Improved stability and immunological potential of tetanus toxoid containing surface engineered bilosomes following oral administration Sanyog Jain, Ph.D.,

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Improved stability and immunological potential of tetanus toxoid containing surface engineered bilosomes following oral administration Sanyog Jain, Ph.D., Harshad Harde, M.S. Pharm., Anura Indulkar, M.S. Pharm., Ashish Kumar Agrawal, M. Pharm. Nanomedicine: Nanotechnology, Biology and Medicine Volume 10, Issue 2, Pages 431-440 (February 2014) DOI: 10.1016/j.nano.2013.08.012 Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 TEM images of TT loaded (A) Niosomes (B) Bilosomes and (C) GM-bilosomes. Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 431-440DOI: (10.1016/j.nano.2013.08.012) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Stability studies. (A) Chemical stability where lanes I to IV represent resolved bands obtained in SDS-PAGE for standard TT and TT extracted from Niosomes, Bilosomes and GM-bilosomes respectively. (B) Conformation stability where overlay CD spectra represent standard TT and TT extracted from different formulations. Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 431-440DOI: (10.1016/j.nano.2013.08.012) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 (A) CLSM image (10×) showing macrophage uptake of (I) free coumarin-6; (II) Niosomes; (III) Bilosomes; and (IV) GM-bilosomes. For each incubation type, the left and right panels represent green fluorescence and overly of green fluorescence and differential interference contrast image. (B) Quantitative determination of macrophage uptake of coumarin-6 loaded vesicular formulations. (***P<0.001). Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 431-440DOI: (10.1016/j.nano.2013.08.012) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Serum IgG titre following administration of TT (oral and i.m.) and TT loaded niosomes, bilosomes and GM-bilosomes. Arrow (↑) represents the day of dosing. Data are presented as mean±SEM (n=6) (***P<0.001, *P<0.05). Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 431-440DOI: (10.1016/j.nano.2013.08.012) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 sIgA titre in (A) saliva (B) intestinal fluid and (C) fecal content following administration of free TT (oral and i.m.) and TT loaded niosomes, bilosomes and GM-bilosomes. Data are presented as mean±SEM (n=6) (***P<0.001, *P<0.05). Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 431-440DOI: (10.1016/j.nano.2013.08.012) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Cytokines level in spleen homogenate following administration of TT (oral and i.m.) and TT loaded niosomes, bilosomes and GM-bilosomes (A) IL-2 (B) IFN-γ. Data are presented as mean±SEM (n=6) (***P<0.001, *P<0.05). Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 431-440DOI: (10.1016/j.nano.2013.08.012) Copyright © 2014 Elsevier Inc. Terms and Conditions

Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 431-440DOI: (10.1016/j.nano.2013.08.012) Copyright © 2014 Elsevier Inc. Terms and Conditions