Nuevos nanosistemas a base de protamina para la liberación de antígenos

En la presente tesis doctoral se plantea el diseño, desarrollo y optimización de sistemas coloidales nanométricos a base de protamina para la vehiculización y liberación de antígenos. En particular los sistemas desarrollados se dividen en (i) nanopartículas, sistema de tipo matricial que se compone de un polisacárido como ácido hialurónico o alginato entrecruzado con protamina y (ii) nanocápsulas, constituidas por un núcleo liquido oleoso (Miglyol®, escualeno o α tocoferol) y una cubierta de protamina. Dependiendo del tipo de estructura, se han asociado distintos antígenos modelo como son el antígeno recombinante de la hepatitis B y el antígeno de influenza H1N1. Los sistemas presentan una adecuada estabilidad en suspensión y en polvo seco a temperatura ambiente. Estudios in vitro han demostrado que ambos tipos de sistemas son eficazmente internalizadas por células inmunocompetentes y estimulan la secreción de diversos tipos de citocinas. Estudios in vivo han demostrado la efectiva acción de los distintos nanosistemas con el antígeno asociado generando una respuesta inmune adecuada y prolongada. Por otra parte, se ha evaluado la posibilidad de que los diferentes sistemas sean formulaciones administradas libres de agujas, a través de la vía nasal, obteniendo respuestas inmunes protectoras frente al antígeno asociado y pudiendo modular el tipo de respuesta obtenida (humoral o celular), dependiendo de la vía de administración. Todos estos resultados ponen en manifiesto el potencial que poseen las estructuras desarrolladas a base de protamina para la vehiculización y liberación de antígenos, consiguiendo avanzar en el desarrollo de nuevos adyuvantes así como en la termo-estabilidad de las diversas formulaciones

Polymeric Nanocapsules for Vaccine Delivery: Influence of the Polymeric Shell on the Interaction With the Immune System

The use of biomaterials and nanosystems in antigen delivery has played a major role in the development of novel vaccine formulations in the last few decades. In an effort to gain a deeper understanding of the interactions between these systems and immunocompetent cells, we describe here a systematic in vitro and in vivo study on three types of polymeric nanocapsules (NCs). These carriers, which contained protamine (PR), polyarginine (PARG), or chitosan (CS) in the external shell, and their corresponding nanoemulsion were prepared, and their main physicochemical properties were characterized. The particles had a mean particle size in the range 250–450 nm and a positive zeta potential (~30–40 mV). The interaction of the nanosystems with different components of the immune system were investigated by measuring cellular uptake, reactive oxygen species production, activation of the complement cascade, cytokine secretion profile, and MAP kinases/nuclear factor κB activation. The results of these in vitro cell experiments showed that the NC formulations that included the arginine-rich polymers (PR and PARG) showed a superior ability to trigger different immune processes. Considering this finding, protamine and polyarginine nanocapsules (PR and PARG NCs) were selected to assess the association of the recombinant hepatitis B surface antigen (rHBsAg) as a model antigen to evaluate their ability to produce a protective immune response in mice. In this case, the results showed that PR NCs elicited higher IgG levels than PARG NCs and that this IgG response was a combination of anti-rHBsAg IgG1/IgG2a. This work highlights the potential of PR NCs for antigen delivery as an alternative to other positively charged nanocarriersThis work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2011-30337-C02-02 and BIO2014-53091-C3-1-R). Financial support from the Xunta de Galicia (Centro singular de investigación de Galicia 2016–2019 and Grupo de referencia competitiva, ED431C 2016041) and the European Union (European Regional Development Fund—ERDF) is gratefully acknowledged. MP acknowledges fellowships from the Spanish Ministry of Education (FPU predoctoral grants program)S

Lower-Sized Chitosan Nanocapsules for Transcutaneous Antigen Delivery

Transcutaneous vaccination has several advantages including having a noninvasive route and needle-free administration; nonetheless developing an effective transdermal formulation has not been an easy task because skin physiology, particularly the stratum corneum, does not allow antigen penetration. Size is a crucial parameter for successful active molecule administration through the skin. Here we report a new core-shell structure rationally developed for transcutaneous antigen delivery. The resulting multifunctional carrier has an oily core with immune adjuvant properties and a polymeric corona made of chitosan. This system has a size of around 100 nm and a positive zeta potential. The new formulation is stable in storage and physiological conditions. Ovalbumin (OVA) was used as the antigen model and the developed nanocapsules show high association efficiency (75%). Chitosan nanocapsules have high interaction with the immune system which was demonstrated by complement activation and also did not affect cell viability in the macrophage cell line. Finally, ex vivo studies using a pig skin model show that OVA associated to the chitosan nanocapsules developed in this study penetrated and were retained better than OVA in solution. Thus, the physicochemical properties and their adequate characteristics make this carrier an excellent platform for transcutaneous antigen delivery

Hyaluronic Acid Nanocapsules as a Platform for Needle-Free Vaccination

Vaccination faces many challenges nowadays, and among them the use of adjuvant molecules and needle-free administration are some of the most demanding. The combination of transcutaneous vaccination and nanomedicine through a rationally designed new-formulation could be the solution to this problem. This study focuses on this rational design. For this purpose, new hyaluronic acid nanocapsules (HA-NCs) have been developed. This new formulation has an oily nucleus with immunoadjuvant properties (due to α tocopherol) and a shell made of hyaluronic acid (HA) and decorated with ovalbumin (OVA) as the model antigen. The resulting nanocapsules are smaller than 100 nm, have a negative superficial charge and have a population that is homogeneously distributed. The systems show high colloidal stability in storage and physiological conditions and high OVA association without losing their integrity. The elevated interaction of the novel formulation with the immune system was demonstrated through complement activation and macrophage viability studies. Ex vivo studies using a pig skin model show the ability of these novel nanocapsules to penetrate and retain OVA in higher quantities in skin when compared to this antigen in the control solution. Due to these findings, HA-NCs are an interesting platform for needle-free vaccination

Dextran Nanocapsules with <i>ω</i>-3 in Their Nucleus: An Innovative Nanosystem for Imiquimod Transdermal Delivery

Transdermal administration of molecules across the skin has gained interest because it can be considered a non-invasive route compared with traditional ones. However, going through the skin is challenging due to the presence of the stratum corneum, the main barrier of substances. For this reason, the goal of this research was the combination of omega-3 (ω-3) and a dextran sulfate assembly in a nanostructure form, which allows passage through the skin and improves the bioavailability and the therapeutic profiles of active molecules, such as imiquimod. Here we report a new colloidal system, named dextran nanocapsules, with ω-3 in its nucleus and a coat made of dextran sulfate with a size ~150 nm, monomodal distribution, and negative zeta potential (~−33 mV). This nanosystem encapsulates imiquimod with high efficacy (~86%) and can release it in a controlled fashion following Korsmeyer–Peppas kinetics. This formulation is stable under storage and physiological conditions. Furthermore, a freeze-dried product could be produced with different cryoprotectants and presents a good security profile in the HaCaT cell line. Ex vivo assays with newborn pig skin showed that dextran nanocapsules promote transdermal delivery and retention 10 times higher than non-encapsulated imiquimod. These promising results make this nanosystem an efficient vehicle for imiquimod transdermal delivery

Design of chitosan nanocapsules with Compritol 888 ATO® for imiquimod transdermal administration. Evaluation of their skin absorption by Raman Microscopy

Purpose Design imiquimod-loaded chitosan nanocapsules for transdermal delivery and evaluate the depth of imiquimod transdermal absorption as well as the kinetics of this absorption using Raman Microscopy, an innovative strategy to evaluate transdermal absorption. This nanovehicle included Compritol 888ATO (R), a novel excipient for formulating nanosystems whose administration through the skin has not been studied until now. Methods Nanocapsules were made by solvent displacement method and their physicochemical properties was measured by DLS and laser-Doppler. For transdermal experiments, newborn pig skin was used. The Raman spectra were obtained using a laser excitation source at 532 nm and a 20/50X oil immersion objective. Results The designed nanocapsules, presented nanometric size (180 nm), a polydispersity index <0.2 and a zeta potential +17. The controlled release effect of Compritol was observed, with the finding that half of the drug was released at 24 h in comparison with control (p < 0.05). It was verified through Raman microscopy that imiquimod transdermal penetration is dynamic, the nanocapsules take around 50 min to penetrate the stratum corneum and 24 h after transdermal administration, the drug was in the inner layers of the skin.Fondo Nacional de Desarrollo Científico y Tecnológico de Chile FONDECYT 1201482 Programa de Equipamiento Científico y Tecnológico FONDEQUIP EQM120021 FONDEQUIP EQM13003

The Importance of Nanocarrier Design and Composition for an Efficient Nanoparticle-Mediated Transdermal Vaccination

The World Health Organization estimates that the pandemic caused by the SARS-CoV-2 virus claimed more than 3 million lives in 2020 alone. This situation has highlighted the importance of vaccination programs and the urgency of working on new technologies that allow an efficient, safe, and effective immunization. From this perspective, nanomedicine has provided novel tools for the design of the new generation of vaccines. Among the challenges of the new vaccine generations is the search for alternative routes of antigen delivery due to costs, risks, need for trained personnel, and low acceptance in the population associated with the parenteral route. Along these lines, transdermal immunization has been raised as a promising alternative for antigen delivery and vaccination based on a large absorption surface and an abundance of immune system cells. These features contribute to a high barrier capacity and high immunological efficiency for transdermal immunization. However, the stratum corneum barrier constitutes a significant challenge for generating new pharmaceutical forms for transdermal antigen delivery. This review addresses the biological bases for transdermal immunomodulation and the technological advances in the field of nanomedicine, from the passage of antigens facilitated by devices to cross the stratum corneum, to the design of nanosystems, with an emphasis on the importance of design and composition towards the new generation of needle-free nanometric transdermal systems