Electrostatic complex of neurotrophin 4 with dendrimer nanoparticles: controlled release of protein in vitro and in vivo.

NT4 has been regarded as a promising therapeutic protein for treatment of damaged retinal pigment epithelium cells. Here, we studied physicochemical parameters of an NT4-polyamidoamine (PAMAM) electrostatic complex, which can provide a sustained concentration of protein in intraocular space over an extended period after delivery. Adsorption/desorption of NT4 molecules to/from positively charged PAMAM dendrimers were precisely determined to control the concentration of bounded/unbounded protein molecules, diffusion coefficient, and size of a protein-laden dendrimer structure. We determined kinetics of NT4 desorption in PBS, vitreous, and damaged retina. Initially, adsorption of NT4 molecules on PAMAM dendrimers was studied in PBS using dynamic light scattering, electrophoresis, solution depletion, ELISA, and atomic force microscopy. This allowed us precisely to determine desorption of NT4 from nanoparticles under in situ conditions. The maximum coverage of irreversibly adsorbed NT4 determined by ELISA allowed us to devise a robust procedure for preparing stable and well-controlled coverage of NT4 on PAMAM nanoparticles. Thereafter, we studied diffusion of nanospheres containing NT4 molecules by injecting them into vitreous cavities of mice exposed to intravenous injections of sodium iodate and evaluated their intraocular desorption kinetics from drug carriers in vivo. Our measurements revealed NT4-dendrimer nanoparticles can be used for continuous neurotrophic factor delivery, enhancing its distribution into mouse vitreous, as well as damaged retina over 28 days of postinjury observation. Understanding of polyvalent neurotrophin interactions with dendrimer nanoparticles might be useful to obtain well-ordered protein layers, targeting future development of drug-delivery systems, especially for neuroprotection of damaged retinal neurons.