nanomedicine formulation

Lipid nanocarriers incorporating glycerides, polyethylene glycol (PEG)-stearates and phospholipids have attracted great attention for in vivo diagnostic, in vivo imaging, activated or non-activated targeted drug delivery. For quality control purposes, the development of appropriate methods for the quantification of their lipid components is needed. In the present study, we developed an analytical method for lipid quantification in formulated nanoparticles. PEG-stearates and glycerides were analyzed in a single run by RP-UPLC-ELSD using a two-step gradient elution program, while the analysis of phospholipids was accomplished by HILIC-UPLC-ELSD after isolation using an SPE silica column. Using both isolated compounds and commercial lipid standards, calibration curves were produced using second-order polynomials to attain the quantitative evaluation of each lipid excipient. Relative standard deviation of all analytes was between 0.9% and 5.3% for intra-day precision and recovery ranged from 83.5% to 112.2%. The presented method was successfully implemented to study the manufacturing process and stability of the formulated lipid excipients during long-term storage and accelerated conditions. The formulation lipid yield was determined and found equal to 82.5%

Electrospinning in water and in situ crosslinking of hyaluronic acid / cyclodextrin nanofibers: Towards wound dressing with controlled drug release

International audienceHyaluronic acid (HA) is widely investigated due to its high potential for wound dressing applications. The fabrication of biomimetic HA-based scaffolds by electrospinning is thus extensively studied. However, HA is often dissolved in toxic organic solvents to allow the efficient production of electrospun nanofibers. Indeed, although HA is soluble in water, its ionic nature leading to long-range electrostatic interactions and the presence of counter ions induce a dramatic increase of the viscosity of aqueous HA solutions without insuring enough chain entanglements necessary for a stable and efficient electrospinning. In this study, biocompatible insoluble HA-based nanofibers were fabricated by electrospinning in pure water. To this end, poly(vinyl alcohol) (PVA) was added as a carrier polymer and it was found that the addition of hydroxypropyl-beta cyclodextrin (HP beta CD) stabilized the process of electrospinning and led to the efficient formation of uniform nanofibrous scaffolds. An in situ crosslinking process of the scaffolds is also proposed, insuring a whole fabrication process without any toxicity. Furthermore, the beneficial presence of HP beta CD in the HA-based scaffolds paves the way for wound dressing applications with controlled drug encapsulation-release properties. As a proof of concept, naproxen (NAP), a non-steroidal anti-inflammatory drug was chosen as a model drug. NAP was impregnated into the scaffolds either in aqueous solution or under supercritical CO2. The resulting functional scaffolds showed a regular drug release profile along several days without losing the fibrous structure. This study proposes a simple approach to form stable HA-based nanofibrous scaffolds embedding HP beta CD using water as the only solvent, enabling the development of safe functional wound dressings

Development and validation of an HPLC-fluorescence method for the quantification of IR780-oleyl dye in lipid nanoparticles

International audienceA reversed-phase (RP) high-performance liquid chromatography (HPLC) method for the content determination of IR780-oleyl (IRO) dye in lipid nanoparticles was developed and validated. Chromatographic separation was performed on a RP C18 column with a gradient program of water and acetonitrile both with 0.1% (v/v) TFA, at a flow rate of 1.0 mL/min and a total run of 21 min. IRO dye detection was made by fluorescence at emission wavelength of 773 nm (excitation wavelength 744 nm). According to ICH guidelines, the developed method was shown to be specific, linear in the range 3-8 mg/ mL (R-2 = 0.9998), precise at the intra-day and inter-day levels as reflected by the coefficient of variation (CV <= 1.98%) at three different concentrations (4, 6 and 8 mg/mL) and accurate, with recovery rates between 98.2-101.6% and 99.2-100.5%. The detection and quantitation limits were 0.41 and 1.24 mg/mL, respectively. Stability studies of sample processing showed that IRO dye was stable after 24 h in the autosampler or after three freeze/thaw cycles. Combined with fluorescence measurements, the developed method was successfully applied to optimize the loading capacity of IRO dye in the core of lipid nanoparticles

Solid Phase Extraction as an Innovative Separation Method for Measuring Free and Entrapped Drug in Lipid Nanoparticles.

Contrary to physical characterization techniques for nanopharmaceuticals (shape, size and zeta-potential), the techniques to quantify the free and the entrapped drug remain very few and difficult to transpose in routine analytical laboratories. The application of Solid Phase Extraction (SPE) technique was investigated to overcome this challenge

Lipidots: competitive organic alternative to quantum dots for in vivo fluorescence imaging.

International audienceThe use of fluorescent nanostructures can bring several benefits on the signal to background ratio for in vitro microscopy, in vivo small animal imaging, and image-guided surgery. Fluorescent quantum dots (QDs) display outstanding optical properties, with high brightness and low photobleaching rate. However, because of their toxic element core composition and their potential long term retention in reticulo-endothelial organs such as liver, their in vivo human applications seem compromised. The development of new dye-loaded (DiO, DiI, DiD, DiR, and Indocyanine Green (ICG)) lipid nanoparticles for fluorescence imaging (lipidots) is described here. Lipidot optical properties quantitatively compete with those of commercial QDs (QTracker(®)705). Multichannel in vivo imaging of lymph nodes in mice is demonstrated for doses as low as 2 pmols of particles. Along with their optical properties, fluorescent lipidots display very low cytotoxicity (IC(50) > 75 nM), which make them suitable tools for in vitro, and especially in vivo, fluorescence imaging applications

Photoinduced effects of m-tetrahydroxyphenylchlorin loaded lipid nanoemulsions on multicellular tumor spheroids

Background Photosensitizers are used in photodynamic therapy (PDT) to destruct tumor cells, however, their limited solubility and specificity hampers routine use, which may be overcome by encapsulation. Several promising novel nanoparticulate drug carriers including liposomes, polymeric nanoparticles, metallic nanoparticles and lipid nanocomposites have been developed. However, many of them contain components that would not meet safety standards of regulatory bodies and due to difficulties of the manufacturing processes, reproducibility and scale up procedures these drugs may eventually not reach the clinics. Recently, we have designed a novel lipid nanostructured carrier, namely Lipidots, consisting of nontoxic and FDA approved ingredients as promising vehicle for the approved photosensitizer m-tetrahydroxyphenylchlorin (mTHPC). Results In this study we tested Lipidots of two different sizes (50 and 120 nm) and assessed their photodynamic potential in 3-dimensional multicellular cancer spheroids. Microscopically, the intracellular accumulation kinetics of mTHPC were retarded after encapsulation. However, after activation mTHPC entrapped into 50 nm particles destroyed cancer spheroids as efficiently as the free drug. Cell death and gene expression studies provide evidence that encapsulation may lead to different cell killing modes in PDT. Conclusions Since ATP viability assays showed that the carriers were nontoxic and that encapsulation reduced dark toxicity of mTHPC we conclude that our 50 nm photosensitizer carriers may be beneficial for clinical PDT applications

Lipid nanoemulsions and liposomes improve photodynamic treatment efficacy and tolerance in CAL-33 tumor bearing nude mice

Background Photodynamic therapy (PDT) as promising alternative to conventional cancer treatments works by irradiation of a photosensitizer (PS) with light, which creates reactive oxygen species and singlet oxygen (1O2), that damage the tumor. However, a routine use is hindered by the PS’s poor water solubility and extended cutaneous photosensitivity of patients after treatment. In our study we sought to overcome these limitations by encapsulation of the PS m-tetrahydroxyphenylchlorin (mTHPC) into a biocompatible nanoemulsion (Lipidots). Results In CAL-33 tumor bearing nude mice we compared the Lipidots to the existing liposomal mTHPC nanoformulation Foslip and the approved mTHPC formulation Foscan. We established biodistribution profiles via fluorescence measurements in vivo and high performance liquid chromatography (HPLC) analysis. All formulations accumulated in the tumors and we could determine the optimum treatment time point for each substance (8 h for mTHPC, 24 h for Foslip and 72 h for the Lipidots). We used two different light doses (10 and 20 J/cm2) and evaluated immediate PDT effects 48 h after treatment and long term effects 14 days later. We also analyzed tumors by histological analysis and performing reverse transcription real-time PCR with RNA extracts. Concerning tumor destruction Foslip was superior to Lipidots and Foscan while with regard to tolerance and side effects Lipidots were giving the best results. Conclusions We could demonstrate in our study that nanoformulations are superior to the free PS mTHPC. The development of a potent nanoformulation is of major importance because the free PS is related to several issues such as poor bioavailability, solubility and increased photosensibility of patients. We could show in this study that Foslip is very potent in destroying the tumors itself. However, because the Lipidots' biocompatibility is outstanding and superior to the liposomes we plan to carry out further investigations and protocol optimization. Both nanoformulations show great potential to revolutionize PDT in the future