Development and Characterization of Colloidally Stable Dendrimer–Botox Conjugates for Targeted Muscle Delivery and Radiolabeled Imaging

Background: Botox’s therapeutic applications are limited by its instability and uncontrolled diffusion into extramuscular tissues. This study aims to develop a polyethylene glycol-based formulation by conjugating Botox to a second-generation anionic linear-globular dendrimer (G2-ALGDs) and evaluate its colloidal stability and muscle localization using technetium-99m radioimaging. Methods: G2-ALGDs were synthesized via N,N′-dicyclohexylcarbodiimide catalysis and conjugated with Botox (D-BTX) using EDC/DCC coupling. Characterization was done by DLS, FTIR, AFM, TEM, and FE-SEM. Stability at 4 °C was monitored for six months using DLS and TEM. Cytocompatibility was assessed by MTT assay in HFF2 cells. D-BTX was radiolabeled with technetium-99m to study purity, stability, and in vivo biodistribution using RT-LC and SPECT imaging. Results: Botox conjugation increased the particle size from 111.6±16.9 nm to 160.0±3.1 nm and shifted the zeta potential from –25.2±1.63 mV to+17.16±1.24 mV. FTIR confirmed amide bond formation at 1649 cm⁻¹. D-BTX was much less cytotoxic than free Botox at 0.001 ng/mL (p=0.0013), but no difference was observed at higher concentrations. The formulation exhibited physical stability for six months at 4°C. The radiolabeled formulation was up to 90.85% pure and remained stable for 24 h. SPECT imaging revealed improved localization of D-BTX within the muscle. After 12 h, D-BTX exhibited maximum signal intensity with minimal dispersion, indicative of controlled diffusion at the injection site. Conclusion: This PEGylated dendrimer-Botox nanoconjugate provides a stable, muscle-targeting, and imageable platform, which surmounts some of the main drawbacks in current clinical applications.