Polyethylenimine-coated PLGA nanoparticles-encapsulated Angelica sinensis polysaccharide as an adjuvant to enhance immune responses
Graphical abstract
Introduction
Vaccination plays a crucial role in preventing infectious and viral diseases because it could induce a vigorous antigen-specific immune response for long-term protection and therapy (Liu, Cao et al., 2016, 2016b; Sarti et al., 2011). Compared to traditional vaccines, the protein subunit vaccines are considered to be safer but with poor immunogenicity. The adjuvants are required to augment resultant humoral and cell-mediated immune responses (Liu, Cao et al., 2016; Zhang, Wang et al., 2014). Aluminum, as a conventional and commercial adjuvant, had used in vaccines for many years. However, the Aluminum has some disadvantages, including local and systemic side effects, poorly cell-mediated immune responses and inefficiency for some antigens (Chen et al., 2014; Courant et al., 2017; Kool et al., 2008). Therefore, it is necessary to design safer and more effective adjuvants to induce strong humoral immune responses and stimulate cell-mediated immune responses.
Angelica sinensis (AS), a Chinese herbal medicine, has been traditionally used to treat anemia, constipation, hepatic fibrosis, cardiovascular disease and gynecological diseases for thousands of years (Hook, 2014; Hua, Xue, Zhang, Wei, & Ji, 2014). Angelica sinensis polysaccharide (ASP), one of the main active components, possess varieties of pharmacological activities, such as hematopoietic effect, hepatoprotective effect, antioxidant potency, antitumor activity, radioprotective effect, and immunologic enhancement (Ji et al., 2014; Lee, Hsieh, Chen, & Chiang, 2012; Zhang et al., 2016; Zhao et al., 2012; Zhuang et al., 2016). It has been reported that the ASP was composed of fucose, galactose, glucose, arabinose, rhamnose, and xylose and in a molar ratio of 1.0:13.6:15.0:8.7:21.3:3.7 (Wang, Ding, Zhu, He, & Fang, 2003), and the neutral ASP was mainly composed of galactose, arabinose and glucose (Zhao et al., 2012), in consistent with the results reported by others (Sun, Tang, Gu, & Li, 2005; Zhang, Cheng, Wang, Zhang, & Wang, 2014). It was found that ASP had a backbone composed of 1,4-α-D-glucopyranosyl residues, with branches attached to O-6 of some residues. The branches were composed of 1,6-α-D-Glcp residues, and terminated with β-L-arabinofuranose residues (Cao, Li, Liu, Yang et al., 2006, 2006b). Recently, ASP has been widely used as immunostimulant and adjuvant to improve the immune activity (Wang, Ge, Li, Guan, & Li, 2016; Yang, Jia, Meng, Wu, & Mei, 2006). However, ASP has some disadvantages such as non-focused action scope, fast-metabolism and low bioavailability, which limit its application.
In recent years, the nanoparticles adjuvant delivery systems have drawn growing attention because the nanoparticles could protect the antigen and drugs from degradation, increase drug bioavailability, facilitate antigen uptake by antigen presenting cells (APCs) and enhance immune responses (Canadas et al., 2016; Divya et al., 2018; Du et al., 2017; Vijayakumar & Vaseeharan, 2018; Vijayakumar et al., 2017; Yue & Ma, 2015). Nanoparticles delivery systems also have the potential to regulate the antigen presentation pathway (Yue & Ma, 2015; Zupancic et al., 2017). Several studies have proved that nanoparticles with positive surface charge could increase the adsorption of antigen, enhance the interaction with negatively charged cell membrane and facilitate the intracellular uptake of antigen by APCs such as dendritic cells (DCs) and macrophages to modulate the immune responses (Chen et al., 2014; Liu, Ma et al., 2016; Salvador et al., 2015; Zupancic et al., 2017). Poly (lactic-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer, has been extensively used to prepare nanoparticles for drug and vaccine delivery with high safety and controlled release (Hafner, Corthesy, Textor, & Merkle, 2016; Salvador et al., 2015). Numerous researches have demonstrated that PLGA nanoparticles delivery systems could protect drug and antigen, provide a controlled and persistent release of drug and antigen and co-deliver drug and antigen to the same APCs (Danhier et al., 2012; Sarti et al., 2011; Wang, Tan, Keegan, Barry, & Heffernan, 2014). However, the PLGA nanoparticles with no modification are often negatively charged, which limits the adjuvant activity of the nanoparticles. Cationic polymers had been widely used as transfection agents and could activate the macrophages and DCs to induce immune responses (Mulens-Arias, Rojas, Perez-Yague, Morales, & Barber, 2015). The polyethylenimine (PEI), one of the widely used cationic polymers, could modify the surface charge of the PLGA nanoparticles (Salvador et al., 2015). It has been proved that the PEI-coated nanoparticles, used as the drug delivery systems, could promote the cellular uptake efficiency and improve immune responses (Chen et al., 2014; Salvador et al., 2015; Yu et al., 2016).
In our previous researches, the ASP was encapsulated into PLGA nanoparticles to develop a drug delivery system (ASP-PLGA nanoparticles) to improve the immunological enhancement of ASP (Gu et al., 2018). In addition, it has been reported the OVA encapsulated into ASP-PLGA nanoparticles as a vaccine delivery system could stimulate strong humoral responses and cellular immune responses (Gu et al., 2019). However, the ASP-PLGA nanoparticles had shown poorly effects on the activation of APCs in our preliminary experiments. In this study, we had developed a stable and well-characterized cationic nanoparticle delivery system (ASP-PLGA-PEI), and we hypothesized that ASP-PLGA-PEI nanoparticles would function as an effective adjuvant and vaccine system to target and activate the APCs, and induce strong humoral and cell-mediated immune responses. This hypothesis was tested by investigating the effects of ASP-PLGA-PEI nanoparticles on activation of macrophages and antigen uptake by macrophages. Furthermore, we utilized the PCV2 antigen adsorbed on ASP-PLGA-PEI nanoparticles to evaluate the ability to induce humoral and cell-mediated immune responses.
Section snippets
Materials
ASP (95%, CY170618) was obtained from Shanxi Ciyuan Biotechnology Co., Ltd (Shanxi, China). PLGA (75:25, MW 18 kDa) was purchased from Jinan Daigang Biomaterial Co., Ltd (Shandong, China). Pluronic F68 (F68) and Sephadex G-50 were obtained from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). PEI (MW 25 kDa) was purchased from Sigma-Aldrich (USA). Anti-Mouse-MHCII-FITC anti-Mouse-CD86-PE antibodies were purchased from eBioscience Inc. (Thermo Fisher Scientific, USA). OVA-FITC and the
FT-IR spectra of ASP
The FT-IR spectra of the ASP were shown in Fig. 1. The band at around 3400 cm−1 was due to the stretching vibration of OH. The weak peak at around 2932 cm−1 was due to the asymmetrical CH stretching and bending vibration. The band at around 1632 cm-1 was attributed to the bound water (Zhang et al., 2016). The absorption peaks at around 1416 cm−1 and 1055 cm-1 were characteristic bands of the carboxylic group. The peaks at 1400–1200 cm-1 were also the presence of the CH bonds. The wave number
Discussion
Numerous studies have shown that Angelica sinensis polysaccharide (ASP) possess the immunomodulatory activity related with T-lymphocyte activation (Jin, Zhao, Huang, Xu, & Shang, 2012; Sun et al., 2005; Yang, Jia et al., 2006). The ASP was biologically safe and had been widely used as immunopotentiator and adjuvant to enhance the immune responses (Wang et al., 2016). Recently, the vaccine delivery and adjuvant systems based PLGA nanoparticles have been intensively investigated due to their
Conclusions
Herein, we successfully engineered a simple, efficient well-characterized and good stability nanoparticles delivery system with positive surface charge. The ASP-PLGA-PEI nanoparticles showed excellent performance in the activation of macrophages. The results indicated that ASP-PLGA-PEI nanoparticles could be a useful strategy for effective antigen adsorption and efficiently enhance the antigen uptake by macrophages. To further investigate the adjuvant activity of the ASP-PLGA-PEI nanoparticles,
Acknowledgments
The project was supported by the National Natural Science Foundation of China (Grant No. 31872509, 31672596), the Fundamental Research Funds for the Central Universities (Grant No. KYZ201844) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We are grateful to all other staff in the Institute of Traditional Chinese Veterinary Medicine of Nanjing Agricultural University for their assistance in this study.
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