The rational design of a synthetic polymer nanoparticle that neutralizes a toxic peptide in vivo

Abstract

Synthetic polymer nanoparticles (NPs) that bind venomous molecules and neutralize their function in vivo are of significant interest as "plastic antidotes." Recently, procedures to synthesize polymer NPs with affinity for target peptides have been reported. However, the performance of synthetic materials in vivo is a far greater challenge. Particle size, surface charge, and hydrophobicity affect not only the binding affinity and capacity to the target toxin but also the toxicity of NPs and the creation of a "corona" of proteins around NPs that can alter and or suppress the intended performance. Here, we report the design rationale of a plastic antidote for in vivo applications. Optimizing the choice and ratio of functional monomers incorporated in the NP maximized the binding affinity and capacity toward a target peptide. Biocompatibility tests of the NPs in vitro and in vivo revealed the importance of tuning surface charge and hydrophobicity to minimize NP toxicity and prevent aggregation induced by nonspecific interactions with plasma proteins. The toxin neutralization capacity of NPs in vivo showed a strong correlation with binding affinity and capacity in vitro. Furthermore, in vivo imaging experiments established the NPs accelerate clearance of the toxic peptide and eventually accumulate in macrophages in the liver. These results provide a platform to design plastic antidotes and reveal the potential and possible limitations of using synthetic polymer nanoparticles as plastic antidotes.

Fig. 1.

Interaction between melittin and NPs synthesized with various feed ratio of TBAm and AAc. (A) AFM image of NPs 9. (B) Neutralization constants of NPs obtained from hemolytic toxicity neutralization assay. C. Apparent binding constant between melittin and NPs obtained from 27-MHz QCM experiments. Red spots on graphs (B) and (C) indicate NPs that did not show neutralization or melittin binding, respectively. Blue spots indicate polymers that precipitated during polymerization or purification. (D) The effect of AAc incorporation on the binding capacities (blue, left axis) and apparent binding constants (green, right axis) of NPs that were polymerized with 40 mol% TBAm. Note that left axis is linear and the right is logarithmic. (E) Effect of AAc incorporation on the stoichiometry between melittin and monomer unit (black, left axis) and melittin and AAc (red, right axis).

Fig. 2.

Biocompatibility of NPs in vitro and in vivo. (A) Cytotoxicity of NPs towards HT-1080 cells, determined by the Alamar Blue® assay. NPs at the indicated concentrations (0.3 μg mL^-1 and 3 μg mL^-1) were incubated with cells for 24 h. The error bars indicate s.d. (B) Amount of NP aggregation that formed by incubation with mouse plasma (37 °C, 1 hour) followed with centrifugation (13,000 rpm, 5 min). (C) Change in body mass of mice injected with NP4 (blue) and NP9 (red)(n = 3, dose = 10 mg kg^-1) compared with control (isotonic glucose solution, n = 3). There is no statistically significant difference in the mass change between control and NPs over a period of 2 weeks. The error bars indicate s.d.

Fig. 3.

Detoxification of melittin in mice by systemic administration of NPs. (A) Survival rates of mice over a 24 h period after intravenous injection of 4.5 mg kg^-1 melittin (green). 30 mg kg^-1 of NP2 (blue), NP4 (red), and NP9 (black) was systemically administrated via a tail vein 20 s after melittin injection. P values are calculated by the Willcoxon test. (B) Level of inflammation of mice quantified by gross pathology (96 h after poisoning). Left two columns; without melittin with/without 30 mg kg^-1 of NP9, right two columns; with 3.8 mg kg^-1 of melittin followed with 30 mg kg^-1 of NP9. (C) Weight change of surviving mice 48 h after melittin injection, followed by (red) and without (black) administration of 30 mg kg^-1 of NP 9. Horizontal bars indicate the mean weight change percentage. (D). Survival rate of mice 24 h after intravenous injection of melittin, followed with (green) or without (blue) administration of 30 mg kg^-1 of NP9. 50% lethal doses (LD₅₀) of each condition are printed on the 50% surviving line.

Fig. 4.

Biodistribution of melittin and NPs. (A) Left; fluorescent images of Cy7-melittin after intravenous injection of Cy7-melittin (0.3 mg kg^-1). 10 mg kg^-1 of NP4 (Center) or NP9 (Right) were injected 20 s after the injection of melittin. (B) Fluorescent ex vivo images of Cy5-melittin (0.3 mg kg^-1, 10 min after injection) of mice followed with (Left) and without (Right) 10 mg kg^-1 NP9. Li, Sp, SI, K, H, and Lu indicate liver, spleen, small intestine, kidney, heart, and lung, respectively. (C) Biodistribution of ¹⁴C-labeled NP9 (30 mg kg^-1) in mice (n = 5) 30 min after administration. (D) Fluorescence histology images of a liver 70 min after injection of Cy5-melittin 0.3 mg kg^-1 and 10 mg kg^-1 of NP9. Green; fluoroscein-NP9, red; Cy5-melittin, yellow; merged. The scale bars; 25 μm.