Immunoenhancement effects of chitosan-modified ginseng stem-leaf saponins-encapsulated cubosomes as an ajuvant

Highlights

• The GSLS loaded in cubosomes modified by CS were prepared by ethanol dispersion method.

• Cub-GSLS^CS significantly promoted the production of various cytokines in vitro.

• Cub-GSLS^CS boosted the uptake of antigens by macrophages.

• Cub-GSLS^CS-OVA induced a long-lasting specific humoral immune response.

Abstract

Nanoparticle delivery of functional molecules and vaccine is a promising method for enhancing the immune response. The objective of this study was to design chitosan (CS)-modified ginseng stem-leaf saponins (GSLS)-encapsulated cubosomes (Cub-GSLS^CS) as a vaccine delivery system and explore its immunologic activity and adjuvanticity. In this study, CS-modified GSLS-encapsulated cubosomes (Cub-GSLS^CS) were prepared. The storage stability of GSLS and that of ovalbumin (OVA) were measured. Additionally, the immunopotentiation of Cub-GSLS^CS were assessed on potentiating macrophage in vitro, and the adjuvant activity was evaluated through immune response triggered by OVA model antigen. The encapsulation efficiency of optimized Cub-GSLS^CS was about 65 % with Im3m nanostructure. The Cub-GSLS^CS showed excellent stability and sustained release for up to 28 days. In vitro, Cub-GSLS^CS nanoparticles improved cellular uptake, stimulated cytokines secretion of IL-6, IL-12, TNF-α, and generated more inducible nitric oxide synthase (iNOS) to produce higher levels of nitric oxide (NO) compared with other groups. Furthermore, the immunoadjuvant effects of OVA encapsulated Cub-GSLSCS nanoparticles (Cub-GSLS^CS-OVA) were observed through immunized mice. Results showed that the ratio of CD4+/CD8 + T lymphocytes was increased in Cub-GSLS^CS-OVA group. In addition, Cub-GSLS^CS-OVA nanoparticles induced dramatically high OVA-specific IgG, IgG1, and IgG2a levels and stimulated the secretion of cytokines. Cub-GSLS^CS may be a potential vaccine delivery system and induce a long-term sustained immunogenicity.

Introduction

Vaccine delivery systems are crucial to ensure that the antigens and immune potentiators included in a vaccine are able to interact with the immune system to enhance the immune response successfully [1]. The aluminum adjuvant and Freund's adjuvant has been generally used as the adjuvant in clinical vaccines. However, these adjuvants also have some disadvantages. Aluminum is a poor adjuvant in many situations, such as when used with subunit vaccine. Furthermore, aluminum cannot stimulate cell-mediated immune response [2]. Freund’s complete and incomplete adjuvant promote a commitment to the Th1 pathway, but has a risk of adverse effects such as local granuloma and inflammation [3]. Therefore, it is extremely urgent to design and prepare more simple, safe, and effective adjuvants, that could contribute to enhancing immune response.

Ginseng stem-leaf saponins (GSLS) have been widely used as immune-modulator and pharmaceuticals [4,5]. The immunoregulatory property discoveries, especially immunological adjuvant activities, have intensified the seeking of immunologic adjuvants which consist of GSLS and other materials to improve vaccination. Yu et al. demonstrated that administration of GSLS significantly improved immune responses to a bivalent Newcastle disease and avian influenza vaccine in chickens [6]. The GSLS combined with thimerosal can improve the vaccination of attenuated pseudorabies virus vaccine [7]. The discovery of biological activities of GSLS is not only limited to immunological activities, but more recently, also in pharmaceutical applications. Multiple pharmaceutical properties of GSLS has been found, such as anti-apoptosis, anti-tumor, anti-fatigue, hypoglycemic, [[8], [9], [10]]. In spite of its excellent immunoregulatory and pharmacological functions, small molecular weight and hydrophilicity of GSLS lead to low bioavailability and a short half-life in vivo. The terminology “cubosome” first arose in a review on cubic lipid/water phases by Kare Larsson in the 1980s [11]. The nanostructure has a tortuous, bicontinuous nature with a non-intersecting network of water channel, the two aqueous channels are in contact with the external environment in the open structure, while the closed cubosomes have one water channel open towards the external environment with the other compartment closed in relation to the outside [12]. This characteristic of cubosomes shows a successful encapsulation efficiency and sustained release as a drug delivery system [13]. Cubosomes based carries have been widely studied in vaccine adjuvant, cancer-directed therapy, infection treatment, treatment and inflammation treatment, and many other diseases [[14], [15], [16], [17], [18]]. However, the cubosomes have been negatively charged which limited their bioactivity. Therefore, cationic modification is required to address the limitations. Chitosan (CS) is a polycationic polysaccharide and has been extensively used in the pharmaceutical process due to it can interact effectively with negatively charged polymers and macromolecules [19]. Based on these characteristics of the cubosomes, CS-modified GSLS cubosomes (Cub-GSLS^CS) were made for the first time, the quality of Cub-GSLS^CS was evaluated in multiple perspectives. And then the response of immune cells to Cub-GSLS^CS was assessed in vitro, including the phagocytosis, and the produce of cytokines, NO, and iNOS. Afterwards, the nanoparticles were used as an adjuvant system injected in mice combined with ovalbumin (OVA). The OVA was encapsulated in Cub-GSLS^CS nanoparticles (Cub-GSLS^CS-OVA) to induce both humoral and cell-mediated immune responses.