Eduardo Ansorena

PLGA-based nanoparticles: an overview of biomedical applications

Poly(lactic-co-glycolic acid) (PLGA) is one of the most successfully developed biodegradable polymers. Among the different polymers developed to formulate polymeric nanoparticles, PLGA has attracted considerable attention due to its attractive properties: (i) biodegradability and biocompatibility, (ii) FDA and European Medicine Agency approval in drug delivery systems for parenteral administration, (iii) well described formulations and methods of production adapted to various types of drugs e.g. hydrophilic or hydrophobic small molecules or macromolecules, (iv) protection of drug from degradation, (v) possibility of sustained release, (vi) possibility to modify surface properties to provide stealthness and/or better interaction with biological materials and (vii) possibility to target nanoparticles to specific organs or cells. This review presents why PLGA has been chosen to design nanoparticles as drug delivery systems in various biomedical applications such as vaccination, cancer, inflammation and other diseases. This review focuses on the understanding of specific characteristics exploited by PLGA-based nanoparticles to target a specific organ or tissue or specific cells.

Effective GDNF brain delivery using microspheres-A promising strategy for Parkinson’s disease

Glial cell line-derived neurotrophic factor (GDNF) has shown promise in the treatment of neurodegenerative disorders of basal ganglia origin such us Parkinsons disease (PD). In this study, we investigated the neurorestorative effect of controlled GDNF delivery using biodegradable microspheres in an animal model with partial dopaminergic lesion. Microspheres were loaded with N-glycosylated recombinant GDNF and prepared using the Total Recirculation One-Machine System (TROMS). GDNF-loaded microparticles were unilaterally injected into the rat striatum by stereotaxic surgery two weeks after a unilateral partial 6-OHDA nigrostriatal lesion. Animals were tested for amphetamine-induced rotational asymmetry at different times and were sacrificed two months after microsphere implantation for immunohistochemical analysis. The putative presence of serum IgG antibodies against rat glycosylated GDNF was analyzed for addressing safety issues. The results demonstrated that GDNF-loaded microspheres, improved the rotational behavior induced by amphetamine of the GDNF-treated animals together with an increase in the density of TH positive fibers at the striatal level. The developed GDNF-loaded microparticles proved to be suitable to release biologically active GDNF over up to 5 weeks in vivo. Furthermore, none of the animals developed antibodies against GDNF demonstrating the safety of glycosylated GDNF use.

Sustained release of bioactive glycosylated glial cell-line derived neurotrophic factor from biodegradable polymeric microspheres

Glial cell-line derived neurotrophic factor (GDNF), a potent neurotrophic factor for dopaminergic neurons, appeared as a promising candidate for treating Parkinsons disease. GDNF microencapsulation could ensure protection against degradation due to the fragile nature of the protein. Poly(lactide-co-glycolide) (PLGA) microparticles loaded with recombinant glycosylated GDNF obtained in a mammalian cell line were prepared by TROMS, a semi-industrial technique capable of encapsulating fragile molecules maintaining their native properties. The effects of several parameters as PLGA copolymer type, PEG 400 quantity co-encapsulated with GDNF or drug loading, on the properties of the particles were investigated. Microparticles showed a mean diameter between 8 and 30 microm, compatible with their stereotaxic implantation. The drug entrapment efficiency ranged from 50.6% to 100% depending on the microsphere composition. GDNF was better encapsulated using hydrophilic polymers with high molecular weight such as RG 503H. In vitro drug release was influenced by the polymer type as well as by the amount of PEG 400 co-encapsulated with GDNF. Microparticles prepared using PLGA RG 503H released 67% of the total protein content within 40 days. Moreover, very low concentrations of poly(vinyl alcohol) were detected after microparticles washing and freeze-drying. Finally, a PC-12 bioassay demonstrated that the in vitro GDNF released was bioactive.

Drug development in Parkinson's disease: from emerging molecules to innovative drug delivery systems.

Current treatments for Parkinsons disease (PD) are aimed at addressing motor symptoms but there is no therapy focused on modifying the course of the disease. Successful treatment strategies have been so far limited and brain drug delivery remains a major challenge that restricts its treatment. This review provides an overview of the most promising emerging agents in the field of PD drug discovery, discussing improvements that have been made in brain drug delivery for PD. It will be shown that new approaches able to extend the length of the treatment, to release the drug in a continuous manner or to cross the blood-brain barrier and target a specific region are still needed. Overall, the results reviewed here show that there is an urgent need to develop both symptomatic and disease-modifying treatments, giving priority to neuroprotective treatments. Promising perspectives are being provided in this field by rasagiline and by neurotrophic factors like glial cell line-derived neurotrophic factor. The identification of disease-relevant genes has also encouraged the search for disease-modifying therapies that function by identifying molecularly targeted drugs. The advent of new molecular and cellular targets like α-synuclein, leucine-rich repeat serine/threonine protein kinase 2 or parkin, among others, will require innovative delivery therapies. In this regard, drug delivery systems (DDS) have shown great potential for improving the efficacy of conventional and new PD therapy and reducing its side effects. The new DDS discussed here, which include microparticles, nanoparticles and hydrogels among others, will probably open up possibilities that extend beyond symptomatic relief. However, further work needs to be done before DDS become a therapeutic option for PD patients.

Long-term neuroprotection and neurorestoration by glial cell-derived neurotrophic factor microspheres for the treatment of Parkinson's disease.

Glial cell‐derived neurotrophic factor is a survival factor for dopaminergic neurons and a promising candidate for the treatment of Parkinsons disease. However, the delivery issue of the protein to the brain still remains unsolved. Our aim was to investigate the effect of long‐term delivery of encapsulated glial cell‐derived neurotrophic factor within microspheres.

Vascular endothelial growth factor‐loaded injectable hydrogel enhances plasticity in the injured spinal cord

We hypothesized that vascular endothelial growth factor (VEGF)-containing hydrogels that gelify in situ after injection into a traumatized spinal cord, could stimulate spinal cord regeneration. Injectable hydrogels composed of 0.5% Pronova UPMVG MVG alginate, supplemented or not with fibrinogen, were used. The addition of fibrinogen to alginate had no effect on cell proliferation in vitro but supported neurite growth ex vivo. When injected into a rat spinal cord in a hemisection model, alginate supplemented with fibrinogen was well tolerated. The release of VEGF that was incorporated into the hydrogel was influenced by the VEGF formulation [encapsulated in microspheres or in nanoparticles or in solution (free)]. A combination of free VEGF and VEGF-loaded nanoparticles was mixed with alginate:fibrinogen and injected into the lesion of the spinal cord. Four weeks post injection, angiogenesis and neurite growth were increased compared to hydrogel alone. The local delivery of VEGF by injectable alginate:fibrinogen-based hydrogel induced some plasticity in the injured spinal cord involving fiber growth into the lesion site.

Production of highly pure human glycosylated GDNF in a mammalian cell line.

The administration of glial cell line-derived neurotrophic factor (GDNF) has emerged as a promising strategy for the treatment of several diseases of the nervous system as Parkinsons disease, amyotrophic lateral sclerosis, spinal cord injury and nerve regeneration as well as ocular diseases and drug addictions. A procedure for the purification of human recombinant glycosylated GDNF using a mammalian expression system as the source of the protein is discussed in the present paper. The neurotrophic factor was purified using cation exchange chromatography and gel filtration. A human cell line was chosen as the source of therapeutic protein, since a recombinant protein with a structure and glycosylation pattern equivalent to the native form is desirable for its prospective therapeutic utilization. The activity of the highly pure protein obtained was confirmed with a cell-based bioassay. The purified protein is suitable for its in vivo evaluation in animals and for possible subsequent clinical application.

Unfolded protein response induced by Brefeldin A increases collagen type I levels in hepatic stellate cells through an IRE1α, p38 MAPK and Smad-dependent pathway.

Unfolded protein response (UPR) triggered as a consequence of ER stress has been shown to be involved in the development of different pathologies, including fibrotic disorders. In the present paper we explore the role played by UPR on a key fibrogenic parameter in the liver: collagen type I levels in activated hepatic stellate cells (HSC). Using Brefeldin A (BFA) as an ER stress inducer we found that UPR correlated with enhanced mRNA and protein levels of collagen type I in a cell line of immortalized non-tumoral rat HSC. Analysis of the three branches of UPR revealed the activation of IRE1α, PERK and ATF6 in response to BFA, although PERK activation was shown not to be involved in the fibrogenic action of BFA. BFA also activated p38 MAPK in an IRE1α-dependent way and the p38 MAPK inhibitor SB203580 prevented the increase in collagen type I mRNA and protein levels caused by BFA, suggesting the involvement of this kinase on this effect. Analysis of Smad activation showed that phosphorylated nuclear levels of Smad2 and 3 were increased in response to BFA treatment. Inhibition of Smad3 phosphorylation by SIS3 prevented the enhancement of collagen type I levels caused by BFA. Pretreatment with IRE1α and p38 MAPK inhibitors also prevented the increased p-Smad3 accumulation in the nucleus, suggesting an IRE1α-p38 MAPK-Smad pathway to be responsible for the fibrogenic action of BFA on HSC.

Brain drug delivery systems for neurodegenerative disorders

Neurodegenerative disorders (NDs) are rapidly increasing as population ages. However, successful treatments for NDs have so far been limited and drug delivery to the brain remains one of the major challenges to overcome. There has recently been growing interest in the development of drug delivery systems (DDS) for local or systemic brain administration. DDS are able to improve the pharmacological and therapeutic properties of conventional drugs and reduce their side effects. The present review provides a concise overview of the recent advances made in the field of brain drug delivery for treating neurodegenerative disorders. Examples include polymeric micro and nanoparticles, lipidic nanoparticles, pegylated liposomes, microemulsions and nanogels that have been tested in experimental models of Parkinsons, Alzheimers and Huntingtons disease. Overall, the results reviewed here show that DDS have great potential for NDs treatment.

A simple and efficient method for the production of human glycosylated glial cell line-derived neurotrophic factor using a Semliki Forest virus expression system

Human glial cell line-derived neurotrophic factor (hGDNF) is a very promising protein for the treatment of Parkinsons disease and other neurodegenerative disorders. The present work describes a quick and simple method to obtain a high amount of purified hGDNF using a mammalian cell-derived system. The method is based on the high expression level provided by a Semliki Forest virus vector and its ability to induce a strong shut-off of host-cell protein synthesis in mammalian cells. As a result, hGDNF is the only protein present in the supernatant and can be efficiently purified by a single chromatographic step. Using this system it was possible to eliminate other secreted proteins from the culture medium, like insulin-like growth factor-5, which are hard to remove using other hGDNF production methods. Purified hGDNF presents a complex glycosylation pattern typical of mammalian expression systems and is biologically active. This protocol could be extended to other secreted proteins and could be easily scaled up for industrial purposes.