Nanoprecipitated catestatin released from pharmacologically active microcarriers (PAMs) exerts pro-survival effects on MSC.

Catestatin (CST), a fragment of Chromogranin-A, exerts angiogenic, arteriogenic, vasculogenic and cardioprotective effects. CST is a very promising agent for revascularization purposes, in "NOOPTION" patients. However, peptides have a very short half-life after administration and must be conveniently protected. Fibronectin-coated pharmacologically active microcarriers (FN-PAM), are biodegradable and biocompatible polymeric microspheres that can convey mesenchymal stem cell (MSCs) and therapeutic proteins delivered in a prolonged manner. In this study, we first evaluated whether a small peptide such as CST could be nanoprecipitated and incorporated within FN-PAMs. Subsequently, whether CST may be released in a prolonged manner by functionalized FN-PAMs (FN-PAM-CST). Finally, we assessed the effect of CST released by FN-PAM-CST on the survival of MSCs under stress conditions of hypoxia-reoxygenation. An experimental design, modifying three key parameters (ionic strength, mixing and centrifugation time) of protein nanoprecipitation, was used to define the optimum condition for CST. An optimal nanoprecipitation yield of 76% was obtained allowing encapsulation of solid CST within FN-PAM-CST, which released CST in a prolonged manner. In vitro, MSCs adhered to FN-PAMs, and the controlled release of CST from FN-PAM-CST greatly limited hypoxic MSC-death and enhanced MSC-survival in post-hypoxic environment. These results suggest that FN-PAM-CST are promising tools for cell-therapy.

Pharmacologically active microcarriers: a tool for cell therapy.

To overcome certain problems encountered in cell therapy, particularly cell survival, lack of cell differentiation and integration in the host tissue, we developed pharmacologically active microcarriers (PAM). These biodegradable particles made with poly(D,L-lactic-co-glycolic acid) (PLGA) and coated with adhesion molecules may serve as a support for cell culture and may be used as cell carriers presenting a controlled delivery of active protein. They can thus support the survival and differentiation of the transported cells as well as their microenvironment. To develop this tool, nerve growth factor (NGF)-releasing PAM, conveying PC12 cells, were produced and characterized. Indeed, these cells have the ability to differentiate into sympathetic-like neurons after adhering to a substrate, in the presence of NGF, and can then release large amounts of dopamine. Certain parameters such as the size of the microcarriers, the conditions enabling the coating of the microparticles and the subsequent adhesion of cells were thus studied to produce optimized PAM.

In vivo evaluation of pharmacologically active microcarriers releasing nerve growth factor and conveying PC12 cells.

Cell therapy will probably become a major therapeutic strategy in the coming years. Nevertheless, few cells survive transplantation when employed as a treatment for neuronal disorders. To address this problem, we have developed a new tool, the pharmacologically active microcarriers (PAM). PAM are biocompatible and biodegradable microparticles coated with cell adhesion molecules, conveying cells on their surface and presenting a controlled delivery of growth factor. Thus, the combined effect of growth factor and coating influences the transported cells by promoting their survival and differentiation and favoring their integration in the host tissue after their complete degradation. Furthermore, the released factor may also influence the microenvironment. In this study, we evaluated their efficacy using nerve growth factor (NGF)-releasing PAM and PC12 cells, in a Parkinson's disease paradigm. After implantation of NGF-releasing or unloaded PAM conveying PC12 cells, or PC12 cells alone, we studied cell survival, differentiation, and apoptosis, as well as behavior of the treated rats. We observed that the NGF-releasing PAM coated with two synthetic peptides (poly-D-lysine and fibronectin-like) induced PC12 cell differentiation and reduced cell death and proliferation. Moreover, the animals receiving this implant presented an improved amphetamine-induced rotational behavior. These findings indicate that PAM could be a promising strategy for cell therapy of neurological diseases and could be employed in other situations with fetal cell transplants or with stem cells.

New thermoluminescent dosimeters (TLD): optimization and characterization of TLD threads sterilizable by autoclave.

To improve the performance of mono-extruded TLD threads as a dosimetric thermoluminescent tool (French Patent 9903729), a new process was developed by co-extrusion methodology leading to threads of 600 microm diameter with a 50 microm homogeneous polypropylene sheath. In this optimization work, study of parameters such as LiF:Mg,Cu,P powder granulometry, load rate and proportion of components led to an increased sensitivity of around 40%. Moreover, the co-extrusion technique allowed the threads to be sterilized by humid steam (134 degrees C/18 min) without significant variation of the linearity response between 0 and 30 Gy after gamma irradiation (60Co).

Therapeutic efficacy of 5-fluorouracil-loaded microspheres on rat glioma: a magnetic resonance imaging study.

The aim of this work was to assess the therapeutic efficacy of an intratumoral bolus injection of 5-fluorouracil (FU) compared to that of drug loaded in biodegradable microspheres, for the treatment of brain tumour. Experiments were carried out using a fast-growing C6-glioma rat model. The therapeutic protocols were performed 12 days after the injection of glioma cells. At this stage, the tumours were installed and the mean volume was 13 +/- 2 microl as measured by proton magnetic resonance (MR) imaging. This technique was used for the follow-up of the tumour volume with respect to time and therapy. In terms of rat survival, both therapies induced a significant 50% increase in animal life span (p < 0.05) compared to animals receiving no drug or unloaded microspheres. Whilst no cure was observed, analysis of the MR images showed that the local and sustained delivery of FU slowed the tumour development in the vicinity of the microspheres by a factor of 3, compared with the bolus intratumoral injection.

Neuroprotection of nerve growth factor-loaded microspheres on the D2 dopaminergic receptor positive-striatal neurones in quinolinic acid-lesioned rats: a quantitative autoradiographic assessment with iodobenzamide.

Huntington's disease (HD) results from the degeneration of striatal neurones, mainly gamma-aminobutyric acid (GABA)ergic projection neurones and lately cholinergic interneurones. The use of trophic factors as agents able to prevent such neural degeneration is a promising strategy. The aim of this study was to validate nerve growth factor-loaded (NGF-loaded) poly-D,L-lactide-co-glycolide (PLGA) microspheres for treatment of HD in a rat model with quinolinic acid lesion using autoradiographic study of D2 dopaminergic receptors (D2R). This target is expressed by about half of striatal neurones and its scintigraphic exploration has already been performed for the follow-up of this degenerative process. Ex vivo autoradiography of D2R performed with iodobenzamide, the widely used ligand for single photo emission computerized tomography, revealed slight neuroprotection. Moreover, tolerance of microspheres was demonstrated by in vitro autoradiography with the marker of gliosis, [(3)H]-PK 11195.

Development of microspheres for neurological disorders: from basics to clinical applications.

Drug delivery to the central nervous system remains a challenging area of investigation for both basic and clinical neuroscientists. Numerous drugs are generally excluded from blood to brain transfer due to the negligible permeability of the brain capillary endothelial wall, which makes up the blood brain barrier in vivo. For several years, we have explored the potential applications of the microencapsulation of therapeutic agents to provide local controlled drug release in the central nervous system. Due to their size, these microparticles can be easily implanted by stereotaxy in discreet, precise and functional areas of the brain without damaging the surrounding tissue. This type of implantation avoids the inconvenient insertion of large implants by open surgery and can be repeated if necessary. We have established the compatibility of poly(lactide-co-glycolide) microspheres with brain tissues. Presently, the most developed applications concern Neurology and Neuro-oncology, with local delivery of neurotrophic factors and antimitotic drugs into neurodegenerative lesions and brain tumours, respectively. The drugs that had been encapsulated by our group included nerve growth factor (NGF), 5-fluorouracil (5-FU), idoxuridine and BCNU. Preclinical studies have been performed with each drug. Studies with NGF are reported as an example. A phase I/II clinical trial has been carried out in patients with newly diagnosed glioblastomas to assess the potentialities of 5-FU-loaded microspheres when intracranially implanted.

Development of 5-iodo-2'-deoxyuridine milling process to reduce initial burst release from PLGA microparticles.

The aim of this study was to prepare 5-iodo-2'-deoxyuridine (IdUrd) loaded poly(d,l-lactide-co-glycolide) (PLGA) microspheres with a reduced initial burst in the in vitro release profile, by modifying the drug grinding conditions. IdUrd particle size reduction has been performed using spray-drying or ball milling. Spray-drying significantly reduced drug particle size with a change of the initial crystalline form to an amorphous one and led to a high initial burst. Conversely, ball milling did not affect the initial IdUrd crystallinity. Therefore, the grinding process was optimized to emphasize the initial burst reduction. A first step allowed us to set qualitative parameters such as ball number (7) and cooling with liquid nitrogen to obtain a mean size reduction and a narrow distribution. In a second step, three parameters including milling speed, drug amount and time were studied by a response surface analysis. The interrelationship between drug amount and milling speed was the most significant factor. To reduce particle size it should be necessary to use a moderate speed associated with a sufficient drug amount (400-500 mg). IdUrd release from microparticles prepared by the o/w emulsion/extraction solvent evaporation process with the lowest crystalline particle size (15.3 microns) was studied. Burst effect could be reduced significantly. Concerning the first phase of drug release, the burst was 8.7% for 15.3 microns compared to 19% for 19.5 microns milled drug particles.

Modulated release of IdUrd from poly (D,L-lactide-co-glycolide) microspheres by addition of poly (D,L-lactide) oligomers.

This paper reports the release characteristics of a radiosensitizer, 5-iodo-2'-deoxyuridine (IdUrd), from poly (D,L-lactide-co-glycolide) 50: 50 (PLGA) microparticles obtained by a phase separation technique. Poly (D,L-lactide) oligomers (D,L-PLA) were incorporated into the PLGA matrix in order to accelerate the overall drug release rate and regulate the triphasic release profile exhibited by the standard PLGA microparticles. For D,L-PLA (800), the burst effect was large and the IdUrd release was complete between 28 and 35 days. These results were attributed to rapid pore formation on the periphery of the microsphere in the early stages of incubation, due to hydrosolubility of the smallest oligomers (D,L-PLA (800)). In the case of D,L-PLA (1,100), drug release occurred over a six week period, the standard time course of conventional radiation therapy. The period during which the radiosensitizer was incorporated in human brain tumor cell nuclei after its entrapment in biodegradable microspheres was determined by using an organotypical tissue culture. The presence of radiosensitizer in the DNA of tumor cell nuclei was detected by immunohistochemical labelling of tumor fragments. IdUrd release from standard microspheres (7+/-0.5 weeks) was longer than from oligomer-containing batches. For D,L-PLA (800)-containing microspheres, the radiosensitizer was entirely released within 4. 5+/-0.5 weeks. The microspheres containing D,L-PLA (1,100) allowed an IdUrd release over a 5 to 6 week period. The ex vivo data were consistent with the in vitro findings in terms of release duration.

NGF release from poly(D,L-lactide-co-glycolide) microspheres. Effect of some formulation parameters on encapsulated NGF stability.

Poly(d,l-lactide-co-glycolide) (PLGA 37.5/25 and 25/50) biodegradable microparticles, which allow the locally delivery of a precise amount of a drug by stereotactic injection in the brain, were prepared by a W/O/W emulsion solvent evaporation/extraction method which had been previously optimized. The aim of this work was to study the influence of two formulation parameters (the presence of NaCl in the dispersing phase and the type of PLGA) on the NGF release profiles and NGF stability during microencapsulation. A honey-comb-like structure characterized the internal morphology of the microspheres. The initial burst was attributed to the rapid penetration of the release medium inside the matrix through a network of pores and to the desorption of weakly adsorbed protein from the surface of the internal cavities. The non-release fraction of the encapsulated protein observed after twelve weeks of incubation was accounted for firstly by the adsorption of the released protein on the degrading microparticles and secondly by the entanglement of the encapsulated protein in the polymer chains. The use of sodium chloride in the dispersing phase of the double emulsion markedly reduced the burst effect by making the microparticle morphology more compact. Unfortunately, it induced in parallel a pronounced NGF denaturation. Finally, it appeared that microparticles made from a hydrophilic uncapped PLGA 37.5/25 in the absence of salt, allowed the release of intact NGF at least during the first 24 h as determined by both ELISA and a PC12 cell-based bioassay.

Internal morphology of poly(D,L-lactide-co-glycolide) BCNU-loaded microspheres. Influence on drug stability.

The solvent extraction/evaporation process has been used to form poly(D,L-lactide-co-glycolide) (PLAGA) BCNU-loaded microspheres designed for use as intracranial controlled-release implants. Their actual payload could reach 25% with a 20-50 microns size distribution. Scanning electron microscopy showed that such carriers had a smooth surface and a spherical geometry. Differential scanning calorimetry analyses carried out on drug-loaded microspheres established that the PLAGA Tg was markedly shifted towards the low temperatures along with the disappearance of the BCNU melting endotherm. Annealing experiments performed at room temperature did not induce any change of the loaded microsphere DSC profiles. These features indicated that the BCNU acted as a plasticizer for the coating material and formed with it a solid solution. Similarly, stability of encapsulated BCNU was assessed in different conditions of storage. It appeared that drug degradation increased with temperature increase: 5.4, 8.8, 32.4 and 51.2% of decomposition after 3 month storage at -18, 4, room temperature (RT) and 37 degrees C respectively. Since the free drug was stable at 4 degrees C and experienced only 10.6% decomposition at RT during the same storage time, the state of solid solution involving the intimate mixing of the drug and the polyester in the matrix favors a progressive decomposition of BCNU. However, keeping the microspheres 6 months at -18 degrees C or 3 months at 4 degrees C prevents a loss of drug superior to 10%.

Preparation, purification and morphology of polymeric nanoparticles as drug carriers.

The aim of this work was to prepare biodegradable poly(D,L-lactic acid-co-glycolic acid) copolymer (PLAGA) nanoparticles by the solvent evaporation process and to incorporate an antifungal antibiotic, amphotericin B. Blank nanoparticles obtained were 130 +/- 27 nm in diameter. When amphotericin B was added in the organic phase, the final suspension showed two populations due to unbound drug. Free amphotericin B was removed by contacting the nanoparticle suspension with an adsorbent polymer. Amberlite XAD16, and subsequently ultrafiltering the medium. The drug payload was between 0.7 and 1.3%. To gain more insight in the cause of this low loading, we studied progesterone-loaded nanoparticles using PLAGA and polystyrene as models because progesterone and these polymers exhibit a degree of miscibility. In the case of polystyrene, nanoparticle drug content reached 8%.

In vitro study of the anti-leishmanial activity of biodegradable nanoparticles.

Leishmania are obligate intracellular parasites, responsible for leishmaniasis. Leishmaniasis are transmitted via insect vector to vertebrate hosts including humans. The infection was reproduced in vitro with promastigotes which can infect murine resident peritoneal cells. Amphotericin B was incorporated into poly(D, L-lactide-co-glycolide) nanoparticles, biodegradable drug carriers, to allow specific targeting inside the cell. The interaction of the drug with infected cells was determined by exposing macrophage cultures to drug carriers. The toxic effects of polymeric drug carriers were defined prior to exposing cells to drug-loaded nanoparticles. For contact times up to 4h, cells tolerated polymer concentrations of 0.01%. The viability of parasites after treatment was determined. Infected macrophages were incubated at 26 degrees C (which allows the transformation of amastigote to promastigote) along with loaded and unloaded nanoparticles, as well as the free drug alone, and a count of the parasites in the medium was recorded. Anti-leishmanial activity was observed with drug-free nanoparticles. This activity may arise through the release of hydrogen peroxide following the activation of macrophages. The incorporation of amphotericin B did not enhance this effect. Interestingly, trehalose, a cryoprotector of the freeze-dried nanoparticles, altered parasite growth and activated macrophages.