Amphiphilic Triazine Polymer Derivatives as Antibacterial And Anti-atopic Agents in Mice Model

Considering the emergence of bacterial resistance and low proteolytic stability of antimicrobial peptides (AMPs), herein we developed a series of ultra-short triazine based amphipathic polymers (TZP) that are connected with ethylene diamine linkers instead of protease sensitive amide bond. The most potent oligomers, TZP3 and TZP5 not only displayed potent antibacterial action on various drug-resistant pathogens but also exhibited a strong synergic antibacterial activity in combination with chloramphenicol against multidrug-resistant Pseudomonas aeruginosa (MDRPA). Since most of atopic dermatitis (AD) infections are caused by bacterial colonization, we evaluated the potency of TZP3 and TZP5 on AD in vitro and in vivo. In vitro AD analysis of these two polymers showed significant inhibition against the release of β-hexosaminidase and tumor necrosis factor (TNF-α) from RBL-2H3 cells. In AD-like skin lesions in BALB/c mice model, these two polymers displayed significant potency in suppressing dermal and epidermal thickness, mast cell infiltration and pro-inflammatory cytokines expression. Moreover, these polymers exhibited remarkable efficacy over the allergies caused by the imbalance of Th1/Th2 by regulating total IgE and IgG2a. Finally, the impact of treatment effects of these polymers was examined through analyzing the weights and sizes of spleen and lymph node of AD-induced mice.


Effect of TZP3 and TZP5 on TNF-α suppression
Therefore, the dimer TZP3 and trimer TZP5 were probed for their potential for inhibiting the TNF-α induced from RBL-2H3 cells in in vitro method.
Calcium ionophore A23187 and PMA induced the TNF-α secretion in RBL-2H3 cells, on the contrary, treatment of which with TZP3 and TZP5 suppressed the TNF-α secretion in a dose-dependent manner, as shown in figure S2. Even though, 15.6 and 31.2 μg/mL of TZP3 displayed almost similar TNF-α suppressions (10%), treatment of 62.5 and 125 μg/mL of TZP3 showed significant effects in TNF-α suppressions, approximately 70% and 90%, respectively. Similarly, 15.6 and 31.2 μg/mL of TZP5 didn't show appreciable TNF-α suppressions, whereas treatment of 62.5 and 125 μg/mL displayed TNF-α suppressions in a dose-dependent manner. Figure S1. Inhibitory effects of the TZP3 and TZP5 on A23187 and PMA-induced TNF-α induced from RBL-2H3 cells. (Full-length blots are included in in the above section).

Effect of TZP3 and TZP5 on β-hexosaminidase suppression
Treatment with calcium ionophore A23187 in the RBL-2H3 cells induced the degranulation by releasing β-hexosaminidase as shown in figure S3.
Treatment of TZP3 and TZP5 against β-hexosaminidase showed a significant effect in a dose-dependent manner. Especially at a concentration of 500 μg/mL, they showed maximum inhibitory effects. concentrations for 24 h followed by MTT assay, and absorbance was measured. Cell viability was calculated as the relative absorbance. Figure S4: Allergic dermatitis model and treatment protocols.
After the addition, the resultant mixture was stirred at 0 o C for three hours and warmed to room temperature, and stirred for 20 h at room temperature.