br The size of liposomes is a critical determining factor
The size of liposomes is a critical determining factor for the lipo-some uptake by DCs. Liposomes with small size (< 100 nm) are rapidly removed from the site of injection, while liposomes with large size (> 200 nm) are shown to enter the surrounding lymph nodes. This is the reason that the size of all of the liposomal formulations were ad-justed below 150 nm and in the same range to insure that the particle size would not be a diﬀerential immunostimulatory factor for the li-posomes in the present study. These liposomes are removed by reticulo-endothelial Muscimol via clathrin-mediated endocytosis (Rejman et al., International Journal of Pharmaceutics 567 (2019) 118492
2004; Saremi et al., 2018; Shen et al., 1997). Moreover, the type of induced immune responses (Th1, Th2 responses) is a key immunologic factor that play important role in immunotherapy. It is worth men-tioning that the size of liposomes influences the type of immune re-sponse and diﬀerent patterns of cytokines. Brewer et al. indicated that liposomes with size of ≥225 nm induce Th1 responses and promote production of IFN-γ and IgG2a; whereas liposomes with a mean dia-meter of ≤155 induce Th2 responses and enhance level of IL-5 cyto-kine. (Brewer et al., 1998). In the current study, all liposomal for-mulation (F1, F2, F3 and F4) have a size ranged from 120 to 135 nm and PdI less than 0.1 that indicates a homogeneous population of li-posomes, able to induce Th2-related immunogenic responses. In con-trast, F1-liposome also induced Th1-related cytokines, indicating that the liposomal composition and surface charge are also the factors that must be taken into account of designing a liposomal platform for a specific immune system polarization.
Since liposomes are mainly taken up by the immune cells especially by macrophages, the impact of liposome on the induction of apoptosis of immune cells is considered as an important factor in the liposome-based immunotherapies (Lechanteur et al., 2018; Peters et al., 2015; Vasievich et al., 2011). The Lipid concentrations of the liposomal for-mulations used in apoptosis assay were similar to those used in the in-vitro and in-vivo therapeutic studies. The result from apoptosis assay revealed that the cationic F1-liposome containing DOTAP and DOPE phospholipid with 0.6 µmole concentration exhibits a relatively high cytotoxicity against splenocytes (Table 2).
Similar to our investigation, several studies evaluated diﬀerential cytotoxicity of liposomal formulations as vaccine delivery systems. For instance, Filion et al. reported that liposomal formulations containing DOPE and cationic lipid DOTAP had toxic eﬀect on macrophages in a relatively low concentrations of DOPE or DOTAP (Filion and Phillips, 1997). Aramaki et al. also reported that cationic liposomes were able to induce apoptosis in macrophages and the macrophage-like cell lines such as RAW264.7 through reactive oxygen species (ROS) pathway, which is associated with the liposomal surface charge (Aramaki et al., 1999). On the other hand, Chen et al. reported that liposomal for-mulations containing DMPC/DMPG/cholesterol had no apoptotic eﬀect on human lymphocytes (Chen et al., 2009).
The most remarkable finding of the present study was that the li-posomal formulations containing DOTAP and DOPE were able to sti-mulate cellular immunity and activated cellular immune responses such as the secretion of IFN-γ, IL-4 and IL-17. As previously reported, posi-tive-charged liposomes are potentially able to bind to the negatively charged surface of APCs in comparison to negative-charge liposomal formulations. This is the reason that liposomes composed of positively charged lipid such as DOTAP can stimulate cellular immune responses by providing antigens through APCs and activating CD8+ T cells (Askarizadeh et al., 2017; Nakanishi et al., 2000).
It suggests that the mentioned positively charged liposomes could stimulate a mixture of immune responses pathways without carrying any specific antigen. In this regard, DOPE, which is a pH-sensitive phospholipid, is shown to destabilize the liposome membrane integrity in the acidic endosomal compartment after liposome endocytosis, re-sulting in antigen escape to cytoplasm and the introduction of the an-tigen to MHC class I pathway (Lechanteur et al., 2018; Peters et al., 2015; Romoren et al., 2005; Vasievich et al., 2011).
In the present study, due to simulation of the generated immune responses by liposomal formulations, i.v and s.c routes were used for administration of liposomes. The idea of the diﬀerent route of lipo-somes injection was that the F1 to F3 liposomes formulations com-monly uses in vaccine researches due to their charge and lipid prop-erties which are desire to antigen delivery to APCs. However, the reason for i.v injection of F4 liposomes in our study is that this rigid, negatively charged liposome is extensively used for i.v administered cytotoxic drug delivery to tumor such as Doxil. Studies reported that various admin-istration routes of Ags with similar particle size leads to diﬀerent levels