Design, Characterization, and Immune Augmentation of Docosahexaenoic Acid Nanovesicles as a Potential Delivery System for Recombinant HBsAg Protein

Abstract

Recombinant HBsAg-loaded docosahexaenoic acid nanovesicles were successfully developed, lyophilized (LRPDNV) and characterized for their physico-chemical properties. The zetapotential (ZP) of LRPDNV was -60.4 ± 10.4 mV, and its polydispersity (PDI) was 0.201, with a % PDI of 74.8. The particle sizes of LRPDNV were 361.4 ± 48.24 z. d.nm and 298.8 ± 13.4 r.nm. The % mass (r.nm) of LRPDNV in a colloidal injectable system was 50, its mobility value was -3.417 µm cm/Vs, while the conductivity of the particles was 0.728 (mS/cm). Transmission electron microscopic (TEM) analysis showed smooth morphological characteristics of discrete spherical LRPDNV. Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) of LRPDNV revealed that LRPDNV is thermostable. The X-ray diffraction (XRD) studies showed a discrete crystalline structure of LRPDNV at 2θ. Nuclear magnet resonance (NMR) studies (1H-NMR and 13C-NMR spectrum showed the discrete structure of LRPDNV. The immunogenicity study was performed by antibody induction technique. The anti-HBs IgG levels were elevated in Wistar rats; the antibody induction was observed more in the product (LRPDNV) treatment group when compared to the standard vaccine group. The level of antibodies on the 14th and 30th day was 6.3 ± 0.78 U/mL and 9.24 ± 1.76 U/mL in the treatment and standard vaccine groups, respectively. Furthermore, the antibody level on the 30th day in the treatment group was 26.66 ± 0.77 U/mL, and in the standard vaccine group, the antibody level was 23.94 ± 1.62 U/mL. The LRPDNV vaccine delivery method released HBsAg sustainably from the 14th to the 30th day. The results of this study indicate the successful formulation of DHA nanovesicles which have great potential as an adjuvant system for the delivery of recombinant HBsAg protein.

Keywords: HBsAg; docosahexaenoic acid; nanovesicles; recombinant proteins; vaccine adjuvants.

Figure 1

The physical characterization of lyophilized recombinant HBsAg–loaded docosahexaenoic acid nanovesicles. (A) Zeta potential of nanovesicles in the injectable formulation; (B) size distribution analysis by intensity in d. nm in the injectable formulation; (C) size distribution analysis in r.nm in the injectable formulation; (D) size distribution analysis by mass in the injectable formulation; (E) cumulative fit of particle size in an injectable formulation (F) distribution fit analysis of particles in the injectable formulation.

Figure 2

Transmission electron microscope study of nanovesicles. (A) Lyophilized recombinant HBsAg loaded docosahexaenoic acid nanovesicles at ×6000 magnification; (B) lyophilized recombinant HBsAg loaded docosahexaenoic acid nanovesicles at ×50,000 magnification; (C) lyophilized recombinant HBsAg loaded docosahexaenoic acid nanovesicles at ×80,000 magnification; (D) lyophilized recombinant HBsAg loaded docosahexaenoic acid nanovesicles at ×200,000 magnification; (E) liquid form of recombinant HBsAg loaded docosahexaenoic acid nanovesicles at ×30,000 magnification; (F) liquid form of recombinant HBsAg loaded docosahexaenoic acid nanovesicles at ×40,000 magnification; (G) liquid form of recombinant HBsAg loaded docosahexaenoic acid nanovesicles at ×50,000 magnification; (H) liquid form of recombinant HBsAg loaded docosahexaenoic acid nanovesicles at ×80,000 magnification.

Figure 3

Physical characterization of lyophilized recombinant HBsAg-loaded docosahexaenoic acid nanovesicles; (A) differential scanning calorimetry study; (B) thermo gravity analysis.

Figure 4

XRD analysis of lyophilized recombinant HBsAg-loaded docosahexaenoic acid nanovesicles.

Figure 5

Nuclear magnetic resonance study of lyophilized recombinant HBsAg-loaded docosahexaenoic acid nanovesicles; (A) ¹H-NMR spectrum; (B) ¹³C-NMR spectrum.

Figure 6

In vitro release profile of HBsAg from lyophilized recombinant HBsAg–loaded docosahexaenoic acid nanovesicles.

Figure 7

In vivo immunogenicity study; (A) standard curve; (B) anti-HBs IgG level of various treatment groups. **** The values are very high significant at p < 0.05 level, ns: non-significant at p < 0.05 level.

Figure 8

Histopathological examination of the liver by using H & E staining. (G1) Normal control without any treatment, (G2) treatment with 0.5 mL (product) immunized by IP without significant changes in hepatic cells, (G3) treatment with vehicle control 0.5 mL (standard) immunized by IP without any significant changes in hepatic cells, (G4) positive control (market standard vaccine) 0.5 mL immunized by IP without any significant changes in hepatic cells.