Seasonal influenza is a highly contagious disease caused by influenza viruses spreading through the respiratory tract, posing a serious threat to human health worldwide [1]. Currently, vaccination by injection is the primary means of preventing influenza viruses from attacking the body [2]. The immune system recognizes the injected exogenous antigen and produces specific IgG antibodies, which can greatly reduce the risk of morbidity [3]. However, influenza virus infection begins in the mucous membranes, and inducing protective immunity at mucosal sites is the most effective way to prevent influenza virus infection. It is difficult for the injected vaccine without inoculation through mucosal tissues to produce adequate IgA antibodies and provide sufficient immune protection against viral respiratory infections [4], [5]. In addition, the production process of injectable vaccines is complex and costly [6]. Therefore, alternative routes of immunization are necessary to achieve better influenza prevention.

It is worth noting that the intestinal tract is at the forefront of the body's immune defense, carrying 70–80 % of the body's immune cells and contributing critically to the body's immune function [7], [8]. Therefore, oral vaccines have attracted increasing attention. Oral vaccines provoke both systemic and mucosal immune responses, generating a dual protective immune response that can be used to fight pathogens infected via either the mucosal or non-mucosal routes [9], [10]. Importantly, oral vaccines can simultaneously establish an immune-protective response at distal mucosal sites, such as respiratory tract, owing to the presence of the common mucosal immune system [11], [12]. Moreover, oral vaccines have high patient compliance and low risk of contamination compared to the injectable route, suggesting that they may become the mainstay of vaccine delivery in the future [13].

However, the complex conditions in the gastrointestinal tract (low pH and protein hydrolysis) present a serious challenge to vaccine activity. Several adjuvants or vaccine delivery systems have been proposed to address the above challenges [6]. For example, immunoadjuvants such as liposomes and lipopolysaccharides proposed in recent years have made some developments in the field of oral vaccines [14], [15]. Unfortunately, the vaccine loading capacity of these organic nanoparticles is low and the vaccines are prone to leak in the gastrointestinal tract [16]. Mesoporous silica nanoparticles (MSN) have been shown to be promising oral vaccines carriers and immune adjuvants [17], [18]. Compared with traditional vaccine adjuvants, MSN have the advantages of stable structure, adjustable pore and particle size, high specific surface area and easy surface modification, providing more suitable conditions for oral vaccine delivery [19], [20], [21]. In particular, large pore mesoporous silica nanoparticles (LMSN), which improve the vaccine loading capacity of conventional MSN, have attracted much attention [22], [23]. When the vaccine is combined with MSN, it can induce a higher level of immune response in the body, triggering humoral and cell-mediated immune responses [6]. Therefore, MSN are favorable contenders for developing novel vaccine delivery systems and immunoadjuvants.

The physicochemical properties of nanoparticles have an important impact on their adjuvant effects and have been extensively studied. For instance, Yang et al. explored that the surface chemistry properties of nanoparticles, such as charge and hydrophobicity, induce different types and intensities of immune responses [24]. In particular, the particle size of nanoparticles is a central factor affecting their absorption and biodistribution, and as vaccine delivery carriers, they will determine how the immune system recognizes foreign antigens [25], [26]. It has been reported that nanoparticles above 150 nm are more inclined to be distributed in the liver and spleen; particles below 5 nm are easily filtered out by the kidney. Our previous research demonstrated that the immune effect of oral BSA/MSN was rely on the architectures of MSNs, including pore size, geometry and particle size [26], [27]. However, this result is caused by the combination of several factors, and the influence of single factor of the carrier on immune effect has not been examined from the aspects of oral absorption and interaction with immune cells.

In this study, the size effect of LMSN regulating the mucosal immune effect of oral influenza vaccines was attempted to be elucidated. In order to load influenza split vaccine (SV) into the pores, the pore size of LMSN was standardized to one around 38 nm. And for correctly evaluating the size effect of LMSN on immunization effect, it is necessary to control the shape, pore size and surface charge of nanocarriers. However, it is a challenge, particularly, for the preparation of nanoparticles with small particle size (<100 nm) and large pore size (about 38 nm). We screened a large number of prescriptions and synthesized three kinds of LMSN (LMSN-S, LMSN-M and LMSN-L) with different small particle sizes (177.1 nm, 252.3 nm and 371.5 nm) as oral vaccines carriers and immunoadjuvants. Significantly, the effect of particle size variation on the immunization effect was explored by achieving the strict control on the shape and surface charge of LMSN. The difference in immunization effect was observed by gavage of SV/LMSN with different particle sizes to BALB/c mice. The results showed that the mucosal IgA antibody level induced by SV/LMSN-M was obviously higher than that induced by SV, SV/LMSN-S and SV/LMSN-L, indicting the adjuvanticity of LMSN-M. To explore in detail the reasons for such difference in immune effect, the relationship between the particle size of carrier and adjuvanticity was investigated in terms of drug loading capacity, release behavior in vitro, intestine absorption and transport, biodistribution, uptake and maturation of immune cells. This study comprehensively analyzed the effect of LMSN particle size towards the immune influence of oral SV and its adjuvant effect, providing a new insight into the rational design of vaccine adjuvants.