3.1 The characters of LPPC
As figure 1a shown, the shape of LPPC was approximately round and the particle size was about 200 nm. In addition, the dark shadow of LPPC was hair-like, which might be PEI and PEG polymers. LPPC is a cationic liposome, and it was found that LPPC can adsorb proteins on its surface. Therefore, DLS was utilized to investigate the particle size of LPPC with or without protein adsorption. The results showed that the diameters of LPPCs with protein adsorption were about 358 ± 16 nm, which was larger than the LPPC without protein adsorption (Figure 1b).
Besides, the previous results have shown the empty LPPCs can be centrifuged and pelleted (Figure 1c) and the further experiments also indicated the protein adsorption did not affect this character. Because centrifugation is available for LPPC, unbound substances could be easily removed.
3.2 The characters of LPPC for protein adsorption
To understand the kinetic for protein adsorption to LPPC, the fluorescence of BSA-FITC was used to evaluate what time the LPPC need to adsorb proteins to their surface. The results showed that LPPC could adsorb 80% of proteins in ten minutes and reach the maximal adsorption in 20 minutes (Figure 2a). Moreover, the protein binding capacity of LPPC was estimated and the results revealed that the maximal adsorption of 40 µg LPPC was about 160 µg BSA (Figure 2b).
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Surprisingly, the pre-adsorbed BSA-FITC on LPPC could not be replaced by the additions of different BSA dose (Figure 2c). The results showed that the pre-adsorbed proteins on LPPC were irreplaceable by the posterior added proteins.
3.3 The activities of immunostimulatory monoclonal antibodies adsorbed on LPPC
3.3.1 The cytotoxicity of LPPC to PBMCs or splenocytes
LPPC could adsorb proteins stably and remain their activities as previous experiments (Table 1). Therefore, to investigate whether LPPC could adsorb immunostimulatory monoclonal antibodies and stimulate immunity was further proceeded. First, the cytotoxicity of LPPC was determined for PBMCs or splenocytes at next experiment. The results indicated that 1 µg LPPC was an appropriate dosage for 105 PBMCs or 2.5×105 splenocytes, because the cells could survive without toxic damage in this concentration (Figure 3).
3.3.2 The effects of the bound antibodies in a dosage-dependent manner
In this study, anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) were utilized to activate T cell, which were used to determine whether the bound protein on LPPC could remain its biofunction. In order to understand the regulatory phenomenon of monoclonal antibodies on LPPC for activities, the different amounts of immunostimulatory mAbs were adsorbed on LPPC to stimulate the proliferation of PBMCs or
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splenocytes. The results showed the cell proliferation rates of PBMCs and splenocytes were higher as anti-CD3 mAbs were increased. It would be more obvious when the anti-CD3 mAbs combined with anti-CD28 mAbs to work on immune cells (Figure 4). Therefore, the bound antibodies on LPPC could activate the cell in a dosage-dependent manner.
Further, whether the secretions of cytokines were triggered by the bound mAbs on LPPC in a dose-dependent manner was investigated and the results indicated that LPPC and the adsorbed immunostimulatory mAbs could stimulate PBMCs or splenocytes to secrete cytokines, such as IL-2, IFN-γ and TNF-α. In addition, the concentrations of cytokines in media were increased as the anti-CD3 mAbs were increased (Figure 5).
Moreover, the expressions of cytokines were increased by the addition of anti-CD28 mAbs which could provide the costimulatory signal to enhance the T-cell response as previously reported. Besides, LPPC alone could activate TNF-α secretion of PBMCs and splenocytes but it could not trigger any the IL-2 and IFN-γ secretions (Figure 5). Therefore, the LPPC reagent was investigated further to analyze whether the inductions of other pro-inflammatory cytokine profiles were. The results showed that the LPPC could stimulate IL-1β, IL-6 and IL-8 secretions of immune cells, except for TNF-α expression (Figure 6).
Comparing to the activities of unbound mAbs, the bound mAbs showed there were no significant differences between the cell proliferation and cytokine secretions (IL-2, IFN-γ) (Figure 7).
Nevertheless, LPPC with mAbs could enhance TNF-α secretion comparing to unbound mAbs, it should be due to the LPPC’s ability to facilitate the TNF-α secretion.
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3.4 The stability of immunostimulatory monoclonal antibodies adsorbed on LPPC in RPMI
To investigate the stability of the bound mAbs on LPPC in 37℃, the activities of the dissociated antibodies in the medium were estimated. The results indicated that the antibodies on LPPC pellet remained their partial activities after 37℃ treatment to induce 90% proliferation for PBMCs or splenocytes (Figure 8). In addition, the antibodies in supernatant only displayed low activities to cause inferior proliferative index for PBMCs or splenocytes. These results revealed that the antibodies bound on LPPC would rather stably adhere than dissociate from the particle.
3.5 The enhancement of uptake protein ability by LPPC
The previous results indicated that LPPC alone has the ability to enhance pro-inflammatory cytokine secretions of PBMCs and splenocytes (Figure 5, 6). To understand whether LPPC has an adjuvant effect for the enhancement of antigen uptake by phagocytosis, the phagocytic rate for the fluorescent antigen was evaluated. As to the ability of phagocytosis, LPPC/BSA-FITC complexes indeed enhanced the uptake ability of P338D1 compared to BSA-FITC without LPPC adsorption (Figure 9a). In addition, as the additional amounts of LPPC were increasing, the uptake abilities were enhanced with the more efficiency (Figure 9b) and the results also showed that the phagocytic rates of P338D1 were in a dose-dependent manner (Figure 9c).
As far as the ability of presentation is also concerned, figure 10
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showed that the addition of BSA alone could be internalized by APC and present to the specific anti-BSA T-cells, which could trigger and increase the cell proliferation and cytokine secretions (including IL-2, IFN-γ, TNF-α, IL-4 and IL-10). By contrast, the results indicated that BSA adsorbed on LPPC could provide more efficacies to induce cell proliferation and cytokine secretions than BSA without LPPC adsorption (Figure 10).
3.6 The specificities of the LPPC-bound proteins
Certain immunostimulatory molecules on cell membrane, such as MHC or B7 molecules could trigger the specific immune response, so that DCs’ proteins on plasma membrane were determined whether they could perform their activities on LPPC surface as same as on the plasma membrane. Before isolated the plasma membrane proteins of DCs, anti-CD11c-PE, anti-MHC II-FITC, and anti-CD86-FITC were used to confirm that the surface markers of DCs performed (Figure 11). The membrane proteins derived from the DCs which were treated with HpHsp60 were bound to LPPC, which could react with the splenocytes derived from the mice had been prior immunized with HpHsp60 to induce the cell proliferation and cytokine secretions (Figure 12). However, neither the splenocytes without Hphsp60-immunized nor the DC’s membrane proteins without Hphsp60-treated could induce the cell proliferation or cytokine releases (Figure 12).
Moreover, YMLDLQPETT peptides (YML) derived from HPV E7 protein were loaded into the HLA-A2 molecules to verify the specificity of the LPPC-bound proteins again. The YML-loaded HLA-A2 molecules
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on LPPC were interacted with the splenocytes derived from naïve or pre-immunizing E7 mice. The results showed that peptide-loaded HLA-A2 molecules on LPPC indeed remained their specificities to activate the splenocytes of pre-immunizing E7 mice and cause the cell proliferation and cytokine expressions but did not react with the naïve splenocytes (Figure 13). In addition, the anti-CD28 mAbs could facilitate the immune responses for the splenocytes of pre-immunizing E7 mice but have no effect on the naïve splenocytes (Figure 13).
3.7 The efficiency of immunization in vivo
The cell proliferations and cytokine expressions of the splenocytes which were i.v. immunized by immuno-LPPC estimated whether the induction of specific immune responses. The induction of the specific anti-HpHsp60 immune responses of splenocytes from membrane proteins /LPPC complex immunized was more efficient than that from HpHsp60 antigen (Figure 14). In addition, the results indicated that the splenocytes form the other immunization methods did not significantly react to HpHsp60.
Furthermore, the splenocytes that the LPPC combined peptide-loaded HLA-A2 molecules with anti-CD28 mAbs immunized were efficiently activated immune responses against YML peptides, such as cell proliferations and cytokine secretions (Figure 15). No apparent immune responses were observed against YML peptides in the other immunization methods.
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