Nazanin Ajdary
1 
, Maryam Davoudi
2, Tahereh Zadeh Mehrizi
3* , Mehdi Shafiee Ardestani
4,5* , Hasti Nequi Marnani
6, Hasan Ebrahimi Shahmabadi
7, Niloofar Ajdary
81 School of Pharmacy, International Campus, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Medical Laboratory Sciences, School of Allied Medical Sciences, Tehran University of Medical Sciences, Tehran, Iran
3 Health Management and Economics Research Center, Health Management Research Institute, Iran University of Medical Sciences, Tehran, Iran
4 Department of Radiopharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
5 Research Center for Nuclear Medicine, Shariati Hospital, North Kargar Ave. 1411713135, Tehran, Iran
6 Vaccine Research Center, Iran University of Medical Sciences, Tehran, Iran
7 Immunology of Infectious Diseases Research Center, Research Institute of Basic Medical Sciences, Rafsanjan University of Medical Sciences, Rafsanjan, Iran
8 Student Research Committee, School of Dentistry, Alborz University of Medical Sciences, Karaj, Iran
Abstract
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.