Natural biodegradable rabies virus glycoprotein derived peptide-conjugated, diethyl sulfide modified zein protein nanoparticles as brain-targeted ROS-responsive nanocarrier for the treatment of ischemic stroke

Natural edible materials from plants have high potential as drug delivery carriers because of their excellent biocompatibility, biodegradability, complete non-toxicity, and zero side effects. They belong to the category that is generally recognized as safe (GRAS) by FDA. As a drug carrier, its clinic trial can be more direct and rapid than other carriers, which is highly appreciated for drug discovery and drug delivery development. A well-selected natural edible biopolymer for drug carrier applications should own optimal chemical, biological, and morphological properties to facilitate the multifunctional drug delivery.

Ischemic stroke is one top leading cause of death and major cause of disability worldwide. After decades of research, due to the side effects, the insufficient protection during drug delivery, and low accumulation of drug at brain ischemic tissue, currently no approved safe and efficient drug-based therapy is available. Targeting and stimuli-responsive drug delivery system, which delivers the drug to specific disease sites only and releases the drugs intracellularly when environmental stimuli are met, has become an important strategy for the treatment of ischemic stroke. A functional peptide derived from the radies virus glycoprotein (RVG), RVG29, has been reported to effectively enhance the brain delivery of various agents. Since a rapid increase in reactive oxygen species (ROS) occurs during and following ischemic stroke, a bioconjugation with RVG29 and ROS-responsive diethyl sulfide (Des) units can be designed to possess both targeting and stimuli-responsive release properties. Further, the conjugated system can be designed to self-assemble into nanoparticles, which have advantages in drug loading and cellular uptake because of their specific endocytosis pathway, size, and surface properties.

We choose zein, an amphiphilic protein from corn, as the base of the drug delivery nanosystem because of its advantages of biocompatibility, biodegradability, none water absorption, and low digestibility over other natural biopolymers. Further, it can self-assemble and be easily modified and conjugated with drugs and functional groups. We propose to make RZDA, the RVG29-conjugated Des modified zein nanoencapsulation of the model drug Ang-(1- 7), for the treatment of ischemic stroke. We have abundant experience on the synthesis and surface modification of zein based bioconjugates and nanoparticles, and the in vitro and in vivo evaluations of zein nanoencapsulations of various compounds. The RZDA will be formed in steps: (i) Synthesis of zein, sulfo-SMCC, and RVG29-Cys to form RVG29-SMCC-zein, (ii) Synthesis of RVG29-SMCC-zein, 4-nitrophenyl chloroformate, 2-2’-thiodiethanol, and 2-2’- thiobis(ethylamine) to form RVG29-SMCC-zein-Des, (iii) Self-assembly of RVG29-SMCC- zein-Des to encapsulate Ang-(1-7) to form RZDA. The prepared RZDA possessed several key features, including: (i) brain targeting via RVG29; (ii) ROS-responsive triggered release because of Des; (iii) prolonged release because of zein nanoparticles; and (iv) biodegradability and biocompatibility. We will examine the in vitro stability and ROS-responsive triggered release of RZDA, and study the in vitro neurotrophic and neuroprotective effects, and cellular uptake of RZDA using the cell model of transient focal ischemic stroke. We will also study the in vivo pharmacokinetics, biodistribution, and neurotrophic and neuroprotective efficacy of RZDA using the animal model of ischemic stroke.