Argaw Kidane

Effects of cellulose derivatives and poly(ethylene oxide)-poly(propylene oxide) tri-block copolymers (Pluronic)surfactants) on the properties of alginate based microspheres and their interactions with phagocytic cells.

The goal of this study was to examine the phagocytosis of alginate based microspheres with different surface properties. Favorable interaction with macrophages is critical for uptake subsequent processing of the microspheres used for oral vaccine delivery. We examined the effects of size of alginate microspheres and hydrophobicity on cellular uptake. We also examined the toxicity of formulation components to phagocytic cells. Alginate microspheres were made by the emulsion-cross-linking technique. Five different formulations of microspheres were evaluated for size, hydrophobicity, cellular uptake and toxicity to macrophages. The formulations examined were: alginate alone (A), alginate with methylcellulose (AA) AA with Pluronic L61 (AA61), alginate with hydroxypropyl methylcellulose (AK3), and AK3 with Pluronic (L61 (AK3 61). Microspheres with without poly-L-lysine (PLL) coating were tested. The mean volume sizes of A, AA, AA61, AK3, AK3 61 microspheres (MS) were 11, 10.5, 3.8, 8.7 and 3.9 mocrom, respectively. After coating them with PLL the mean volume sizes were 10.4, 10, 3.7, 8.8 and 3.5 microm, respectively. Hydrophobicity of the microspheres was evaluated by measuring contact angle on a glass slide coated with the microspheres. The contact angles measured using a goniometer on A, AA, AA61, AK3, AK3 61 MS were 20, 34.8, 71, 29 and 80 degrees, respectively whereas those MS coated with PLL were 49.7, 55.8, 91, 48.25 and 84.4 degrees, respectively. Cellular uptake studies using flow cytometery revealed that AA61 MS coated with PLL were phagocytosed most often by mouse macrophages. There was no statistically significant difference in cellular uptake among those MS without PLL coating. Toxicity to macrophages was shown to depend on the ratio of microspheres to cells. These studies suggest that formulation can dramatically affect the physical characteristics of alginate MS in ways that can affect how they will interact with cells in the body when administered as a vaccine delivery system.

The efficacy of oral vaccination of mice with alginate encapsulated outer membrane proteins of Pasteurella haemolytica and One-Shot.

The goal of this study was to examine the efficacy of oral delivery of alginate encapsulated outer membrane proteins (OMP) of Pasteurella haemolytica and a commercial One-Shot vaccine in inducing protection in mice against lethal challenge with virulent P. haemolytica. We examined two alginate microsphere formulations and compared them with oral unencapsulated and subcutaneously administered vaccines. Alginate microspheres were made by the emulsion-cross-linking technique. They were examined for size, hydrophobicity, and antigen loading efficiency before they were used in the study. Mice were vaccinated by administering 200 microg of antigens in 200 microl of microspheres suspension orally or subcutaneously. One group of mice received blank microspheres and a second group was given unencapsulated antigen orally. A third and a fourth group received different formulations of alginate encapsulated antigens by oral administration. Three groups received subcutaneous inoculations (alginate encapsulated, non-adjuvanted and unencapsulated antigens, and adjuvanted One-Shot), and one group received water (naïve group). Mice were vaccinated orally for four consecutive days and challenged with P. haemolytica 5 weeks after the first vaccination. Weekly serum and feces samples were assayed for antigen specific antibodies. The number of dead mice in each group 4 days post challenge was used to compare the efficacy of the various vaccination groups. The mean volume sizes of blank alginate microsphere formulations A, and AA were 15.9, 16 and 9.2 microm, respectively. Hydrophobicity of the microspheres was evaluated by measuring contact angle on a glass slide coated with the microspheres. The contact angles on A and AA were 37.8 and 74.3 degrees, respectively. Antigen concentration in a 1:1 w/w suspension of microspheres in water was 0.9 mg/ml. Rate of death for the blank group was 42.8% whereas for groups vaccinated with antigens encapsulated in A and AA the death rates were 40 and 33.33%, respectively. The death rate in mice vaccinated with unencapsulated antigens was 55.6%. Groups vaccinated by subcutaneous inoculation showed the lowest death rate. These results show that encapsulating OMP and One-Shot in alginate microspheres improves their performance as an oral vaccine.