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Releasing a protein according to a zero-order profile without protein denaturation during the polymeric microparticle degradation process is very challenging. The aim of the current study was to develop protein-loaded microspheres with new PLGA based penta-block copolymers for a linear sustained protein release. Lysozyme was chosen as model protein and 40 µm microspheres were prepared using the solid-in-oil-in-water solvent extraction/evaporation process. Two types of PLGA-P188-PLGA penta-block copolymers were synthetized with two PLGA-segments molecular weight (20 kDa or 40 kDa). The resulting microspheres (50P20-MS and 50P40-MS) had the same size, an encapsulation efficiency around 50-60% but different porosities. Their protein release profiles were complementary: linear but non complete for 50P40-MS, non linear but complete for 50P20-MS. Two strategies, polymer blending and microsphere mixing, were considered to match the release to the desired profile. The (1:1) microsphere mixture was successful. It induced a bi-phasic release with a moderate initial burst (around 13%) followed by a nearly complete linear release for 8 weeks. This study highlighted the potential of this penta-block polymer where the PEO block mass ratio influence clearly the Tg and consequently the microsphere structure and the release behavior at 37 °C. The (1:1) mixture was a starting point but could be finely tuned to control the protein release., Competing Interests: Declaration of Competing Interest The authors declared that there is no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Hypothesis: Prilling process is one of advanced techniques for manufacturing microspheres of controlled and uniform size. In this process, homogenous polymer droplets fall into an extraction medium. The aim of this study was to identify the key parameters influencing the behavior of PLGA polymer-based droplets falling into a complex extraction medium, to select appropriate conditions for prilling., Experiments: Polymer solutions and extraction media were characterized by determining their viscosity, density and surface tension. A simple model simulating the prilling process was developed to study droplet behavior. Particle shape and velocity at the air-liquid interface and during sedimentation in the container were analyzed step by step. The correlations between the variables studied were visualized by principal component analysis (PCA)., Findings: Droplet deformation at the interface greatly affected the recovery and final particle shape. It depended on the viscosity ratio of polymer solution/extraction medium. The particle shape recovery depended on the viscosity and density of extraction media and polymer solutions. The solidification speed is also an important parameter. In media which the solvent diffused slowly, particles were able to relax and recover their shape, however, they can also deform during sedimentation and collision with the bottom of the cuvette., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Here, we aimed to develop protein loaded microspheres (MSs) using penta-block PLGA-based copolymers to obtain sustained and complete protein release. We varied MS morphology and studied the control of protein release. Lysozyme was used as a model protein and MSs were prepared using the solid-in-oil-in-water emulsion solvent extraction method. We synthesized and studied various penta-block PLGA-based copolymers. Copolymer characteristics (LA/GA ratio and molecular weight of PLGA blocks) influenced MS morphology. MS porosity was influenced by process parameters (such as solvent type, polymer concentration, emulsifying speed), whereas the aqueous volume for extraction and stabilizer did not have a significant effect. MSs of the same size, but different morphologies, exhibited different protein release behavior, with porous structures being essential for the continuous and complete release of encapsulated protein. These findings suggest strategies to engineer the morphology of MSs produced from PLGA-based multi-block copolymers to achieve appropriate release rates for a protein delivery system., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Stem cells combined with biodegradable injectable scaffolds releasing growth factors hold great promises in regenerative medicine, particularly in the treatment of neurological disorders. We here integrated human marrow-isolated adult multilineage-inducible (MIAMI) stem cells and pharmacologically active microcarriers (PAMs) into an injectable non-toxic silanized-hydroxypropyl methylcellulose (Si-HPMC) hydrogel. The goal is to obtain an injectable non-toxic cell and growth factor delivery device. It should direct the survival and/or neuronal differentiation of the grafted cells, to safely transplant them in the central nervous system, and enhance their tissue repair properties. A model protein was used to optimize the nanoprecipitation conditions of the neuroprotective brain-derived neurotrophic factor (BDNF). BDNF nanoprecipitate was encapsulated in fibronectin-coated (FN) PAMs and the in vitro release profile evaluated. It showed a prolonged, bi-phasic, release of bioactive BDNF, without burst effect. We demonstrated that PAMs and the Si-HPMC hydrogel increased the expression of neural/neuronal differentiation markers of MIAMI cells after 1week. Moreover, the 3D environment (PAMs or hydrogel) increased MIAMI cells secretion of growth factors (b-NGF, SCF, HGF, LIF, PlGF-1, SDF-1α, VEGF-A & D) and chemokines (MIP-1α & β, RANTES, IL-8). These results show that PAMs delivering BDNF combined with Si-HPMC hydrogel represent a useful novel local delivery tool in the context of neurological disorders. It not only provides neuroprotective BDNF but also bone marrow-derived stem cells that benefit from that environment by displaying neural commitment and an improved neuroprotective/reparative secretome. It provides preliminary evidence of a promising pro-angiogenic, neuroprotective and axonal growth-promoting device for the nervous system., Statement of Significance: Combinatorial tissue engineering strategies for the central nervous system are scarce. We developed and characterized a novel injectable non-toxic stem cell and protein delivery system providing regenerative cues for central nervous system disorders. BDNF, a neurotrophic factor with a wide-range effect, was nanoprecipitated to maintain its structure and released in a sustained manner from novel polymeric microcarriers. The combinatorial 3D support, provided by fibronectin-microcarriers and the hydrogel, to the mesenchymal stem cells guided the cells towards a neuronal differentiation and enhanced their tissue repair properties by promoting growth factors and cytokine secretion. The long-term release of physiological doses of bioactive BDNF, combined to the enhanced secretion of tissue repair factors from the stem cells, constitute a promising therapeutic approach., (Copyright © 2016 Acta Materialia Inc. All rights reserved.)

In recent years, cell-based therapies using adult stem cells have attracted considerable interest in regenerative medicine. A tissue-engineered construct for cartilage repair should provide a support for the cell and allow sustained in situ delivery of bioactive factors capable of inducing cell differentiation into chondrocytes. Pharmacologically active microcarriers (PAMs), made of biodegradable and biocompatible poly (D,L-lactide-co-glycolide acid) (PLGA), are a unique system which combines these properties in an adaptable and simple microdevice. This device relies on nanoprecipitation of proteins encapsulated in polymeric microspheres with a solid in oil in water emulsion-solvent evaporation process, and their subsequent coating with extracellular matrix protein molecules. Here, we describe their preparation process, and some of their characterization methods for an application in cartilage tissue engineering.

The challenge of tissue engineering of the infarcted heart is how to improve stem cell engraftment, survival, homing, and differentiation for myocardial repair. We here propose to integrate human adipose-derived stem cells (ADSCs) and pharmacologically active microcarriers (PAMs), a three-dimensional (3D) carrier of cells and growth factors, into an injectable hydrogel (HG), to obtain a system that stimulates the survival and/or differentiation of the grafted cells toward a cardiac phenotype. PAMs are biodegradable and non-cytotoxic poly(lactic-co-glycolic acid) (PLGA) microspheres conveying cells on their 3D surface that deliver continuously and in a controlled manner a growth factor (GF) acting on the transported cells and on the microenvironment to improve engraftment. The choice of the appropriate GF and its protection during the formulation process and delivery are essential. In this study two GFs, hepatocyte growth factor (HGF) and insulin-like growth factor (IGF-1), have been encapsulated under a solid state in order to limit their interaction with the polymer and conserve their integrity. GF precipitation conditions and release profile from PAMs have been first investigated before combining them to ADSCs. The released IGF-1 and HGF induced the protein synthesis of cardiac differentiation markers GATA4, Nkx2.5, cTnI and CX43 after 1week in vitro. Moreover, the GFs accelerated cell cycle progression, as suggested by the increased expression of Cyclin D1 mRNA and the widespread distribution of Ki67 protein. Integrating PAMs within the thermosensitive P407 hydrogel increased their elastic properties but decreased the transcription of most cardiac markers. In contrast, CX43 expression increased in ADSC-PAM-GF complexes embedded within the hydrogel compared to the ADSCs cultured alone in the absence of P407. These results suggest that particulate scaffolds releasing HGF and IGF-1 may be beneficial for applications in tissue-engineering strategies for myocardial repair and the association with a P407 hydrogel can increase substrate elasticity and junction connections in ADSCs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The use of injectable scaffolding materials for in vivo tissue regeneration has raised great interest in various clinical applications because it allows cell implantation through minimally invasive surgical procedures. In case of cartilage repair, a tissue engineered construct should provide a support for the cell and allow sustained in situ delivery of bioactive factors capable of inducing cell differentiation into chondrocytes. Pharmacologically active microcarriers (PAMs), made of biodegradable poly(d,l-lactide-co-glycolide acid) (PLGA), are a unique system, which combines these properties in an adaptable and simple microdevice. However, a limitation of such scaffold is low and incomplete protein release that occurs using the hydrophobic PLGA based microspheres. To circumvent this problem, we developed a novel formulation of polymeric PAMs containing a P188 poloxamer, which protects the protein from denaturation and may positively affect chondrogenesis. This poloxamer was added as a free additive for protein complexation and as a component of the scaffold covalently linked to PLGA. This procedure allows getting a more hydrophilic scaffold but also retaining the protective polymer inside the microcarriers during their degradation. The novel PLGA-P188-PLGA PAMs presenting a fibronectin-covered surface allowed enhanced MSC survival and proliferation. When engineered with TGFβ3, they allowed the sustained release of 70% of the incorporated TGF-β3 over time. Importantly, they exerted superior chondrogenic differentiation potential compared to previous FN-PAM-PLGA-TGF-β3, as shown by an increased expression of specific cartilage markers such as cartilage type II, aggrecan and COMP. Therefore, this microdevice represents an efficient easy-to-handle and injectable tool for cartilage repair., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A promising strategy to repair injured organs is possible by delivering a growth factor via poly-(d,l lactide-co-glycolide) (PLGA) microspheres; the latter are coated with adhesion molecules that serve as a support for cell delivery. At present, PLGA is not the optimal choice of polymer because of poor or incomplete protein release. The use of a more hydrophilic PLGA-PEG-PLGA (A-B-A) copolymer increases the degree of protein release. In this work, the impact of different combinations of (B) and (A) segments on the protein-release profile has been investigated. Continuous-release profiles, with no lag phases, were observed. The triblock ABA with a low molecular weight of PEG and a high molecular weight of PLGA showed an interesting release pattern with a small burst (<10% in 48 h) followed by sustained, protein release over 36 days. Incomplete protein release was found to be due to various causes: protein adsorption, protein aggregation and protein denaturation under acidic conditions. Interestingly, cell viability and cell adhesion on microspheres coated with fibronectin highlight the interest of these polymers for tissue engineering applications., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The aim of this work was to develop a new microparticulate system able to form a complex with radionuclides with a high yield of purity for diagnostic or therapeutic applications. Owing to its properties potato starch was chosen as starting material and modified by oxidization and coupling of a ligand (polyamine) enabling modified starch to chelate radionuclides. The choice of suitable experiments was based on a combination of a Rechtschaffner experimental design and a surface response design to determine the influence of experimental parameters and to optimize the final product. Starch-based microparticle formulations from the experimental plans were compared and characterized through particle size analysis, scanning electron microscopy, elemental analysis and, for the most promising formulations, by in vitro labeling stability studies and determination of free polyamine content or in vivo imaging studies. The mechanism of starch-based microparticle degradation was identified by means of size measurements. The results of the Rechtschaffner design showed the positive qualitative effect of the temperature and the duration of coupling reaction whereas surface response analysis clearly showed that, by increasing the oxidization level and starch concentration, the nitrogen content in the final product is increased. In vitro and in vivo characterization led to identification of the best formulation. With a size around 30 μm, high radiochemical purity (over 95%) and a high signal-to-noise ratio (over 600), the new starch-based microparticulate system could be prepared as ready-to-use kits and sterilized without modification of its characteristics, and thus meet the requirement for in vivo diagnostic and therapeutic applications., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Drug delivery via biodegradable microparticles benefits from both the protection of the encapsulated drug from hazardous conditions and the controlled release of the encapsulated drug, thereby reducing the administration frequency and improving patient compliance. Microsphere-size particle distribution is considered as being an important factor that affects the choice of the administration route and the drug-release rate. Significant research efforts have been directed towards the production of monodispersed "designer" particles. Amongst various techniques, some have been examined from lab-scale to industrial-scale. This review provides a global overview of monodispersed microparticle production methods and then focuses on recent processes being used to produce biodegradable microparticles applied in the pharmaceutical field. Further discussion about the choice of process according to the microparticle objectives of use is suggested., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The prognosis of patients with malignant glioma remains extremely poor, despite surgery and improvements in radio- and chemo-therapies. Nanotechnologies hold great promise in glioma therapy as they protect the therapeutic agent and allow its sustained release. However, new paradigms permitting tumor-specific targeting and extensive intratumoral distribution must be developed to efficiently deliver nanoparticles. Modifications and functionalizations of nanoparticles have been developed to specifically track tumor cells. However, these nanoparticles have yielded few clinical results due to intra-patient heterogeneity and inter-patient variability. Stem cells with a specific tropism for brain tumors could be used as delivery vehicles for nanoparticles. Indeed, these cells have a natural tendency to migrate and distribute within the tumor mass and they can also incorporate nanoparticles. Stem cell therapy combined with nanotechnology could be a promising tool to efficiently deliver drugs to brain tumors., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The prognosis of patients with malignant glioma remains extremely poor, despite surgery and improvements in radio- and chemo-therapies. Nanotechnologies represent great promise in glioma therapy as they protect therapeutic agent and allow its sustained release. However, new paradigms allowing tumor specific targeting and extensive intratumoral distribution must be developed to efficiently deliver nanoparticles (NPs). Knowing the tropism of mesenchymal stem cells (MSCs) for brain tumors, the aim of this study was to obtain the proof of concept that these cells can be used as NP delivery vehicles. Two types of NPs loaded with coumarin-6 were investigated: poly-lactic acid NPs (PLA-NPs) and lipid nanocapsules (LNCs). The results show that these NPs can be efficiently internalized into MSCs while cell viability and differentiation are not affected. Furthermore, these NP-loaded cells were able to migrate toward an experimental human glioma model. These data suggest that MSCs can serve as cellular carriers for NPs in brain tumors., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Cartilage engineering using mesenchymal stem cells (MSC) will require the use of a scaffold which will act as a support for cell adhesion keeping the cells in the cartilage defect. Optimally, a tissue engineered construct should allow sustained delivery of bioactive factors capable of inducing MSC differentiation into chondrocytes and should be easily injected inside the cartilage lesions to avoid surgical operations. We therefore developed pharmacologically active microcarriers (PAM) made of poly-lactic-co-glycolic acid (PLGA) produced using an oil-in-water (o/w) emulsion method. The microspheres were coated with a biomimetic surface of fibronectin (FN) and engineered to release TGF-beta3 as a chondrogenic differentiation factor. When human MSCs were incubated in vitro with TGF-beta3 releasing FN-coated PAMs in chondrogenic medium, they firmly adhered onto the surface of PAMs rapidly forming cell aggregates. After 3 weeks, strong up-regulation of cartilage-specific markers was observed both at the mRNA and protein level whereas osteogenic or adipogenic genes could not be detected. Importantly, implantation of MSC/TGF-beta3 releasing PAM complexes in SCID mice resulted in the formation of histologically resembling cartilage which stained positive for chondrocyte markers, collagen II and aggrecan. The present study demonstrated that functionalized PLGA-based microparticles can provide an appropriate environment for chondrogenic differentiation of MSCs and should contribute to injectable biomedical device development improving in vivo cartilage engineering., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Incomplete protein release from PLGA-based microspheres due to protein interactions with the polymer is one of the main issues in the development of PLGA protein-loaded microspheres. In this study, a two-dimensional adsorption model was designed to rapidly assess the anti-adsorption effect of formulation components (additives, additives blended with the polymer or modified polymers). Lysozyme was chosen as a model protein because of its strong, non-specific adsorption on the PLGA surface. This study showed that PEGs, poloxamer 188 and BSA totally inhibited protein adsorption onto the PLGA37.5/25 layer. Similarly, it was emphasised that more hydrophilic polymers were less prone to protein adsorption. The correlation between this model and the in vitro release profile was made by microencapsulating lysozyme with a low loading in the presence of these excipients by a non-denaturing s/o/w encapsulation technique. The precipitation of lysozyme with the amphiphilic poloxamer 188 prior to encapsulation exhibited continuous release of active lysozyme over 3 weeks without any burst effect. To promote lysozyme release in the latter stage of release, a PLGA-PEG-PLGA tribloc copolymer was used; lysozyme was continuously released over 45 days in a biologically active form., (Copyright 2010 Elsevier B.V. All rights reserved.)

Proteins were precipitated to ensure their stability upon subsequent encapsulation within PLGA microspheres. Spherical, nanosized protein particles were formed by the addition of a salt (sodium chloride) and a water-miscible organic solvent (glycofurol) to protein solutions. Various process parameters were modified to optimize the precipitation efficiency of four model proteins: lysozyme, alpha-chymotrypsin, peroxidase and beta-galactosidase. As monitored by enzymatic activity measurement of the rehydrated particles, conditions to obtain more than 95% of reversible precipitates were defined for each protein. The study of the structure of the rehydrated particles by absorbance spectroscopy, fluorescence spectroscopy and circular dichroism showed an absence of structural-perturbation after precipitation. Protein particles were then microencapsulated within PLGA microspheres using s/o/w technique. The average encapsulation yield was around 80% and no loss of protein activity occurred after the encapsulation step. Additionally, a lysozyme in vitro release study showed that all of the released lysozyme was biologically active. This method of protein precipitation is appropriate for the encapsulation in PLGA microspheres of various proteins without inactivation.

One of the most challenging tasks in the delivery of therapeutic proteins from PLGA-based microparticles is the sustained and complete release of the protein in its native form. The mechanisms responsible for incomplete protein release from these devices are numerous and complex; the beneficial effect of different formulations has often been evaluated in vitro. Strategies employed for overcoming protein destabilization during the release step are reviewed in this paper. Proteins have been protected in the deleterious environment by adding stabilizers to the formulation, or by modifying the protein or the polymer. Alternatively, some strategies have aimed at avoiding the formation of the destabilizing environment. As experimental conditions may influence the results from in vitro release studies, we initially report precautions to avoid adverse effects.

The objective of the work was to prepare chitosan-Ca-alginate microparticles that can effectively deliver 5-ASA to the colon after peroral administration. For these requirements, a spray-drying technique was applied to 5-ASA/sodium alginate aqueous solution to obtain spherical particles having a mean diameter less than 10microm. The microparticles formed were cross-linked and coated into solution of CaCl(2) and chitosan to obtain stable microsystem. (1)H NMR and UV-vis spectra of 5-ASA have shown no degradation when working in adequate conditions, such as light protection, freshly prepared solution and use of nitrogen to prevent the oxidative self-coupling of 5-ASA moieties. By imaging with SEM, acceptable spherical morphology was observed, but also flattened, disk-shaped particles of smooth surface and low porosity. CLSM imaging showed dominant localization of chitosan in the particle wall, while for alginate, a homogeneous distribution throughout the particle was observed giving the particles negative charge. In the FTIR spectra of 5-ASA loaded Ca-alginate microparticles the characteristic peaks of 5-ASA were not altered indicating no covalent interaction between the drug and the polymer. DSC and X-ray diffraction studies revealed that 5-ASA was molecularly dispersed within the chitosan-Ca-alginate microparticles during the production process.

Purpose: To evaluate the potential delay of the retinal degeneration in rd1/rd1 mice using recombinant human glial cell line-derived neurotrophic factor (rhGDNF) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microspheres., Methods: rhGDNF-loaded PLGA microspheres were prepared using a water in oil in water (w/o/w) emulsion solvent extraction-evaporation process. In vitro, the rhGDNF release profile was assessed using radiolabeled factor. In vivo, rhGDNF microspheres, blank microspheres, or microspheres loaded with inactivated rhGDNF were injected into the vitreous of rd1/rd1 mice at postnatal day 11 (PN11). The extent of retinal degeneration was examined at PN28 using rhodopsin immunohistochemistry on whole flat-mount retinas, outer nuclear layer (ONL) cell counting on histology sections, and electroretinogram tracings. Immunohistochemical reactions for glial fibrillary acidic protein (GFAP), F4/80, and rhodopsin were performed on cryosections., Results: Significant delay of rod photoreceptors degeneration was observed in mice receiving the rhGDNF-loaded microspheres compared to either untreated mice or to mice receiving blank or inactivated rhGDNF microspheres. The degeneration delay in the eyes receiving the rhGDNF microspheres was illustrated by the increased rhodopsin positive signals, the preservation of significantly higher number of cell nuclei within the ONL, and significant b-wave increase. A reduction of the subretinal glial proliferation was also observed in these treated eyes. No significant intraocular inflammatory reaction was observed after the intravitreous injection of the various microspheres., Conclusions: A single intravitreous injection of rhGDNF-loaded microspheres slows the retinal degeneration processes in rd1/rd1 mice. The use of injectable, biodegradable polymeric systems in the vitreous enables the efficient delivery of therapeutic proteins for the treatment of retinal diseases.

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.

Purpose: To develop biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles prepared by an original emulsion-extraction process, with glycofurol, a nontoxic excipient, as polymer solvent., Methods: The preparation of microparticles consisted in dissolving polymer in glycofurol. This solution was emulsified in a vegetable oil, and then amphiphilic agent was added into the emulsion to extract glycofurol and lead to microparticle formation. Physicochemical studies were carried out, and an experimental design was prepared in order to elucidate the impact of the formulation composition on the microparticle characteristics. Finally, encapsulation tests were made with a model protein., Results: In a ternary diagram, a small feasibility area allowing particle formation was located. The resulting microparticles were spherical with a homogeneous, polymeric matrix structure. They exhibited a variable size from 3 to 15 microm, which was controlled by the different formulation parameters. Differential scanning calorimetry (DSC) analysis made it possible to detect their composition. Preliminary results showed that these particles were able to encapsulate a protein model, lysozyme., Conclusions: This simple and convenient technique enabled us to obtain spherical, biodegradable microparticles from acceptable excipients. Moreover, the process conditions made possible the encapsulation of drugs, including proteins.

Purpose: To determine (i) the efficiency of radiosensitizing 5-FU-loaded microspheres and (ii) the impact of microparticle formulation on response to treatment., Methods: C6 tumor-bearing rats were stereotactically implanted with microspheres and/or allocated to: control groups (untreated) or treatment (only radiotherapy; fast-release 5-FU microspheres + radiotherapy; slow-release 5-FU microspheres + radiotherapy). The next day, fractionated radiotherapy, limited to the hemibrain, was initiated in all treated animals. The irradiation cycle included 36 Gy, given in 9 sessions for 3 consecutive weeks. Tumor development was assessed by T2-weighted MRI., Results: 5-FU microspheres associated with radiotherapy caused a 47% complete remission rate (9/19) as opposed to the 8% rate (1/12) when radiotherapy alone or 0% in control animals. Drug delivery for 3 weeks produced better survival results (57%) compared to one-week sustained release (41%). MR images showed exponentially increasing tumor volumes during the first half of the radiotherapy cycle, followed by a decrease, and the disappearance of the tumor if survival exceeded 120 days., Conclusions: 5-FU controlled delivery is a promising strategy for radiosensitizing gliomas. Drug delivery system formulation is unambiguously implicated in both the response to treatment and the limitation of toxic side effects.

Only a few studies mention the existence of tyrosine hydroxylase immunoreactive neurons in the striatum. These neurons are known to be increased following lesion of the dopaminergic nigrostriatal pathway. Recently it has been shown that glial cell line-derived neurotrophic factor treatment was able to increase the number of these neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intoxicated primate. Here we report that, in the rat, these neurons are responsive to intrastriatal injection of 6-hydroxydopamine and that following the lesion their number tends to increase with time. Moreover, we have shown that L-DOPA treatment for 2 weeks or nerve growth factor treatment for 8 weeks are able to dramatically augment their number., (Copyright 2004 Elsevier Ireland Ltd.)

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).

Glial cell line-derived neurotrophic factor (GDNF) is a protein with potent trophic actions on dopaminergic neurons, which is under investigation as a therapeutic agent for the treatment of neurodegenerative disorders, including Parkinson's disease. The aim of this work was to develop GDNF-loaded microspheres, which could be implanted by stereotaxy in the brain and could offer an alternative strategy in the treatment of Parkinson's disease. A w/o/w extraction-evaporation technique was chosen to prepare protein-loaded microspheres. An in vitro release study of the protein was required to assess the retention of integrity and the performance of the microsphere formulation with regard to sustained release. In order to assess the in vitro release profile of the GDNF-loaded microspheres, a preliminary study was performed to select an appropriate buffer for GDNF stabilization, using experimental designs. GDNF was measured by both enzyme-linked immunosorbant assay (ELISA) and radioactivity using (125)I-GDNF. The GDNF-loaded microsphere release profile was assessed in a low continuous flow system, and showed a sustained release over 56 days of biologically active GDNF at clinically relevant doses.

Glial cell line-derived neurotrophic factor (GDNF) offers the possibility to stimulate axonal regeneration of mesencephalic dopaminergic neurons, which are affected in Parkinson's disease. Nevertheless, a safe and efficient GDNF delivery system that may be used in clinical trials is still lacking. In a previous study, we showed that GDNF-releasing microspheres can deliver the neurotrophic factor for 2 months, allowing in a partial rat model of Parkinson's disease a sprouting of the preserved dopaminergic fibers and functional improvement 8 weeks after the treatment. The present study confirms these previous observations and shows that the amphetamine-induced rotation score is still decreased 24 weeks after the end of GDNF delivery. Nevertheless, the improvement was not statistically significant at the latest time point due to the spontaneous reinnervation observed in the model used.

The recent identification of neurotrophic factors, such as the glial cell line derived neurotrophic factor (GDNF), acting on mesencephalic dopaminergic neurons, offers the possibility to stimulate the axonal regeneration of these cells which are affected in Parkinson's disease. Nevertheless, a safe and efficient GDNF delivery system that may be used in clinical trials is still lacking. We have developed GDNF-releasing microspheres capable of releasing the neurotrophic factor for at least 2 months in vivo. In this study we demonstrate that these microspheres, when implanted in the brains of 'Parkinsonian' rats, were well tolerated, and were able to induce sprouting of the preserved dopaminergic fibers with synaptogenesis. Moreover, this neural regeneration was accompanied by functional improvement. The implantation of GDNF-releasing microspheres could be a promising strategy in the treatment of Parkinson's disease.

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.

A formulation process, based on the inversion phase of an emulsion, was used to prepare lipid nanocapsules. Triglycerides, lecithin, salted water and hydroxy stearate of poly(ethylene glycol) were used in the preparation. The amounts of each that allowed nanocapsules to be formed described a feasibility domain within a ternary diagram. The size distribution of various nanoparticulate carriers has already been shown to influence their applications. An experimental mixture design inside the feasibility domain has been used in order to approximate, through an empirical model, the influence of the quantitative composition of nanocapsules on their size distribution. Reduced cubic polynomial equations successfully modelled the evolution of responses in terms of particle average diameters and coefficients of variation. The results were presented using an analysis of response surface showing a scale of possible particle sizes between 20 and 95 nm and a coefficient of variation between 11 and 40%. Furthermore, this technique showed that the proportion of hydrophilic surfactant had a major influence on the average diameter and the size distribution of the particles decreasing when its proportion increased. On the contrary, the coefficient of variation and the average diameter slightly increase with the proportion of triglycerides. Such a tool offers major advantages to design the formulation of particles as a function of the required size distribution.

Background: Biodegradable loaded systems are promising devices for controlled and sustained release of anticancer drugs to brain tumours. We investigated the influence of drug-release profiles of 5-fluorouracil-loaded microspheres designed for the treatment of malignant gliomas., Materials and Methods: 2.5 mg 5-FU delivered by either fast. (1 formulation) or slow-(2 formulations) 5-FU release microspheres (MS) were tested in C6-glioma rat brains. Tumor response was assessed by T2-weighted MRI., Results: All treated animals, whatever the release profile considered, displayed a comparable 50% increase in life span versus controls. Delays in C6-glioma development appeared to correspond to the in vitro release periods of MS. In terms of curative prospect, complete remission was only observed in 11% of 5-FU-treated animals (4 out of 38)., Conclusion: Formulation was unambiguously implicated in the response observed after local delivery of 5-FU to glioma.

Purpose: The development of a novel in vitro system is required to assess the stability and release kinetics of a protein microsphere formulation used for drug delivery to the brain., Methods: Microspheres containing lysozyme as model protein were prepared using a (w/o/w) emulsion-solvent evaporation process. Both the active and total (active + inactive) encapsulation efficiencies and release profiles were determined. The biologic activity of lysozyme was measured using bacterial cell lysis; total protein content was measured using a 125I-radiolabel. A novel in vitro apparatus was developed to determine kinetics over a sustained time period (>30 days)., Results: The microencapsulation technique allowed an entrapment of active lysozyme at 80 +/- 4% and a sustained (>42 days) in vitro release. The kinetics study showed that the novel in vitro system was able to detect the release of low amounts (ng) of protein. To improve the stability of the protein within microspheres and allow the release of biologically active lysozyme, a basic additive ( Mg(OH)2 ) was successfully encapsulated., Conclusions: This novel in vitro system was appropriate to study protein microsphere release kinetics. In addition, the model is cost-effective and mimes brain physiological conditions more closely than previous models.

The use of glial cell line-derived neurotrophic factor (GDNF) appears to be a promising strategy to promote survival and function of the nigrostriatal dopaminergic pathway damaged in Parkinson's disease (PD). However, effective intracerebral administration is required for optimal therapeutic benefit and tools to evaluate such therapies must be developed. A rodent model of PD was therefore developed using striatal injection of 6-hydroxydopamine (6-OHDA) with simultaneous implantation of GDNF-delivering microspheres. The effects of GDNF released from microspheres were assessed by classical methods such as amphetamine-induced rotating behavior and tyrosine hydroxylase (TH) immunoreactivity, as well as by quantitative autoradiography using PE2I, a dopamine transporter (DAT) radiotracer, which is also suitable for SPET imaging in humans. 6-OHDA-lesioned animals that received microspheres without GDNF were used as controls. During the first 3 weeks after simultaneous lesion and implantation, the amphetamine-induced rotating behavior of GDNF-treated rats was improved compared to controls and an increase in TH expression (+26%) was measured in the striatum 6 weeks after lesion. In accordance with these results, an increase in striatal PE2I-labeled DAT density was obtained (+17%) after 3 and 6 weeks of treatment. In conclusion, this study demonstrates the neuroprotective action of GDNF delivered by microspheres and suggests that PE2I may be an appropriate radiotracer for use in SPET scintigraphy to follow up treatment of PD in humans., (Copyright 2002 Wiley-Liss, Inc.)

This study was performed (i) to monitor the diffusion of the anti-cancer drug 5-fluorouracil (5-FU) and (ii) to elucidate the fate of poly(lactide-co-glycolide) (PLGA) based microspheres within living rat brain tissue upon intracranial implantation. Drug-loaded microparticles were prepared using a solvent emulsion/extraction process and administered into healthy and C6 glioma-bearing Sprague-Dawley rats. The same surgical procedure was carried out with magnetite-loaded microspheres. To monitor 5-FU diffusion from the implantation site, tissue combustion was performed on animals implanted with tritiated drug microspheres. T2-weighted nuclear magnetic resonance imaging was undertaken on animals implanted with magnetite-loaded microspheres to determine microsphere localization after deposit. Results show that an important microparticle backflow occurs in healthy rats, whereas the microspheres remain at the site of administration in C6 glioma-bearing rats. Drug diffusion is limited to the vicinity of the implantation site.

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., (Copyright 2001 John Wiley & Sons, Ltd.)

The aim of this study was to determine the bioburden of PLGA microspheres produced by the solvent emulsion/extraction process as a means of determining an appropriate gamma-irradiation dose for sterilization. Bioburden was evaluated on the basis of ISO specifications. The analysis of initial microbial contamination was performed on blank microspheres, prepared by a non-aseptic laboratory scale process. A mean bioburden of 36.04 CFU (colony forming units)/110 mg microspheres was determined. Most of the detected germs originated from human commensal flora. According to the ISO dose-selection method, a gamma-irradiation dose of 19.6 kGy was found sufficient to ensure a sterility level of 10(-6). The effect of the selected irradiation dose on both the molecular weight of the polymer and the kinetics of 5-fluorouracil drug release from the microspheres was compared to the European Pharmacopeia recommended irradiation dose (25 kGy). This 20% reduced dose showed a lower extent of molecular weight reduction of PLGA and a better control of 5-FU release from microparticles. This can be related to reduce polymer radiation damage.

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.

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.

Purpose: The aim of this work was to understand the mechanism by which co-encapsulated PEG 400 improved the stability of NGF and allowed a continuous release from PLGA 37.5/25 microspheres., Methods: Microparticles were prepared according to the double emulsion method. PEG 400 was added with NGF in the internal aqueous phase (PEG/PLGA ratio 1/1 and 1.8/1). Its effect was investigated through interfacial tension studies. Protein stability was assessed by ELISA., Results: A novel application of PEG in protein stabilization during encapsulation was evidenced by adsorption kinetics studies. PEG 400 limited the penetration of NGF in the interfacial film of the primary emulsion. Consequently, it stabilized the NGF by reducing the contact with the organic phase. In addition, it avoided the NGF release profile to level off by limiting the irreversible NGF anchorage in the polymer layers. On the other hand, the amount of active NGF released in the early stages was increased. During microparticle preparation, NaCl could be added in the external aqueous phase to modify the structure of microparticles. This allowed to reduce the initial release rate without affecting the protein stability always encountered in the absence of PEG., Conclusions: PEG 400 appeared of major interest to achieve a continuous delivery of NGF over seven weeks from biodegradable microparticles prepared by the double emulsion technique.

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.

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.

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.

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%.

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%.

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.