Expresión y purificación de GDNF para su microencapsulación y aplicación en la enfermedad de Parkinson

La enfermedad de Parkinosn (EP) es un proceso neurodegenerativo del sistema nervioso central que afecta a las neuronas de dopamina de la sustancia negra, núcleo mesoencefálico del control motor. La perdida en el cerebro de este neurotransmisor vital causa los síntomas de la enfermedad. La EP afecta actualmente a 200 de cada 100.000 personas y a 2 de cada 100 entre los mayores de 60 años. En España hay unos 110.000 enfermos. Además, hoy por hoy no se conoce nada que pueda prevenir o curar la enfermedad, ni existe ninguna prueba de laboratorio que permita diagnosticarla. Recentiemente se ha demostrado que el GDNF, factor neurotrófico derivado de las células gliales, es capaz de proteger las neuronas dopaminérgicas e incluso inducir la regeneración del tejido dopaminérgico dañado in vivo. El objetivo del trabajo fue diseñar y desarrolar un método de expresión y purificación de GDNF bioactivo para su posterior microencapsulación y aplicación en la enfermedad de Parkison. El sistema escogido para expresar el GDNF fue el sistema de células eucariotas de mamífero. El vector utilizado para la producción del GDNF en células eucariotas fue el pDEST26 (Tecnología Gateway de Invitrogen). Como sistema de expresión de GDNF se utilizaron las líneas celulares eucariota BHK, 293 y COS 7. Estas células fueron cultivadas en medio D-MEM (Invitrogen) complementado con un 10% de suero fetal bovino (FBS) y Penicilina/Streptomicina (100u/ml) (Invitrogen). La transfección se realizó con Lipofectamine Plus (Invitrogen). Se analizó la expresión de GDNF a nivel de mRNA mediante PCR y a nivel de proteína mediante Western Blot del medio condicionado. Los clones positivos se crecieron en botellas de cultivo de 850 cm2 (Corning) y se realizaron ciclos de recolección del medio. Cada ciclo fue analizado por SDS-PAGE y Western Blot. Para evaluar la actividad de la proteína se ha desarrollado un ensayo de actividad en el que se demuestra la diferenciación morfológica de células PC-12 inducida por GDNF. La presencia de los receptores GFRa1 y RET, necesarios para que el GDNF ejerza su acción, fue determinada por PCR. Las conclusiones obtenidas de este estudio son la obtención de GDNF recombinante a partir de un sistema de expresión en células eucariotas, el desarrollo de un protocolo para su posterior purificación y la obtención de GDNF recombinante biológicamente activo

Lipid nanoparticles for cyclosporine a administration: Development, characterization, and in vitro evaluation of their immunosuppression activity

Cyclosporine A (CsA) is an immunosuppressant commonly used in transplantation for prevention of organ rejection as well as in the treatment of several autoimmune disorders. Although commercial formulations are available, they have some stability, bioavailability, and toxicity related problems. Some of these issues are associated with the drug or excipients and others with the dosage forms. With the aim of overcoming these drawbacks, lipid nanoparticles (LN) have been proposed as an alternative, since excipients are biocompatible and also a large amount of surfactants and organic solvents can be avoided. CsA was successfully incorporated into LN using the method of hot homogenization followed by ultrasonication. Three different formulations were optimized for CsA oral administration, using different surfactants: Tween® 80, phosphatidylcholine, taurocholate and Pluronic® F127 (either alone or mixtures). Freshly prepared Precirol nanoparticles showed mean sizes with a narrow size distribution ranging from 121 to 202 nm, and after freeze-drying were between 163 and 270 nm, depending on the stabilizer used. Surface charge was negative in all LN developed. High CsA entrapment efficiency of approximately 100% was achieved. Transmission electron microscopy was used to study the morphology of the optimized LN. Also, the crystallinity of the nanoparticles was studied by X-ray powder diffraction and differential scanning calorimetry. The presence of the drug in LN surfaces was confirmed by X-ray photoelectron spectroscopy. The CsA LN developed preserved their physicochemical properties for 3 months when stored at 4°C. Moreover, when the stabilizer system was composed of two surfactants, the LN formulations were also stable at room temperature. Finally, the new CsA formulations showed in vitro dose-dependent immunosuppressive effects caused by the inhibition of IL-2 levels secreted from stimulated Jurkat cells. The findings obtained in this paper suggest that new lipid nanosystems are a good alternative to produce physicochemically stable CsA formulations for oral administration

Microencapsulación de factores neurotróficos: aplicación al tratamiento de la enfermedad de Parkinson

La enfermedad de Parkinson (EP) es una enfermedad degenerativa, lentamente progresiva caracterizada por temblor de reposo, cara inexpresiva, rigidez, lentitud al iniciar y practicar movimientos voluntarios. Neuropatológicamente se caracteriza por pérdida de células dopaminérgicas en la sustancia nigra lo cual conlleva déficit en el suministro de dopamina a nivel de ganglios basales. Los factores de crecimiento nervioso, o factores neurotróficos, que respaldan la supervivencia, crecimiento y desarrollo de las células cerebrales, son un tipo de terapia prometedora para la enfermedad de Parkinson. Se ha demostrado que el GDNF, factor neurotrófico derivado de la línea celular glial, protege las neuronas de dopamina y promueve su supervivencia en los modelos animales de la enfermedad de Parkinson. Sin embargo, la administración de proteínas en el cerebro no está exenta de dificultades, por ello, el sistema elegido para administrar el GDNF en el cerebro será uno de los puntos clave para el éxito del tratamiento. En este sentido, el uso de micropartículas formuladas a partir de polímeros biodegradables parece ser la estrategia más apropia. En nuestro grupo de investigación hemos desarrollado un protocolo de expresión y purificación de GDNF en células eucariótas de mamífero. El objetivo de este estudio es la microencapsulación de la proteína en partículas biodegradables

Topical application of acyclovir-loaded microparticles: quantification of the drug in porcine skin layers.

The goal of this work was to increase the amount of acyclovir (ACV) in the basal epidermis, site of Herpes virus simplex infections, using microparticles as carriers. Poly(d,l-lactic–co-glycolic acid) microparticles loaded with ACV were prepared using a solvent evaporation technique. ACV distribution into porcine skin after topical application of microparticles for 6, 24 and 88 h, was determined by horizontal slicing of the skin. An ACV suspension served for comparison. The results showed that, at 6 and 24 h, the quantity of the drug in the basal epidermis with the microparticles, is similar to that obtained with the ACV suspension. However, after 88 h, the ACV reservoir in the basal epidermis was higher with the microparticles compared with the control suspension. This could be explained by the controlled drug release produced by the vector in the basal epidermis. Besides, at 88 h the amount of ACV detected in the receptor chamber of the diffusion cells was much lower with the microparticles than with the suspension. This type of carrier can improve acyclovir topical therapy since it increases drug retention in the basal epidermis and consequently increases the time intervals between doses

Terapias neuroprotectoras y neurorestauradoras en el tratamiento de la enfermedad de Parkinson

Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. Current therapies are symptomatic and, although these therapies are efficacious during the early stages of the disease, they present important side effects when they are used for a long time. The ideal therapy would be the one that would slow down or stop the progression of the disease. This can be achieved, for instance, with neuroprotective and neurorestorative therapies. Among them, cell therapy and therapy with trophic factors such as glial cell line derived neurotrophic factor (GDNF) are the most challenging and promising ones for the scientific community. Although the use of GDNF as a treatment for Parkinson s disease was proposed several years ago, it is necessary to develop alternative strategies to deliver GDNF appropriately to concrete areas of the brain. Here, the use of microspheres as the most suitable tool for the administration of this neurotrophic factor is discussed

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

BACKGROUND: Glial cell-derived neurotrophic factor is a survival factor for dopaminergic neurons and a promising candidate for the treatment of Parkinson's 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. METHODS: A single dose of microspheres containing 2.5 μg of glial cell-derived neurotrophic factor was implanted intrastriatally in animals 2 weeks after a 6-hydroxydopamine lesion. RESULTS: The amphetamine test showed a complete behavioral recovery after 16 weeks of treatment, which was maintained until the end of the study (week 30). This effect was accompanied by an increase in dopaminergic striatal terminals and neuroprotection of dopaminergic neurons. CONCLUSIONS: The main achievement was the long-term neurorestoration in parkinsonian animals induced by encapsulated glial cell-derived neurotrophic factor, suggesting that microspheres may be considered as a means to deliver glial cell-derived neurotrophic factor for Parkinson's disease treatment. © 2011 Movement Disorder Society

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 Parkinson's 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

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 Parkinson's 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 μm, 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

Geomorphological evolution of the calcareous coastal cliffs in North Iberia (Asturias and Cantabria regions)

This paper presents an analysis of the main morphologies observed in the coastal cliffs of northern Spain (Asturias and Cantabria regions). The objective of this contribution is to establish a hypothesis on the origin and evolution of this rocky coast, as well as to present a detailed inventory, to characterise quantitatively and qualitatively singular morphologies and to highlight the geological heritage of this protected coast. The evolution process starts with the formation of an ancient coastal planation surface characterised by a flat morphology caused by regional mainly uplift and to relative sea level falls. Afterwards, wave erosion processes would have started eroding the cliff foot and simultaneously, karst activity produced some exokarst morphologies (sinkholes, karren, etc.) through stratification and fracturing network, while the underground drainage systems produced some caves and chasms. In the following step, corresponding to the last glaciation from the paleoclimatic point of view, sea level fall together with a deepening of the fluvial network caused the preservation of the existing caves and chasms and the generation of new ones at a lower level. On the other hand, dissolution processes on limestones created sinkholes in those areas characterised by alternating layers of limestones and marls, generating collapses. When the sea level reached the maximum height during the Holocene a new erosion cycle of the coastal cliffs began. As a consequence, new landforms and processes were produced, like bays, caves fillings, and intrusion of new sediments in small confined estuaries. In these areas, other types of morphologies associated with the last sea level rise can be observed, such as closed beaches, uncommon closed estuaries developed inside a sinkhole, blowholes produced by mixed wave action and widening of prevailing vertical pipes inside the limestones (including the second largest in the world), total or partial sedimentary filling of small confined estuaries, as well as a tombolo deposit. It is important to point out, that some sites described are included in the Spanish Inventory of Sites of Geological Interest (IELIG). Due to the evolution model here proposed, a portion of the coastal sector described are included in the Global Geosites Project

Optimization of topical cidofovir penetration using microparticles.

Edelfosine is the prototype molecule of a family of anticancer drugs collectively known as synthetic alkyl-lysophospholipids. This drug holds promise as a selective antitumor agent, and a number of preclinical assays are in progress. In this study, we observe the accumulation of edelfosine in brain tissue after its oral administration in Compritol® and Precirol® lipid nanoparticles (LN). The high accumulation of edelfosine in brain was due to the inhibition of P-glycoprotein by Tween® 80, as verified using a P-glycoprotein drug interaction assay. Moreover, these LN were tested in vitro against the C6 glioma cell line, which was later employed to establish an in vivo xenograft mouse model of glioma. In vitro studies revealed that edelfosine-loaded LN induced an antiproliferative effect in C6 glioma cell line. In addition, in vivo oral administration of drug-loaded LN in NMRI nude mice bearing a C6 glioma xenograft tumor induced a highly significant reduction in tumor growth (p < 0.01) 14 days after the beginning of the treatment. Our results showed that Tween® 80 coated Compritol® and Precirol® LN can effectively inhibit the growth of C6 glioma cells in vitro and suggest that edelfosine-loaded LN represent an attractive option for the enhancement of antitumor activity on brain tumors in vivo