Killer Peptide-containing polyelectrolytic nano complexes to fight toxoplasma gondii infection

Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is usually asymptomatic in immunocompetent individuals but can cause severe complications in immunocompromised patients and during pregnancy. Current treatments (e.g., pyrimethamine or sulfadiazine) are effective against acute infections but fail to eradicate encysted bradyzoites and have significant side effects. Antimicrobial peptides, such as the Killer Peptide (KP), are investigated as alternative therapeutics due to their antimicrobial and immunomodulatory activities, showing efficacy against fungal and parasitic infections, such as those by Candida albicans and T. gondii1,2. However, intracellular delivery is challenging. Cells infected by T. gondii overexpress CD44, the receptor for hyaluronic acid3. On these premises, hyaluronate-based nanoparticles containing KP were prepared to enhance its targeted delivery and therapeutic potential in infected cells. Polyelectrolyte complexes made of chitosan and hyaluronate were produced by microfluidic technology. KP was dissolved at 3.5 mg/mL in chitosan solution (410 μg/mL) and mixed with a hyaluronate solution (70 μg/mL) in a herringbone mixer at a flow rate ratio of 1:6 and a total flow rate of 280 μL/min (KP-NPs). Blank nanoparticles without KP were analogously prepared (BLK-NPs). Dynamic light scattering revealed an average size of 344 ± 169 nm and 394 ± 129 nm for BLK-NPs and KP-NPs, respectively. Z-potential was -10 ± 7 mV for BLK-NPs and -12 ± 7 mV for KP-NPs, suggesting the presence of hyaluronate on the outer layer, potentially aiding cell uptake via CD44 receptors. Encapsulation efficiency (EE%) was estimated in 76 ± 9%. The stability of NPs was assessed over one week at 4°C, 25°C or 37°C showing no significant variations in size or EE%. Antimicrobial activity of free KP or KP-NPs was tested against C. albicans at 2.5 μg/mL, leading in both cases to a 100% killing of the yeast, confirming that encapsulation did not alter KP efficacy. Analogously, with T. gondii, comparable IC₅₀ values were estimated for free KP and NP-KPs (120–200 μg/mL) using a Toxo-β-gal assay. Infected cell reduction ranged from 67–86%, with intracellular tachyzoite reduction from 71–93%. Interestingly, BLK-NPs alone showed antiparasitic activity, prompting further investigation into their intrinsic effects. References 1. Polonelli et al Sci Rep 2017, 7, 10896 2. Giovati et al Frontiers in Microbiology 2018, 9:753 3. Unno et al., Bioch and Bioph Res Comm 2010, 391, 477–480