Iontophoresis influence on transcutaneous immunization using liposomes and metal nanoparticle

Author(s)

Daniela Spuri Bernardi

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2015-09-24

Examining board members

Renata Fonseca Vianna Lopez; Hamilton Cabral; Mônica Cristina de Oliveira; Maria Virginia Costa Scarpa

• Advisor: Renata Fonseca Vianna Lopez

Abstract

Transcutaneous immunization (TI) is a promising strategy for vaccine in which the antigen-containing formulation is applied to the skin to induce immune response. The success of this type of immunization occurs due to the presence of antigen presenting cells (APCs) in the viable epidermis, which are potent stimulator of T lymphocytes. However, the antigen should transpose the barrier imposed by the stratum corneum to reach the viable epidermis in appropriate concentrations so that an effective immune response is induced. In this work, the influence of iontophoresis, a physical method that uses a weak electrical current to increase skin penetration of drugs, was investigated for the first time in TI. Its association with liposomes, to target the antigen release to the viable epidermis, and with silver nanoparticles (NPAg), to enhance the immune response, was also evaluated for the first time. Ovalbumin (OVA) was used as a model antigen and OVA subcutaneous injection as a positive control of immunization. Anionic and cationic liposomes containing 5 mg/ml and 0.25 mg/mL of OVA, respectively, were obtained and were added or not by NPAg. Anionic liposomes had an average size of approximately 120 nm and 78,4% OVA encapsulation efficiency. Cationic liposomes had an average size of approximately 260 nm and 83,08% OVA encapsulation efficiency. The addition of NPAg did not significantly alter the size of the liposomes, but changed the zeta potential (-7.5 mV to - 14 mV for anionic liposomes and +41 mV to +36 mV for cationic liposomes). The atomic force microscopy and transmission electron microscopy (TEM) confirmed the presence of liposomal vesicles; TEM showed NPAgs dispersed in the external aqueous medium of the anionic liposomes and in the internal aqueous medium of the cationic liposomes. The OVA and the liposomes were stable during the preparation process and in front of the electric current. In the in vitro skin penetration studies it was observed that the encapsulation of OVA into liposomes directed their release to the viable epidermis. The association with iontophoresis increased 108-fold the release of OVA in the viable epidermis when anionic liposomes containing NPAg were administered and 92-fold when cationic liposomes containing NPAg were administered. The presence of NPAg in the formulations increased the amount of OVA released in the viable epidermis when iontophoresis was applied, but decreased OVA passive penetration. The amount of OVA penetrated by anodic and cathodic iontophoresis of anionic liposomes was similar. The OVA penetration coefficient in the viable epidermis from the iontophoresis of anionic liposomes was 1.6-fold (in the absence of NPAg) and 39- fold (in the presence of NPAg) greater than the iontophoresis of cationic liposomes. In transcutaneous immunization in vivo experiments, it was observed that the amount of OVA that penetrated the skin by iontophoresis was sufficient to induce similar humoral immune response than that induced by subcutaneous injection of OVA when anionic liposomes, in the presence and absence of NPAg, and cationic liposome, only in the presence of NPAg, were administered. These results suggest that NPAg in the presence of low concentrations of OVA acted as immunological adjuvants. Altough induced humoral immune response; anodal iontophoresis of the formulations as well as subcutaneous injection of OVA did not stimulate significant cellular immune response. Cathodic iontophoresis, on the other hand, induced both humoral and cellular immune response, as well as stimulating the production of IFN-? and the recruitment of antigen presenting cells, both in spleen and in inguinal lymph nodes. Therefore, iontophoresis of liposomes containing OVA and NPAg was able to target the release of OVA to the epidermis, induce high titers of IgG1 and activate the cellular immune response, being a promising strategy for TI. (AU)

• FAPESP's process: 11/13307-1 - Influence of iontophoresis on transcutaneous immunization using liposomes and metalic nanoparticles
• Grantee: Daniela Spuri Bernardi
• Support Opportunities: Scholarships in Brazil - Doctorate