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Controlled release and immunogenic efficacy of fattigated vaccine antigen nanoparticles

  • 김보미 (아주대학교 일반대학원)

초록/요약

The aims of this work were to investigate release rate and immunogenic efficacy of human serum albumin (HSA) nanoparticles (NPs) encapsulating free or fattigated vaccine antigens. Influenza hemagglutinin (Heg) or thermally stabilized Heg-oleic acid (OA) conjugates (HOC) were used as vaccine antigen models. HSA NPs were prepared via a de-solvation technique. HSA NP encapsulating Heg or HOC (Heg- HSA NPs, HOC-HSA NPs) appeared spherical, having 209.92 ± 2.66 nm or 217.1 ± 12.2 nm in size, respectively. Vaccine antigens were also successfully loaded into NPs, confirming by size exclusion (SEC)-HPLC, bicinchoninic acid (BCA) assay, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Interestingly, the loading content and encapsulation efficiency of HOC was approximately twice higher compared with highly water-soluble Heg due to the increased hydrophobicity of HOC via OA conjugation and binding affinity with HSA. The cellular cytotoxicity of HSA NPs or GLU cross-linked HSA NPs was not observed in the Mouse myoblast cell (C2C12) line. The amount of vaccine antigen, Heg bound was determined using ELISA, which detect the number of specific antibodies to an antigen. The powdered fattigated HOC released Heg rapidly over 90% for 30 min and then slowly degraded while Heg rapidly released and degraded at an elevated temperature (37°C). In contrast, Heg- or HOC-HSA NPs released Heg in a controlled manner. The initial release rate of Heg-HSA was much faster but was reduced due to the low stability of Heg in solution. Most of all, HOC-HSA NPs showed the controlled release with minimized initial burst release and enhanced thermal stability. ELISA results, which detect the number of specific antibodies to an antigen. It was proven that HOC-HSA NPs could bind to more specific antibody titers on the plate, resulting in higher absorbance. Overall, utilizing a fattigation platform and controlled release of vaccine antigens in biocompatible HSA NPs could provide a versatile strategy to increase thermal stability and immunogenic efficacy in the future vaccine delivery and therapeutic applications.

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목차

1. Introduction 1
2. Materials and Methods 4
2.1. Materials 4
2.2. Preparation of Heg- and HOC-HSA NPs 4
2.2.1. HOC synthesis 4
2.2.2. Preparation of HSA NP 5
2.3. Physicochemical characterization of HOC 6
2.3.1. Fourier transform-infrared (FT-IR) spectroscopy 6
2.3.2. Free amine quantification of oleic acid conjugates 6
2.4. Physicochemical characterization of nanoparticles 7
2.4.1. Dynamic light scattering (DLS) measurement 7
2.4.2. Field emission scanning electron microscopy (FE-SEM) 7
2.4.3. Field emission transmission electron microscopy (FE-TEM) 7
2.4.4. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) 7
2.4.5. Bicinchoninic acid (BCA) assay 8
2.4.6. Size exclusion chromatography HPLC (SEC-HPLC) 8
2.5. Cell Cytotoxicity assay 9
2.5.1. Cell culture and maintenance 9
2.5.2. Cell viability 9
2.6. Liquid or solid-state stability of nanosuspensions during storage 10
2.7. Determination of specific antibody titers 11
2.8. In vitro release test 11
3. Results and discussions 13
3.1. Hemagglutinin-Oleic acid conjugate (HOC) and HSA NPs 13
3.1.1. Physicochemical characterization of HOC 13
3.1.2. Physicochemical characterization of the nanoparticles 15
3.2. Morphological Characterization and drug loading capacity of NP 17
3.3. In vitro cytotoxicity 22
3.4. Stability of nanoparticles 24
3.5 Specific antibody titer to Hemagglutinin antigens 27
3.6. In vitro release test 29
4. Conclusions 31
5. References 32
국문초록 35

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