New strategies based on nanosystems to facilitate the crossing through biological barriers

Abstract

During the past years working in Ernest Giralt’s group, I’ve been involved in three different subprojects that constitute one of the three chapter of my thesis and have the same common link: the use of peptides combined with nanoparticles to facilitate the transport through different biological barriers. They can be described as:

1. Gold nanorods (NRs) decorated with peptides for endosomal release

2. Polymeric nanoparticles for gene therapy through the blood brain barrier (BBB)

3. Nanoparticles containing insulin for oral delivery

In the first of the subprojects, we tried to transport our NRs to the cytosol by crossing the cellular membrane and then escaping from the endosome. For cell internalization, rods were functionalized with molecules able to transport cargoes from the outside towards the inside. These molecules are named Cell Penetrating Peptides (CPPs). In our case, we selected the D form of octaarginine (r8). Once NRs are internalized by the endocytic route, were irradiated with a near infrared laser which is in the range where rods absorb and convert the light into heat. We hypothesized that by applying a specific power intensity to gold NRs, they can disrupt the endosome and release its content to the cytosol. Synthesis of NRs was optimized in order to obtain a high yield of rods (more than 93%) compared with some byproducts. Rods were

50.7 ± 7.7 nm in length and 9.0 ± 0.7 nm in width. These measures conferred to the rods a longitudinal Plasmon band around 1000nm which correspond to near infrared and has the advantage of deeper tissue penetration by the laser. Peptides were synthesized by solid-phase peptide synthesis using Fmoc/t-Bu strategy. A fluorescent molecule was attached to the peptide in order to visualize it by fluorescence microscopy. For rod functionalization, a two steps approach was selected: first, polyethylene glycol (PEG) was attached to the rods surface and then the peptide was coupled to PEG moiety. This construct was used for cell internalization assays. Transmission electron microscopy and confocal images confirmed that our rods were able to be internalized into HeLa cells. Finally, preliminary results irradiating with NIR laser showed that rods could disrupt the endosomes/lysosomes arriving to the cytosol. In order to confirm this result, siRNA was attached to the rods surface and delivered to the cytoplasm. The effect was detected by western blot and preliminary results showed protein silencing, achieving thus the goal of crossing the endosome barrier.

The aim of the second subproject was DNA encapsulation into poly (lactic-co-glycolic acid) (PLGA) nanoparticles for gene therapy through the BBB. Plasmid DNA encoding frataxin protein was used for the treatment of Friedreich’s Ataxia. PLGA nanoparticles were synthesized and characterized by DLS. The particles were lower than 200nm in size and had low polydispersity. First, DNA was complexed with PEI (positive polymer) in order to increase transfection efficiency. Then PLGA modified with a BBB-shuttle was added on the complex to form the final nanoparticles. Glioblastoma, HeLa and fibroblasts cells were successfully transfected by our formulations, indicating the great potential of PLGA NPs as gene therapy agents. Finally, NPs were assayed in a BBB cellular model.in order to detect transport through the BBB membrane. Preliminary results showed minor transport.

In last subproject and in collaboration with Prof. Brayden (Dublin), CPPs were modified in order to act as intestinal permeation enhancers. The final goal was to increase insulin transport through the intestinal barrier by the use of these peptide permeation enhancers. Medium chain fatty acids and cholesterol were attached covalently to the CPP in order to increase insulin transport. Insulin was complexed by these compounds and assayed in Caco-2 cell models and Ussing chambers (Dublin). Finally, the main problem was complexes instability and transport could not be calculated. Future experiments will be addressed to increase complexes stability.

La present tesi estava dividida en tres capítols els quals tenien en comú l’ús de pèptids inclosos en nanopartícules per tal de millorar el transport de molècules d’interès a través de barreres biològiques.

El primer capítol es va centrar en la modificació, mitjançant pèptids penetradors de membrana (CPPs), de nanovaretes d’or per tal de ser internalitzades a dintre de les cèl·lules. Les partícules van ser capaces d’internalitzar-se per via endocítica. Un cop a dintre, aquestes nanovaretes van ser irradiades amb un làser d’infraroig proper, el qual son capaces d’absorbir, emetent calor. Per tal de veure si aquesta calor era suficient per trencar l’endosoma, un siRNA va ser adherit al sistema. Resultats preliminars van mostrar cm la proteïna en concret va poder ser silenciada per l’efecte d’alliberament endosomal gràcies a la irradiació de les nanovaretes d’or.

L’objectiu del segon capítol va ser el de transportar DNA encapsulat a dintre de nanopartícules d’àcid poli làctic-glicòlic (PLGA) a través de la barrera hematoencifàlica (BHE). Primer, vam complexar el DNA amb un polímer positiu (PEI) per tal d’augmentar la seva transfecció. Després, el PLGA va ser afegit formant les nanopartícules. Aquestes partícules de PLGA es van provar en tres línies cel·lulars donant alta transfectivitat. Finalment i com a resultat preliminar, es van assajar les partícules en un model cel·lular de BHE detectant DNA a la part acceptora. Aquest resultat indicava que les partícules havien sigut capaces de travessar el model de BHE.

L’últim capítol estava enfocat en el descobriment de nous pèptids potenciadors del transport a traves de la barrera intestinal. En aquest cas, un CPP va ser modificat amb diferents àcids grassos per tal de veure un millor transport de la nostra molècula d’interès: la insulina. Es van formar complexes barrejant insulina amb els CPPs modificats i es van assajar en dos models de barrera intestinal: Caco-2 i Ussing Chambers. Tot i que alguns dels compostos tenien potencial per incrementar el transport d‘insulina, els complexes van precipitar en la majoria dels casos. Una millor estabilitat s’estudiarà en un futur per tal d’aconseguir aquesta millora en el transport d’insulina.