Fabrication, characterization and evaluation of a new designed botulinum toxin-cell penetrating peptide nanoparticulate complex

Abstract

Background: To have a better and longer effect, botulinum neurotoxin (BoNT) is injected several times in a treatment course, which could increase side effects and cost. Some of the most cutting-edge strategies being investigated for proteins to their physiologic targets involve the reformulation of BoNT based on peptide-based delivery systems. For this purpose, cell-penetrating peptides (CPPs) are of particular interest because of their capacity to cross the biological membranes.

Objectives: A short and simple CPP sequence was used as a carrier to create nanocomplex particles from BoNT/A, with the purpose of increasing toxin entrapment by target cells, reducing diffusion, and increasing the duration of the effect.

Method: CPP-BoNT/A nanocomplexes were formed by polyelectrolyte complex (PEC) method, considering the anionic structure of botulinum toxin and the cationic CPP sequence. The cellular toxicity, and absorption profile of the complex nanoparticles were evaluated, and the digit abduction score (DAS) was used to assess the local muscle weakening efficacy of BoNT/A and CPP-BoNT/A.

Results: The provided optimized polyelectrolyte complex nanoparticles had a 244 ± 20 nm particle size and 0.28 ± 0.04 PdI. In cellular toxicity, CPP-BoNT/A nanocomplexes as extended-release formulations of BoNT/A showed that nanocomplexes had a more toxic effect than BoNT/A. Furthermore, the comparison of weakening effectiveness on muscle was done among nanoparticles and free toxin on mice based on the digit abduction score (DAS) method, and nanocomplexes had a slower onset effect and a longer duration of action than toxin.

Conclusion: Using PEC method allowed us to form nanocomplex from proteins, and peptides without a covalent bond and harsh conditions. The muscle-weakening effect of toxin in CPP-BoNT/A nanocomplexes showed acceptable efficacy and extended-release pattern.

Keywords: Botulinum toxin; Cell-penetrating peptides; Dermatology; Digit abduction score; Drug delivery.

Fig. 1

Pep-1-BoNT/A nanocomplexes characterization: a) SEM, b) Particle size distribution

Fig. 2

DP-BoNT/A nanocomplexes characterization: a) SEM, b) Particle size distribution

Fig. 3

SEM of lyophilized a) Pep-1-BoNT/A nanocomplexes and b) DP-BoNT/A nanocomplexes

Fig. 4

In vitro cell studies of DP peptide sequence after 48 h and 72 h (Green bars for 48 h and blue bars for 72 h)

Fig. 5

In vitro cell studies: a) MTT assay of BoNT/A and DP-BoNT/A nanocomplex particles on NIH-3 T3 cells after 48 h incubation (blue bar for BoNT/A, and green bar for DP-BoNT/A nanocomplex particles), b) MTT assay of BoNT/A and DP-BoNT/A nanocomplex particles on NIH-3 T3 cells after 72 h incubation (blue bar for BoNT/A and green bar for DP-BoNT/A nanocomplex particles)

Fig. 6

uptake images of fibroblast NIH-3 T3 cells a) control, b) BoNT/A, c) DP-BoNT/A, d) Dansyl tagged DP, e) Dansyl tagged DP-BoNT/A.

Fig. 7

Uptake images of fibroblast NIH-3 T3 cells incubated with DP-BoNT/A nanocomplexes after a) 1 h b) 2 h c) 3 h

Fig. 8

Mice with BoNT/A (12.5 U/kg) and DP-BoNT/A nanocomplexes on treated right foot and untreated left foot after; a) 12 h, b) 24 h, c) 48 h, d) 3 days, e) 4 days, f) 5 days and g) 6 days