Sustained in vitro release and cell uptake of doxorubicin adsorbed onto gold nanoparticles and covered by a polyelectrolyte complex layer.

Gold nanoparticles functionalized with doxorubicin and stabilized with multilayers of degradable polyelectrolyte were allowed to age in aqueous medium in vitro in order to show the possibility of drug release in cellular environment. The chemico-physical characteristics of the nanoparticles are reported. The observed release of doxorubicin (DOX) was pH-dependent, and it increased in acidic environment. Cell uptake of nanoparticles and drug release were monitored by laser scanning confocal microscopy. Data showed that drug-bearing nanoparticles delivered DOX into the nuclei of A549 cells, leading to pronounced cytotoxic effects to this lung tumor cells. Our results suggest that gold nanoparticles conjugated with doxorubicin could be used as a pH-triggered drug releasing carrier for tumor drug delivery.

Tissue healing during degradation of a long-lasting bioresorbable gamma-ray-sterilised poly(lactic acid) mesh in the rat: a 12-month study.

AIM: The purpose was to evaluate soft-tissue healing after poly(lactic acid) (PLA(94)) mesh implantation in a rat model. METHODS: Full-thickness abdominal wall defects were created in 108 Wistar rats, and reconstructed with 83 PLA(94) and 25 lightweight polypropylene (PPL) meshes. The meshes were previously gamma-ray sterilised with 25, 75 or 125 kGy to accelerate PLA(94) degradation. RESULTS: The inflammatory response in PLA(94) was significantly less pronounced and collagen organisation significantly better than in PPL. The higher the level of gamma-radiation, the higher the incidence of abdominal wall herniation (22.2, 31.3 and 52.6% with 25, 75 and 125 kGy, respectively). No herniation occurred in the PPL group. Tensile strength was dramatically reduced after gamma-ray-sterilised PLA(94) mesh implantation. CONCLUSION: The gamma-ray-sterilised PLA(94) mesh was poor in preventing abdominal wall hernia recurrences in a rat model.

[Development of new skin substitutes based on bioresorbable polymer for treatment of severe skin defects]. / Développement de nouveaux substituts cutanés à base de polymères biorésorbables pour la prise en charge des affections cutanées sévères.

Over the last years, increasing attention has been paid to skin engineering due to its predominant function in body integrity. Thus, many laboratories are attempting to develop a dermal-epidermal complex. The aim of this study was to evaluate the potential of poly(alpha-hydroxyacid)s in the development of biocompatible and bioresorbable dermal scaffold combining human fibroblasts and keratinocytes, in order to obviate the drawbacks of using natural polymers such as collagen, hyaluronic acid and fibrin. We first confirmed the interest of poly(d,l-lactic acid) (PLA(50)) during the reconstitution of epidermis and next, we investigated the potential of poly(d,l-lactic acid)-poly(ethylene glycol)-poly(d,l-lactic acid) (PLA(50)-PEG-PLA(50)) for either skin cytocompatibility or scaffold processing. Data showed that PLA(50)-PEG-PLA(50) is compatible with the culture of human skin cells (fibroblasts and keratinocytes) and the development of a porous scaffold; two requirements compulsory for being considered as an adequate skin substitute. In fine, this material could represent the first generation of new skin dressings, i.e. a new concept taking advantage of both implantable devices and current dressings.

Wet or dry mechanochemical synthesis of calcium phosphates? Influence of the water content on DCPD-CaO reaction kinetics.

Mechanosynthesis of calcium phosphates can be performed under wet or dry conditions. In most papers and patents, grinding under wet conditions was selected. So far, only a few papers were devoted to dry mechanosynthesis of calcium phosphates. To understand why wet mechanosynthesis was preferred, the influence of water addition on the kinetics of the mechanochemical reaction of dicalcium phosphate dihydrate with calcium oxide was investigated. The DCPD disappearance rate constant k and the final reaction time t(f) were determined in each case and correlated with the water content present in the slurry. Results showed that the addition water (i) slowed down the reaction rate and (ii) increased the powder contamination by mill material (hard porcelain) due to ball and vial erosion; and that (iii) wet milling did not generate the expected products, in contrast to dry grinding, because porcelain induced hydroxyapatite decomposition with the formation of beta-tricalcium phosphate and silicon-stabilized tricalcium phosphate. Consequently, dry mechanosynthesis appears preferable to wet milling in the preparation of calcium phosphates of biological interest.

Novel amphiphilic poly(epsilon-caprolactone)-g-poly(L-lysine) degradable copolymers.

As part of the search of novel degradable polymers, amphiphilic and cationic poly(epsilon-caprolactone)-g-poly(l-lysine) (PCL-g-PlL) copolymers have been synthesized following a grafting "onto" or a grafting "from" method both applied to a macropolycarbanionic PCL derivative. The first approach led to PCL-g-PZlL containing 36% of epsilon-caprolactone and 64% of N-epsilon-Z-l-lysine units, by reaction of activated poly(N-epsilon-Z-l-lysine) on the macropolycarbanion derived from PCL. The second route was based on the anionic ring opening polymerization of N-carboxyanhydride of N-epsilon-benzyloxycarbonyl-l-lysine initiated by the macropolycarbanion derived from PCL and led to a similar copolymer containing 45% of of epsilon-caprolactone and 55% of N-epsilon-Z-l-lysine units. After deprotection of the lysine units, PCL-g-PlL copolymers were obtained. These copolymers are water-soluble and form nanometric micelle-like objects with mean diameters between 60 and 500 nm in distilled water depending on the synthesis route.

Fabrication using a rapid prototyping system and in vitro characterization of PEG-PCL-PLA scaffolds for tissue engineering.

In the field of tissue engineering new polymers are needed to fabricate scaffolds with specific properties depending on the targeted tissue. This work aimed at designing and developing a 3D scaffold with variable mechanical strength, fully interconnected porous network, controllable hydrophilicity and degradability. For this, a desktop-robot-based melt-extrusion rapid prototyping technique was applied to a novel tri-block co-polymer, namely poly(ethylene glycol)-block-poly(epsilon-caprolactone)-block-poly(DL-lactide), PEG-PCL-P(DL)LA. This co-polymer was melted by electrical heating and directly extruded out using computer-controlled rapid prototyping by means of compressed purified air to build porous scaffolds. Various lay-down patterns (0/30/60/90/120/150 degrees, 0/45/90/135 degrees, 0/60/120 degrees and 0/90 degrees) were produced by using appropriate positioning of the robotic control system. Scanning electron microscopy and micro-computed tomography were used to show that 3D scaffold architectures were honeycomb-like with completely interconnected and controlled channel characteristics. Compression tests were performed and the data obtained agreed well with the typical behavior of a porous material undergoing deformation. Preliminary cell response to the as-fabricated scaffolds has been studied with primary human fibroblasts. The results demonstrated the suitability of the process and the cell biocompatibility of the polymer, two important properties among the many required for effective clinical use and efficient tissue-engineering scaffolding.

Interactions of GRF(1-29)NH2 with plasma proteins and their effects on the release of the peptide from a PLAGA matrix.

The administration of the GRF(1-29)NH2 Growth Hormone Releasing Hormone analog is known as relevant of the concept of drug delivery system using a bioresorbable matrix. However, the release of this peptide from poly(dl-lactic acid-co-glycolic acid) matrices is affected by its insolubility at neutral in salted media and in plasma as well. In order to investigate the origin and the nature of the insolubility in these media in more details, the precipitates collected when the peptide was set in contact with saline, isotonic pH=7.4 phosphate buffer and plasma were analyzed by various techniques, namely weighting, gel chromatography, 1D- and 2D-immunoelectrophoresis, and dialysis to discern the soluble from the insoluble or aggregated fractions. It is shown that precipitation in protein-free salted media is due to a salting out phenomenon complemented by the neutralization of the solubilizing electrostatic charges in the isotonic buffer. In contrast, the precipitation in plasma is due to inter polyelectrolyte-type complexation that involved polyanionic proteins having a rather low isoelectric point like albumin, transferin, haptoglobulin and IgG immunoglobulins. When a rather large quantity of GRF(1-29)NH2 was entrapped in bioresorbable pellets working at a percolating regime after subcutaneous implantation in rats, the peptide was slowly released despite the complexation with plasma proteins. However only a very small part of the peptide was found in blood, this small part being still large enough to cause a detectable increase of the circulating growth hormone concentration. Attempts made to increase the solubility of the peptide in plasma were successful when the peptide was combined with arginine, an amino acid known to promote the poor hormonal activity of injected GRF(1-29)NH2 solutions under clinical conditions.

Biodegradation of [(3)H]poly(epsilon-caprolactone) in the presence of active sludge extracts.

Poly(epsilon-caprolactone), PCL, is a commercial biodegradable and biocompatible polyester that can be bioassimilated by outdoor microorganisms. For biomedical and environmental applications, monitoring the fate of degradation products in vivo or under environmental conditions is one of the critical steps to evaluate degradation characteristics. [(3)H] radiolabeling is the best method to monitor the fate of degradable polymer chains in contact with complex living systems and to show bioassimilation. Therefore, tritiated PCL was synthesized by chemical modification using anionic activation by reaction with lithium diisopropylamide. The resulting radioactive polymer was characterized and allowed to degrade at 37 degrees C under aerobic conditions in the presence of active sludge. Comparison was made with abiotic hydrolytic degradation in pH = 7.4, 0.13 M phosphate buffer at 37 degrees C. Water-soluble degradation products were assessed by measuring radioactivity in the solution phase. It was shown that biodegradation of PCL started after a few hours and proceeded up to the ultimate stage over ca. 72 days, giving tritiated water (80-90%) and biomass. Radioactivity detection appeared much more sensitive than measurement of CO(2) production or consumption to monitor degradation phenomena. In particular, it showed that the onset of biodegradation occurs earlier than that reported using respirometry.

Biodistribution of long-circulating PEG-grafted nanocapsules in mice: effects of PEG chain length and density.

PURPOSE: To study the pharmacokinetics and biodistribution of novel polyethyleneglycol (PEG) surface-modified poly(rac-lactide) (PLA) nanocapsules (NCs) and to investigate the influence of PEG chain length and content. METHODS: The biodistribution and plasma clearance in mice of different NC formulations were studied with [3H]-PLA. PLA-PEG copolymers were used in NC preparations at different chain lengths (5 kDa and 20 kDa) and PEG contents (10% and 30% w/w of total polymer). In vitro and in vivo stability were also checked. RESULTS: Limited [3H]-PLA degradation was observed after incubation in mouse plasma for 1 h, probably because of to the large surface area and thin polymer wall. After injection into mice. NCs prepared with PLA-PEG copolymers showed an altered distribution compared to poloxamer-coated PLA NCs. An increased concentration in plasma was also observed for PLA-PEG NCs. even after 24 h. A dramatic difference in the pharmacokinetic parameters of PLA-PEG 45-20 30% NCs compared to poloxamer-coated NCs indicates that covalent attachment, longer PEG chain lengths, and higher densities are necessary to produce an increased half-life of NCs in vivo. CONCLUSIONS: Covalently attached PEG on the surface of NCs substantially can reduce their clearance from the blood compartment and alter their biodistribution.

Protein release from physically crosslinked hydrogels of the PLA/PEO/PLA triblock copolymer-type.

A series of PLA/PEO/PLA triblock copolymers was prepared by ring opening polymerization of rac-lactide in the presence of various di-hydroxyl poly (ethylene glycol)s, using CaH2 as a biocompatible initiator. Hydrogels were prepared by a phase separation method consisting of introducing small amounts of water over solutions of the copolymers in a biocompatible organic solvent, namely tetraglycol [poly(ethylene glycol monotetrahydrofurfuryl ether)]. The resulting hydrogels appeared much more hydrophilic than the rather tough hydrogels formed by swelling of dry tablets or films processed from the same copolymers. The phase separation-derived hydrogels were soft enough to be injected through a trochar. Two proteins, namely bovine serum albumine (BSA) and fibrinogen, were physically entrapped in these hydrogels by mixing with the polymer solutions before gel formation. This procedure appeared to be protein-respecting according to circular dichroism analysis on the released BSA. Dramatically different release profiles were obtained for the two proteins. In the case of BSA, the release depended on the quantity of protein incorporated in the hydrogel and presented a parabolic-type profile, in agreement with the behaviors of diffusion-controlled monolitic drug delivery devices. In contrast, almost linear release profiles were observed in the case of fibrinogen, the hydrogels behaving like a reservoir drug delivery system. These findings are tentatively interpreted in terms of gel-protein compatibility in the case of BSA and gel-protein incompatibility in the case of fibrinogen.

Novel degradable polymers combining D-gluconic acid, a sugar of vegetal origin, with lactic and glycolic acids.

To synthesize functionalized poly(lactic acid-co-glycolic acid)-based polyesters for biomedical and pharmaceutical applications such as controlled drug delivery, D-gluconic acid was considered as an interesting source of comonomer. Accordingly D-gluconic acid was used to synthesize novel 1,4-dioxane-2,5-diones with protected hydroxyl groups, namely 3-(1,2:3,4-tetraoxobutyl-di-O-isopropylidene)-dioxane-2,5-dione (5a) and 3-methyl-6-(1,2:3,4-tetraoxobutyl-di-O-isopropylidene)-dioxane-2,5-dione (5b). The ring-opening homopolymerization and copolymerization of these cyclic dilactones with DL-lactide provided novel degradable polyesters with higher glass transition temperatures than poly(lactic acid-co-glycolic acid) polymers.

Labile conjugation of a hydrophilic drug to PLA oligomers to modify a drug delivery system: cephradin in a PLAGA matrix.

The physical entrapment of a hydrophilic drug within degradable microspheres is generally difficult because of poor entrapment yield and/or fast release, depending on the microsphere fabrication method. In order to counter the effects of drug hydrophilicity, it is proposed to covalently attach the drug to lactic acid oligomers, with the aim of achieving temporary hydrophobization and slower release controlled by the separation of the drug from the degradable link within the polymer matrix. This strategy was tested on microspheres of the antibiotic cephradin. As the prodrug form, the entrapment of the drug was almost quantitative. The prodrug did degrade in an aqueous medium, modelling body fluids, but cleavage did not occur at the drug-oligomer junction and drug molecules bearing two lactyl residual units were released. When the prodrug is entrapped within a PLAGA matrix, no release was observed within the experimental time period. However, data suggest that conjugation via a bond more sensitive to hydrolysis than the main chain PLA ester bonds should make the system work as desired.

Synthesis, characterisation and in vivo behaviour of a norfloxacin-poly(L-lysine citramide imide) conjugate bearing mannosyl residues.

With the aim of promoting the targeting of macrophage mannose receptors and the internalisation of the norfloxacin antibiotic, which is active against some intracellular bacteria, a macromolecular prodrug was synthesised where the antibiotic and mannosyl moieties were coupled to a polymeric carrier, namely poly(L-lysine citramide imide). This carrier, which derived from two metabolites, citric acid and L-lysine, is known to be biocompatible and slowly degradable under slight acidic conditions. Norfloxacin was coupled onto the acid groups present along the polymer chains, and conjugates were characterised by UV, TLC and SEC. The mannosyl groups selected to promote the targeting of the mannose-specific lectin present on the outer membrane of macrophages were incorporated through a biodegradable glycolic spacer arm. Two different strategies were considered to synthesise the full conjugates, namely coupling norfloxacin onto mannosylated conjugates, and coupling mannose onto PLCAI/Nflx conjugates. The second pathway led to better results regarding mannosylation. The presence of norfloxacin and mannose caused chain aggregation, especially for conjugates with a high content of mannosyl residues. The targeting ability of the prodrug was investigated using a method based on the competition between the mannosylated macromolecules and glucose oxidase, a mannosyl-bearing non-human protein. This method showed that prodrug macromolecules competed effectively with glucose oxidase and thus should be able to bring the drug up to the mannosyl receptor-bearing membranes of macrophages infected by intracellular bacteria.

In vitro delivery of a sparingly water soluble compound from PLA50 microparticles.

The administration of a sparingly soluble drug is always problematic, especially when the drug has to be released from the degradable matrix of a polymeric drug delivery system. Attempts were made to achieve the complete release of 1-[2-(fluorobenzoyl) aminoethyl]-4-(7-methoxynaphtyl)piperazine (FAMP), a potential anxiolytic and antidepressor hydrophobic compound, from racemic poly(lactic acid) (PLA50)-based microparticles, 100% release was required at a low rate in order to allow monthly repeated S.C. or I.M. injections of this potent compound. FAMP-polymer combinations were made in the form of microspheres by the solvent evaporation technique. Release profiles were investigated under dynamic conditions by using a constant flow rate of pH 7.4 0.15 M phosphate buffer, used as a model of body fluids. Under these conditions, none of the microsphere compositions led to total release within a month, even when hydrophilic excipients, namely fructose and PEG were added. PLA50-FAMP microparticles with compositions and sizes similar to those of the microspheres, were then made by direct blending in dichloromethane, evaporation of the solvent, grinding and sieving. These formulations also failed in providing total drug release within 30 days, even at a high drug load. FAMP/PLA50/water-soluble additive, ternary grounded particles were finally prepared with fructose, PLA50 oligomers or poly(ethylene glycol) (PEG) as the additive. Only PLA50 grounded particles with percolating FAMP-PEG microdomains allowed 100% release of FAMP over a 30 day period, at a quasi constant rate which depended primarily on solubility and channelling provided the flow was slow enough. Data are discussed in terms of the accessibility of the entrapped drug to the aqueous medium.

The use of additives to modulate the release of a sparingly water soluble drug entrapped in PLA50 microparticles: in vivo investigation.

Sustained and total release of the sparingly water soluble compound, namely 1-[2-(4-fluorobenzoyl)aminoethyl]-4-(7-methoxynaphthyl) piperazine hydrochloride (FAM), from poly (DL-lactic acid) (PLA50) microparticles was previously shown to be feasible if the particles are obtained by grinding a solid mixture composed of the polymer and a percolating array of the compound mixed with an additive. Such microparticles, where the additive was poly (ethylene glycol) (PEG), dimyristoylphosphatidylcholine (DMPC), or Poloxamer 6800, were administrated subcutaneously to rats either as depot or using a liquid vehicle. The variations of the plasma concentration vs time determined by high pressure liquid chromatography and fluorometric detection, were plotted for the various microparticle systems, blood being taken twice from each animal and each measurement being triplicated. Data were analysed by non-compartmental analysis, in order to evaluate the elimination constant, the half-life, the area under the curve and the bioavailability for each system. Kinetics experiments were performed over 24h and also for 7 days. It was found that, for the selected formulations, the release of the sparingly water soluble compound depends on the dissolution rate in vivo and on the physicochemical characteristics of the additive, including solubility and micelle formation. Data correlated well with the results of previous in vitro investigation.

The use of additives to modulate the release of a sparingly water soluble drug entrapped in PLA50 microparticles.

One of the major problems raised by the microencapsulation of drugs which are sparingly soluble in water is the difficulty to achieve a controlled and total release of the drug. It was previously shown that the microencapsulation of a model water insoluble drug, namely 1-[2-(4-fluorobenzoyl)aminoethyl]-4-(7-methoxynaphthyl) piperazine hydrochloride (FAMP) with a hydrophilic additive like low molar mass poly(ethylene glycol)s (PEG) can fulfil these requirements, provided all the drug + additive matter is in contact with the surrounding liquid medium via open pores and percolating channels. In this paper, PEG was replaced by other additives, selected because of their potential ability to increase the solubility of FAMP in pH = 7.4 isosomolar phosphate buffer (PBS). The idea was that increasing the solubility locally in microparticles could allow the drug to be released, despite its poor solubility in aqueous media like body fluids, and be absorbed before recrystallization. The solubility in PBS of FAMP mixed with additive, in the form of solid dispersions, was determined for various additives, namely citric acid, dimyristoyl DL-alpha-phosphatidyl choline (DMPC), poloxamer copolymers of different compositions and poly(dodecyl L-lysine citramidate) (PLCAC12(100)), an aggregate-forming hydrophilic polyelectrolyte containing 100%, hydrophobizing ester groups which can accommodate lipophilic compounds in hydrophobic pockets present in the aggregates. PEG was taken as a reference. It was found that DMPC, some poloxamers and the hydrophobized polyelectrolyte do increase the solubility of FAMP in PBS. Investigation was made of the release of FAMP from ground microparticles, whose loads were composed of FAMP combined with these solubilization-promoting additives. It was found that the release rate of FAMP from such systems can be increased and modulated to achieve an in vitro sustained release over a 20-30 day period and secure exhaustion of the particles at the end of this period.