Angelica sinensis polysaccharide encapsulated into PLGA nanoparticles as a vaccine delivery and adjuvant system for ovalbumin to promote immune responses
Graphical abstract
Introduction
Vaccines had been widely used to prevent many infectious diseases for many years because they could provide a strong and long-term protection and therapy (Sarti et al., 2011). Compared to the traditional inactivated and live attenuated microbe vaccines, the new types of vaccines based on subunit antigens and recombinant proteins, become safer but with poor immunogenicity (Hanson et al., 2014, Liu et al., 2016a, Wang et al., 2014). Thus, the adjuvants were required to increase the immunogenicity and augment immune responses caused by vaccines with the development of vaccines (Hanson et al., 2014, Liu et al., 2016a). It is reported that Aluminum was the most conventional adjuvant, which could effectively induce humoral immune responses but poorly elicit the cell-mediated immunity (Chen et al., 2014, Kool et al., 2008), which plays an important role in immune responses to limit disease severity (Liu et al., 2015). Also, aluminum adjuvants may cause some side effects and safety concerns, such as inflammation and induce serious immunological disorders in humans (Tomljenovic and Shaw, 2011). Similarly, the FCA (Freund complete adjuvant) composed of inactivated mycobacteria, could effectively stimulate cell-mediated immunity and induce stronger humoral immune responses. However, as a potent and efficient adjuvant, FCA was considered unsuitable for human use due to its local and systemic toxicity (Aguilar and Rodriguez, 2007). Therefore, it is necessary to develop the safe and efficient adjuvants to induce strong and long-term immune responses.
In the last few years, several studies had revealed that Nanoparticles (NPs) delivery systems could increase the effects of vaccines in preclinical animal studies when the antigens were encapsulated into NPs (Siefert et al., 2016, Wang et al., 2014). Poly(lactic-co-glycolic acid) (PLGA), a synthetic biodegradable polymer accepted by FDA and EMA for use in humans, has drawn much attention and has been widely used as adjuvants in vaccination to carry antigens and drugs due to their advantaged characteristics, such as the excellent biodegradability and biocompatibility, good colloidal stability and the capacity to control the release of the encapsulated antigens and drugs (Desai and Schwendeman, 2013, Rose et al., 2015, Salvador et al., 2015). A number of studies have demonstrated that PLGA NPs with encapsulated antigens have the potential to enhance immune responses due to the protection and controlled release of antigens for a long time (Desai and Schwendeman, 2013, Jiang et al., 2005, Rosas et al., 2001).
Angelica sinensis (Oliv.), a kind of traditional Chinese herbal medicine, has been widely used to treat anemia, gastrointestinal disease, cardiovascular disease and other diseases for many years (Ai et al., 2013, Wei et al., 2016, Zhang et al., 2016). Angelica sinensis polysaccharide (ASP) is one of the main effective chemical substances of Angelica sinensis. It is reported that ASP has a backbone composed of (1 → 3)-linked Galp, (1 → 6)-linked Galp and 2-OMe-(1 → 6)-linked Galp with three branches attached to O-3 of 2-OMe-(1 → 6)-linked Galp and terminated with GlcpA and Araf and the ASP consists of glucuronic acid, glucose, arabinose, galactose with a molar ratio of 1.00:1.70:1.85:5.02 (Zhang et al., 2016). Many studies have proved that ASP was biologically safe and possess a variety of pharmacological effects and bioactivities, such as immunologic enhancement, anti-tumor, hematopoiesis, hepatoprotective effect, anti-oxidation and radioprotection (Lee et al., 2012, Wang et al., 2016a, Wang et al., 2016b, Zhuang et al., 2016, Zhao et al., 2012, Zhang et al., 2010). Currently, ASP has been widely studied as immunopotentiator and it has been demonstrated its immuno-enhancement activity (Wang et al., 2016a). However, ASP has some disadvantages such as fast-metabolism, unconcentrated action scope and low bioavailability, which restrict its application in the clinic.
In this study, the ASP and model protein antigen ovalbumin (OVA) were encapsulated into PLGA NPs to construct a novel vaccine delivery system (ASP-PLGA/OVA NPs). The NPs were characterized regarding the size, distribution, surface charge, morphology and encapsulation efficiency of OVA. Also, the stability of ASP-PLGA/OVA NPs was evaluated. In order to investigate the magnitude and kinetics of antibody and cellular immune responses to the vaccine delivery system, the ASP-PLGA/OVA NPs was subcutaneously administered to mice on Day 0 and boosted with equivalent doses at Day 14. The immune responses were characterized by measuring antibody levels and cytokines expression in serum from vaccinated mice. We also tested the spleen lymphocytes proliferation and the immunophenotype of T cells with re-stimulated by OVA ex vivo. These results suggested that the ASP-PLGA/OVA NPs could stimulate strong and long-term immune responses and a mixed Th1/Th2 immune response. The ASP-PLGA NPs has the potential to be an efficient and safe adjuvant.
Section snippets
Materials
PLGA (75:25, MW 18 KDa) was obtained from Jinan Daigang Biomaterial Co., Ltd (Shandong, China). OVA was purchased from Sigma-Aldrich (USA). ASP (95%, CY170618) was purchased from Shanxi Ciyuan Biotechnology Co., Ltd (Shanxi, China). Pluronic F68 (F68) was obtained from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). The bicinchoninic acid (BCA) Protein Assay Kit was obtained from Solario (Beijing Solarbio&Technology Co., Ltd, Beijing, China). Freund complete adjuvant (FCA) was
Characterization of OVA-encapsulated NPs
The particle size, PDI, zeta potential and OVA-EE (%) of the NPs were presented in Table 1. The mean hydrodynamic size of PLGA/OVA NPs was 188.3 ± 0.92 nm, while the mean particle size of ASP-PLGA/OVA NPs was 225.2 ± 2.66 nm. The average size of ASP-PLGA/OVA NPs was a little larger than blank PLGA/OVA NPs, which may because the ASP was encapsulated in the NPs. The PDI of PLGA/OVA NPs was 0.123 ± 0.008, and 0.213 ± 0.024 for ASP-PLGA/OVA NPs, both presented a low polydispersion. The results
Discussion
Designing safe and efficacious vaccines for cancer and infectious diseases remains a major goal in global public health. In order to enhance the effects of the vaccines, the novel vaccine adjuvants were developed. Compared with traditional vaccine adjuvants, nanoparticles (NPs) delivery vaccine systems such as PLGA, could potentially induce a long-lasting adoptive immune responses due to their biocompatibility, stability and slow release of encapsulated antigens (Liu et al., 2016a, Pashine et
Conclusions
In this study, our goal was to design a stable, safe and efficacious adjuvant to deliver the vaccine antigens. The ASP-PLGA/OVA NPs was constituted with ASP and OVA encapsulated into PLGA NPs. We found that ASP-PLGA/OVA NPs had good colloidal stability at 4 °C and could provide a sustained and controlled release of OVA from the NPs at 37 °C. It has shown that mice immunized with ASP-PLGA/OVA NPs promoted the lymphocyte proliferation and increased the ratio of CD4+ to CD8+ T cells, thereby
Acknowledgments
The project was supported by National Natural Science Foundation of China (Grant no. 31872509, 31672596), the Fundamental Research Funds for the Central Universities (Grant no. KYZ201844), Special Fund for Agro-scientific Research in the Public Interest (Grant no. 201403051) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We are grateful to all other staffs in the Institute of Traditional Chinese Veterinary Medicine of Nanjing
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