Zn- and Mg- containing tricalcium phosphates-based adjuvants for cancer immunotherapy

Abstract

Zn-, and Mg-containing tricalcium phosphates (TCPs) loaded with a hydrothermal extract of a human tubercle bacillus (HTB) were prepared by immersing Zn-TCP and Mg-TCP in HTB-containing supersaturated calcium phosphate solutions. The in vitro and in vivo immunogenic activities of the HTB-loaded Zn-, and Mg-TCPs (Zn-Ap-HTB and Mg-Ap-HTB, respectively) were evaluated as potential immunopotentiating adjuvants for cancer immunotherapy. The Zn-Ap-HTB and Mg-Ap-HTB adjuvants showed no obvious cytotoxicity and more effectively stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion by macrophage-like cells than unprocessed HTB or HTB-loaded TCP (T-Ap-HTB) in vitro. Zn-Ap-HTB and Mg-Ap-HTB mixed with liquid-nitrogen-treated tumor tissue markedly inhibited the in vivo development of rechallenged Lewis lung carcinoma (LLC) cells compared with T-Ap-HTB and the unprocessed HTB mixed liquid-nitrogen-treated tumor tissue. Zn-Ap-HTB and Mg-Ap-HTB contributed to eliciting potent systemic antitumor immunity in vivo.

Figure 1. XRD patterns of TCP, Mg-TCP (A) and Zn-TCP (B) powders.

Figure 2. Distributions of hydrodynamic radius of Mg6.8 (A) and Zn6.8 (B) dispersed in PBS(-) obtained by dynamic light scattering measurement at various incidence angles of laser (from 30–100°).

Figure 3. Electrical mobility distributions of TCP, Mg1.5 and Zn1.5 particles in PBS(-).

Figure 4. FESEM images of TCP (A-1), Mg1.5 (B-1), Zn1.5 (C-1), T-Ap-HTB (A-2), Mg1.5-Ap-HTB (B-2) and Zn1.5-Ap-HTB (C-2).

Figure 5. GM-CSF secretion by macrophage-like cells after cultured in the original culture medium, culture media containing HTB, Mg-Ap-HTB (A), and Zn-Ap-HTB (B) adjuvants (n = 8).

Figure 6. In vitro cell viability of macrophage-like cells after cultured in the original culture medium, culture media containing T-Ap-HTB, Mg-Ap-HTB (A), and Zn-Ap-HTB (B) adjuvants (n = 3*2).

Figure 7. Percent of mice without development of re-challenged tumor on the right flank of mice over time after re-challenge of LLC cells.

(* P<0.05 vs.HTB group, n = 12).

Figure 8. Contents of cytokines relating to cell-mediated immunity in mice spleen after 29 days from re-challenge of LLC cells.

GM-CSF (A), IL-2 (B), TNFα (C), IL-12 (D) and IFNγ (E). Red solid circles with error bars show the mean value and standard deviation (* p<0.05, n = 12).