Development of multifunctional polymeric nanoparticles by nano-emulsion templating as advanced nanocarriers targeting the blood-brain barrier

Abstract

Multifunctional polymeric nanoparticles (NP) represent a promising alternative for the treatment of neurodegenerative diseases using the intravenous route (i.v.). In current treatments, the effects of the intravenously injected drugs are systemic, requiring high drug doses to achieve therapeutic effects, thus causing severe side effects. NP can act specifically in a tissue provided that they are properly designed. An interesting approach is the preparation of NPs by nano-emulsion templating. Nano-emulsions (NE) are fine emulsions, with droplet sizes typically between 20 – 200nm. They can be prepared by the phase inversion composition (PIC) method, a low-energy emulsification method appropriate for pharmaceutical applications, since it can be performed at mild process conditions. Nanoparticles are obtained from nano-emulsions by solvent evaporation. To target the central nervous system (CNS), a specific targeting moiety on the nanoparticle surface is required to cross the blood-brain barrier (BBB), which is a current key goal under intense investigations. The aim of this work was to obtain multifunctional polymeric NP as advanced delivery systems able to cross the BBB. O/W polymeric NE were prepared by the PIC method and polymeric NPs were obtained by solvent evaporation. Polymeric NP with appropriate sizes for the i.v. administration (<1milimicron) were obtained. With the aim to design imaging systems, a model fluorescent dye and magnetic nanoparticles were encapsulated in polymeric NPs. An analgesic and an antiapoptotic drugs were also encapsulated into PLGA NP for therapeutic purposes. High encapsulation efficiencies were found for all tested compounds, attributed to the method of nanoparticle preparation as well as to low solubility of the components in the aqueous dispersion media. In addition, a sustained and controlled release of fluorescent dyes / drugs was achieved. NP surface was functionalized using various elements. On the one hand, it was functionalized with a monoclonal antibody against the transferring receptor, overexpressed in the BBB, to achieve an active targeting to the BBB. On the other hand, NPs were functionalized with oligonucleotides, to be used as non-viral gene delivery systems. Firstly, carbosilane cationic dendrons were covalently attached to nanoparticle surface to achieve a cationic surface. In a further step, antisense oligonucleotides, siRNA and plasmids were electrostatically bound to cationized nanoparticles. In vitro tests showed that the formulated NP did produce neither cytoxicity nor hemolysis. In addition, they were weak activators of the immune system and produced only a slight adsorption of blood proteins. Therefore, they are appropriate to be used by the i.v. route. NPs functionalized with oligonucleotides enhanced gene transfection in cell cultures, up to values comparable to those of commercial values (up to 90%). These NPs are advantageous in terms of toxicity issues over the commercial formulations. Therefore, they represent promising non-viral gene delivery systems. In vivo tests, which measured the central analgesia produced by an encapsulated drug into nanoparticles (loperamide) that is not able to cross the BBB, confirmed a central analgesic effect, reaching a potency of analgesia comparable to positive controls when nanoparticles were functionalized with the antibody. Therefore, the antibody functionalized nanoparticles efficiently crossed the BBB. In conclusion, the designed polymeric nanoparticles, functionalized with the antitransferrin receptor antibody, are able to cross the BBB with high efficiency. These nanoparticles represent promising nanosystems to deliver actives to the central nervous system.

Les nanopartícules polimèriques multifuncionals (NPs) representen una alternativa prometedora pel tractament de malalties neurodegeneratives, a través de l’administració intravenosa (i.v.), ja que els tractaments actuals provoquen molts efectes secundaris. Les NPs, en canvi, si estan correctament dissenyades, poden actuar específicament en el teixit diana. Ja que l’òrgan diana és el cervell, és necessari un element de vectorització per poder creuar la barrera hemato-encefàlica (BBB). En aquest context, l’objectiu de la present tesi és l’obtenció de NPs com a sistemes avançats d’alliberament de principis actius que travessin la BBB. Es van obtenir NPs a partir de nano-emulsions (NE) plantilla, emprant l’àcid poli-(làctic-co-glicòlic) com a polímer i el mètode d’inversió de fases a temperatura constant per emulsionar, seguit d’evaporació de solvent per obtenir NPs. Les NPs obtingudes tenen mides apropiades per l’administració i.v.. Es va aconseguir encapsular un fluorescent i NPs magnètiques dins les NPs polimèriques, per fer-les servir com a sistemes d’imatge. També es van encapsular fàrmacs per usar-les com a sistemes terapèutics. En tots els casos, es van aconseguir eficiències d’encapsulació molt elevades i un alliberament del fàrmac controlat i prolongat en el temps. A més, es va aconseguir funcionalitzar la superfície de les NPs amb diferents elements. Es van unir covalentment dendrons catiònics per posteriorment unir oligonucleòtids electrostàticament. També es va afegir una coberta exterior de polietilenglicol per protegir el material genètic. Per altra banda, es va funcionalitzar la superfície de les NPs amb un anticòs específic contra el receptor de la transferrina, sobreexpressat a la BBB. A continuació, es van fer assajos in vitro, que van posar de manifest que les NPs no són citotòxiques ni hemolítiques. També es va estudiar l’eficiència de transfecció cel•lular del material genètic, arribant a eficiències de transfecció equivalents a les dels vectors comercials. Assajos in vivo van permetre confirmar el pas a través de la BBB, sobretot de les NPs funcionalitzades amb l’anticòs. Els resultats obtinguts permeten concloure que s’ha aconseguit dissenyar noves NPs polimèriques a partir de NE, apropiades per l’administració i.v. i amb capacitat de travessar la BBB.