Gilles Spenlehauer

In vitro and In vivo degradation of poly(D,L lactide/glycolide) type microspheres made by solvent evaporation method

Microspheres of different poly(alpha-hydroxy acids) were prepared by solvent evaporation to study the effects of gamma-sterilization on stability and to establish the degradation process in vitro and in vivo. gamma-Irradiation dramatically decreases polymer molecular weight and this degradation continues on storage. gamma-Irradiation modifies the controlled release pattern of cisplatin-loaded microspheres. After embolization of rat livers by microspheres, a histological study of the inflammatory response was made, along with gel permeation chromatography analysis of degrading polymers. The degradation rate of the polymers increased with the glycolic unit content in the lactic chains. Scanning electron microscopy of microsphere degradation in vitro correlated with the former observations.

Non-stealth(poly(lactic acid/albumin))and stealth(poly(lactic acid-polyethylene glycol))nanoparticles as injectable drug carriers

Abstract Stealth liposomes and, today, stealth nanoparticles, constitute a new generation of parenteral therapeutic systems. PLA/ abumin nanoparticles are of particular interest because they constitute fully biodegradable and well tolerated colloidal suspensions. Solvent evaporation and microfluidisation did not damage the albumin molecules; therefore, PLA/albumin nanoparticles are no more immunogenic than native albumin in solution. However, rapid albumin exchanges on the nanoparticle surface probably does not prevent C3-complement binding and phagocytosis by the liver Kupffer cells. Because of their possible intracellular accumulation and toxicity, PLA/albumin nanoparticles are presumably limited to subcutaneous or intramuscular administration. Poly( d,l -lactide)-poly (ethylene glycol) (PLA-PEG) is a new biodegradable hydrophobic dibloc copolymer. The oriented PEG layer, coating the nanoparticle surface, dramatically increases the plasma half-life of the colloidal carrier (‘stealth nanoparticles’ ). In this way, the PLAPEG nanoparticle half-life is about 6 h instead of a few minutes as for PLA/ albumin or PLA/poloxamer 188-coated nanoparticles. The plasma clearance of a water-insoluble hydrophobic drug encapsulated in stealth nanoparticles and administered intravenously, decreases very significantly in comparison with non-stealth nanoparticles. PLAPEG nanoparticles can be considered as a sustained release parenteral (intravenous) dosage form.

Body distribution of fully biodegradable [14C]-poly(lactic acid) nanoparticles coated with albumin after parenteral administration to rats

Fully biodegradable polylactic acid (PLA) nanoparticles (90-250 nm) coated with human serum albumin (HSA) were prepared by high-pressure emulsification and solvent evaporation, using the protein as surfactant. A new analytical tool was developed, based on Mies law and size exclusion chromatography, to establish that, after evaporation of the solvent, the protein saturates the surface of the nanoparticles, masking the PLA core. According to this technique, no HSA is encapsulated in the polymer matrix. A radiolabelled [14C]-PLA50 was synthesized to follow the fate of this new drug carrier after i.v. administration to rats. The time necessary to clear the albumin-coated nanoparticles from the plasma was significantly longer than for the uncoated ones but not extended enough to target cells other than mononuclear phagocytes. As deduced from whole-body autoradiography and quantitative distribution experiments, the 14C-labelled polymer is rapidly captured by liver, bone marrow, lymph nodes, spleen and peritoneal macrophages. Nanoparticle degradation was addressed following 14C excretion. The elimination of the 14C was quick on the first day (30% of the administered dose) but then slowed down. In fact, if the metabolism of the PLA proceeds to lactic acid which is rapidly converted into CO2 via the Krebs cycle (80% of the total excretion was fulfilled by the lungs), anabolism from the lactic acid may also have taken place leading to long-lasting radioactive remnants, by incorporation of 14C into endogenous compounds.

Effect of PEO surface density on long-circulating PLA-PEO nanoparticles which are very low complement activators.

The rapid uptake of injected nanoparticles by cells of the mononuclear phagocytes system (MPS) is a major obstacle when a long blood circulation time is needed. Whereas nanoparticles made from PLA and stabilized by surfactants (PLA-F68) are rapidly phagocytized, the rate of phagocytosis is strongly reduced in case of nanoparticles made from a diblock copolymer (PLA-PEO). Because of the role of the complement system in opsonization, this difference of phagocytosis was hypothesized to be related to this system. An important complement consumption was obtained in 5 min in the presence of PLA-F68 particles. In the presence of a higher surface area of PLA-PEO particles possessing a high PEO surface density, the consumption remained very low. When the average PEO surface density was decreased on such particles below a given threshold, a fast and strong complement consumption occurred again. These experimental data support the concept of steric repulsion towards proteins, by surfaces covered with terminally attached PEO chains and emphasize the prime importance of PEO surface density in such an effect. The major, but probably not exclusive, role of complement as an opsonin capable of inducing a fast phagocytosis by MPS should be taken into account concerning the in vitro evaluation of nanoparticles as candidates for a long blood circulation.

Biodegradable cisplatiim microspheres prepared by the solvent evaporation method: Morphology and release characteristics

Abstract Various cisplatin-loaded microsphere systems have been prepared from d,l -lactic acid stereocopolymers and d,l -lactic acid/glycolic acid copolymers using a solvent evaporation process. The morphology of these systems depends very much on different factors: addition of extra cisplatin in the dispersing phase, drug loading, polymer molecular weights and polymer concentration in the organic phase. In vitro release profiles have been determined for the various systems. It has been found that drug release depends on the amount of entrapped drug, on the presence of extra cisplatin in the dispersing phase, and on polymer molecular weights. In contrast, the release of cisplatin seems to be independent of the presence ofglycolic units within polylactic chains, and of radiosterilisation, although irradiation dramatically affected polymer molecular weights of completed microspheres. Results are discussed with respect to microsphere processing and morphology, especially cisplatin crystal distributions within the microspheres. It is concluded that the viscosity of the organic phase, in which the polymer is dissolved at the processing stage, is a critical factor. Data collected are discussed in terms of a predominantly diffusional release mechanism by either polymer matrices or dissolution in channels depending upon cisplatin crystal distribution in microspheres and, to some extent, upon polymer degradation.

Degradation of poly(d,l-lactic acid) nanoparticles coated with albumin in model digestive fluids (USP XXII)

Entirely biodegradable poly(D, L-lactic acid) (PLA50) nanoparticles coated with albumin were prepared by the solvent evaporation technique. Their degradative properties were investigated in simulated gastric and intestinal fluids (USP XXII). The degradation of the albumin coating was monitored by HPLC, whereas PLA50 degradation was determined by size exclusion chromatography (SEC) as well as by the detection of lactate in bulk solution by enzymatic assay. As expected, the coating effect of albumin, a readily digestible protein, rapidly disappeared in both gastric and intestinal media, thus exposing albumin-free PLA50 cores to hydrolytic processes. In pepsin-rich simulated gastric fluid, no degradation of the PLA50 core was observed over 8 h incubation time. In contrast, in pancreatin-rich simulated intestinal fluid, the PLA50 nanoparticles were rapidly converted into lactate. The results showed that the PLA50 degradation was mainly due to an enzymatic cleavage process. Further experiments showed the involvement of lipases in the degradation of the PLA50 core in simulated intestinal fluid.

Interactions of poly(lactic acid) and poly(lactic acid-co-ethylene oxide) nanoparticles with the plasma factors of the coagulation system

When surfactant-stabilized biodegradable poly(lactic acid) (PLA) particles are injected into rats, the rate of clearance from blood is fast. The rate can be strongly reduced by using particles made from diblock copolymers of PLA and poly(ethylene oxide) (PLA-PEO), resulting in an increased duration of contact with the components of the coagulation system. Thus, possible adverse effects such as activation of the coagulation cascade could occur. In this paper, the interactions of surfactant-stabilized PLA and PLA-PEO nanoparticle suspensions with the plasma factors of the coagulation system are presented. PLA suspensions stabilized by sodium cholate (PLA-Ch) interact with thrombin, factor V and calcium ions. Formation of complexes and aggregates is induced by addition of calcium ions to PLA-Ch suspensions in the presence or in the absence of plasma. On the contrary, PLA-PEO suspensions are remarkably inert towards the coagulation factors and calcium ions, even when cholate is present. Steric repulsion owing to the high surface density of PEO is sufficient to avoid strong interations with the proteins and formation of aggregates between particles.

Stabilizers against heat-induced aggregation of RPR 114849, an acidic fibroblast growth factor (aFGF)

In an effort to optimize stabilization conditions for RPR 114849, a wide variety of known stabilizers were screened for their effects on the stability of the protein against thermal denaturation. For the screening purpose, the effects of excipients on aggregation rate were examined employing UV spectrophotometric turbidity measurements at 50°C and pH 7.4. The protein is sensitive to aggregation near its isoelectric point. Higher concentrations of the protein promote faster aggregation. Reducing agents do not decrease the aggregation rate indicating that oxidation of thiol groups to intermolecular disulfide bonding is not a rate-limiting factor in the aggregation process. In addition to well-known heparin, a wide variety of sulfated and phosphorylated anionic polymers have shown to be powerful stabilizers for the protein. The chain length of a polymeric anion is a critical factor in stabilizing the protein aggregation. The stabilizing effect approaches a constant value asymptotically as the chain length increases. The combined action of enoxaparin and sodium citrate is additive indicating that the stabilizers act independently and do not affect each others mode, degree, or efficacy of action. High concentrations of non-specific stabilizers, such as sugars and polyols, are capable of suppressing aggregation of the protein to a minor extent. Surfactants, gelling and microencapsulating agents were found to have no practical utility in stabilizing the protein.

Release of the fluorescent marker Prodan® from poly(D,L-lactic acid) nanoparticles coated with albumin or polyvinyl alcohol in model digestive fluids (USP XXII)

Prodan® release from poly(D,L-lactic acid) (PLA50) nanoparticles coated with albumin or polyvinyl alcohol (PVA) in simulated gastric and intestinal fluids (USP XXII) was investigated. Prodan® is a fluorescent marker, whose fluorescence properties enabled the concurrent determination of the free and bound compound without prior separation of the nanoparticles. In simulated gastric fluid, where the PLA50 matrix of both PLA50 nanoparticle systems coated with albumin or PVA was not degraded, the nanoparticles coated with albumin or PVA showed similar Prodan® release properties: Prodan® release from both nanoparticles coated with either albumin or PVA was shown to follow kinetics consistent with diffusion through the intact polymeric matrix. One hundred percent release was obtained within 120 min. In simulated intestinal fluid, however, the Prodan® release from the PLA50 nanoparticles was different depending on the agent coating the nanoparticles: when PVA was used as a coating agent, e.g. when no degradation of the PLA50 matrix was observed, Prodan® was released by diffusion through the intact PLA50 matrix. Due to its very low affinity for water at this pH, only 44% of Prodan® was released. By contrast, Prodan® release from PLA50 nanoparticles coated with albumin was driven by both matrix erosion and diffusion processes. Accordingly, 100% release occurred over 480 min, despite the low affinity of Prodan® for the aqueous phase at the pH of the simulated intestinal fluid.

Modelling of drug release from pellets coated with an insoluble polymeric membrane

Abstract In this work, a model is proposed to accurately describe drug release kinetics from coated pellets where the membrane does not entirely control the release. To validate the model, ketoprofen pellets prepared using the extrusion-spheronisation process, were coated with a membrane comprising acrylic resins (either 100% Eudragit RL or 50% Eudragit RL and 50% Eudragit RS) and different plasticizers. The effect of the membrane composition on drug release from coated pellets was investigated using the USP dissolution paddle method. In addition, permeation kinetics through free films made with the coating polymers were performed. The model permitted to quantitatively correlate the release kinetics from coated pellets with permeation properties of free films by taking into account the physical parameters of the systems. The advantage of this model is the possibility of ab initio optimization, from easy experiments not including coated pellets: drug release from uncoated matrices and permeation kinetics through free films.