Document Type

Dissertation - University Access Only

Award Date

2012

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

First Advisor

Omathanu P. Perumal

Abstract

Transcutaneous vaccine delivery is a promising alternative to conventional needle vaccination via subcutaneous and intramuscular routes. However, vaccine delivery through skin is limited by the barrier properties of skin. To achieve effective transcutaneous immunization (TCI), three main components are required: i) a vaccine delivery vehicle, ii) a potent vaccine adjuvant, and iii) skin penetration enhancement. This thesis work investigates the feasibility of TCI using different combinations of delivery vehicles, vaccine adjuvants, and skin penetration enhancement techniques. The overall goal of this thesis was to develop effective transcutaneous vaccine delivery systems. Ovalbumin was used as the model vaccine antigen. The first objective was to investigate the feasibility of transcutaneous vaccine delivery using calcium phosphate (CAP) nanoparticles as the delivery vehicle/vaccine adjuvant, and tape stripping as the physical penetration enhancement technique. CAP nanoparticles coated with sugar and antigen were prepared using nano-precipitation method. X-Ray diffraction spectrum of CAP nanoparticles showed characteristic peaks at 25°, 31-32°, and 50-55°, indicating the formation of hydroxyapatite core predominantly. Among the three different sugars used, cellobiose was selected as the optimum sugar for coating onto the CAP nanoparticles. The ovalbumin and cellobiose coated CAP nanoparticles were 350±22.Snm in size, with a zeta potential of -12.93±1.02mV. Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) confirmed the adsorption of sugars and ovalbumin on CAP nanoparticles. The adsorption efficiency of ovalbumin on CAP nanoparticles was 25±3%. Around 60% ovalbumin was released from nanoparticles within 24 hrs. In-vivo immunization studies were carried out in Balb/C mice. Application of ovalbumin adsorbed CAP nanoparticles on tape stripped mice skin induced significantly higher total IgG and IgG 1 antibody titers in mice compared to topical application of ovalbumin alone. These results suggest the feasibility of TCI using combined application of CAP nanoparticles and tape stripping. Intradermal injection of CAP-cellobiose-ovalbumin nanoparticles was used as the positive control. As expected, it resulted in significantly higher antibody titers compared to topical application of nanoparticles. In the second objective, cationic liposome was used as the delivery vehicle, Cytosine Phosphate Guanosine (CpG) oligonucleotide (ODN) was used as the immune potentiator, and iontophoresis (0.47mA/cm 2 , 4 hrs) was used as the skin penetration enhancement technique. Ovalbumin was entrapped inside the cationic liposomes, while CpG was complexed to the positively charged surface at 10: 1 Nitrogen/Phosphate (N/P) charge ratio. The size and zeta potential of liposomes-ovalbumin-CpG was 264.03±18.49nm and 6±2.00mV respectively. Agarose gel electrophoresis confirmed the complex formation between cationic liposomes and CpG ODN. The in-vitro porcine skin penetration of fluorescently labeled antigen (FITC-Albumin) and adjuvant (Rhodamine CpG) was studied using confocal laser scanning microscope (CLSM). The combined application of cationic liposomes and iontophoresis significantly enhanced the depth and extent of penetration of antigen and adjuvant into the viable epidermis compared to the passive delivery of liposomes and corresponding aqueous solutions (FITC-Albumin and Rho-CpG). ATR-FTIR showed shift in skin lipid peaks after treatment with cationic liposomes suggesting alteration and fluidization of the lipid bilayer. In-vivo immunization studies in mice with iontophoretic application of liposomes-ovalbumin-CpG resulted in significantly higher antibody titers (upto 300-1500 times) compared to the passive delivery. Presence of CpG in the liposomes significantly enhanced the total IgG, IgG 1 and especially lgG2a antibody titers compared to iontophoretic application of liposomesovalbumin. Iontophoretic application of liposomes-ovalbumin-CpG resulted in activation of Langerhans cells, migration of ovalbumin to the local inguinal lymph nodes and enhanced in-vitro T cell proliferation of splenocytes. These results suggest the effectiveness of combined application of cationic liposomes and iontophoresis for TCI. To further investigate the influence of lipid vesicle composition on TCI, modified forms of liposomes which are more elastic, flexible, and ultradeformable were studied. To this end, the third objective was to use combined application of highly elastic vesicles ( ethosomes) and iontophoresis for transcutaneous delivery of antigen and adjuvant. Ethosomes significantly enhanced the penetration of vaccine antigen and adjuvant compared to the corresponding control (30%v/v ethanol:HEPES buffer). The skin penetration of ovalbumin and CpG loaded ethosomes was 9-25 times higher compared to the passive application of cationic liposomes. Application of iontophoresis further enhanced the skin penetration of molecules in the viable epidermis by at-least 3 times compared to the passive delivery of ethosomes. The in-vivo immunization studies in Balb/C mice demonstrated that application of iontophoresis enhanced the antibody titers upto 100-130 times compared to passive delivery of ethosomes. The titers after iontophoretic application of ethosomes were at-least two times higher compared to iontophoretic application of liposomes. The fourth objective was to use ultradeformable vesicles (Transfersomes) for TCI. Transfersomes were prepared using three different surfactants including Tween 80, Span 80, and Sodium deoxycholate. Transfersomes significantly enhanced the penetration of antigen and adjuvant compared to the corresponding surfactant solution. The penetration of molecules was further enhanced by 2-3 times after application of iontophoresis. Iontophoretic application of Span 80 transfersomes-ovalbumin-CpG resulted in highest antibody titers. The titers were at-least 1. 75 times and 3.5 times higher compared to iontophoretic application of ethosomes and liposomes respectively. Overall, the outcomes from this thesis demonstrate the feasibility of transcutaneous vaccine delivery using combined application of an antigen and adjuvant using a delivery vehicle and a skin penetration enhancement technique.

Library of Congress Subject Headings

Vaccines
Immunological adjuvants
Skin

Publisher

South Dakota State University

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Rights Statement

In Copyright