Behavior of poly(d,l-lactic-co-glycolic acid) (PLGA)-based droplets falling into a complex extraction medium simulating the prilling process.

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.

Modulation of protein release from penta-block copolymer microspheres.

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.

Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres.

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.

Penta-block copolymer microspheres: Impact of polymer characteristics and process parameters on protein release.

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.

Pharmacologically active microcarriers delivering BDNF within a hydrogel: Novel strategy for human bone marrow-derived stem cells neural/neuronal differentiation guidance and therapeutic secretome enhancement.

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 & ß, 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.

Intravitreous injection of PLGA microspheres encapsulating GDNF promotes the survival of photoreceptors in the rd1/rd1 mouse.

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.

Scaffolds for Controlled Release of Cartilage Growth Factors.

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.

Striatal implantation of GDNF releasing biodegradable microspheres promotes recovery of motor function in a partial model of Parkinson's disease.

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.

In vitro study of GDNF release from biodegradable PLGA microspheres.

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.

Long-term effect of intra-striatal glial cell line-derived neurotrophic factor-releasing microspheres in a partial rat model of Parkinson's disease.

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.

Striatal tyrosine hydroxylase immunoreactive neurons are induced by L-dihydroxyphenylalanine and nerve growth factor treatment in 6-hydroxydopamine lesioned rats.

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.

Release kinetics of 5-fluorouracil-loaded microspheres on an experimental rat glioma.

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.

Anti-cancer drug diffusion within living rat brain tissue: an experimental study using [3H](6)-5-fluorouracil-loaded PLGA microspheres.

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 influence of lipid nanocapsule composition on their size distribution.

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.

Pharmacologically active microcarriers associated with thermosensitive hydrogel as a growth factor releasing biomimetic 3D scaffold for cardiac 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.

New PLGA-P188-PLGA matrix enhances TGF-ß3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells.

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 TGFß3, they allowed the sustained release of 70% of the incorporated TGF-ß3 over time. Importantly, they exerted superior chondrogenic differentiation potential compared to previous FN-PAM-PLGA-TGF-ß3, 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.

Protein-loaded PLGA-PEG-PLGA microspheres: a tool for cell therapy.

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.

Why and how to prepare biodegradable, monodispersed, polymeric microparticles in the field of pharmacy?

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.

The potential of combinations of drug-loaded nanoparticle systems and adult stem cells for glioma therapy.

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.

Mesenchymal stem cells as cellular vehicles for delivery of nanoparticles to brain tumors.

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.