Tunable Polyglycerol-Based Redox-Responsive Nanogels for Efficient Cytochrome C Delivery

Abstract

The sensitivity of therapeutic proteins is a challenge for their use in biomedical applications, as they are prone to degradation and opsonization, thus limiting their potential. This demands for the development of drug delivery systems shielding proteins and releasing them at the site of action. Here, we describe the synthesis of novel polyglycerol-based redox-responsive nanogels and report on their potential as nanocarrier systems for the delivery of cytochrome C (CC). This system is based on an encapsulation protocol of the therapeutic protein into the polymer network. NGs were formed via inverse nanoprecipitation using inverse electron-demand Diels–Alder cyclizations (iEDDA) between methyl tetrazines and norbornenes. Coprecipitation of CC led to high encapsulation efficiencies. Applying physiological reductive conditions of l-glutathione (GSH) led to degradation of the nanogel network, releasing 80% of the loaded CC within 48 h while maintaining protein functionality. Cytotoxicity measurements revealed high potency of CC-loaded NGs for various cancer cell lines with low IC50 values (up to 30 μg·mL−1), whereas free polymer was well tolerated up to a concentration of 1.50 mg·mL−1. Confocal laser scanning microscopy (CLSM) was used to monitor internalization of free and CC-loaded NGs and demonstrate the protein cargo’s release into the cytosol.

pH-Degradable Polyglycerol-Based Nanogels for Intracellular Protein Delivery

“…Full functionality of the released CC after coprecipitation and in vitro release was proven via ABTS assays (Figure S23). , …”

“…Subsequent amide coupling of metTet-COOH gave dPG-NH-metTet in an overall yield of 30% (Scheme ). , For dPG-AC-Norb, the reactive dimethyl acetal of the commercially available 5-norbornene-2-carboxaldehyde was formed followed by trans -acetalization with dPG-OH in the absence of water to give the desired product in two steps with an overall yield of 65%. The synthetic approach to the three benzacetals started from the commercially available p -hydroxybenzaldehydes.…”

“…Full functionality of the released CC after coprecipitation and in vitro release was proven via ABTS assays (Figure S23). 35,49 Cellular Uptake and Cell Viability. Cell internalization of the particles is mandatory to ensure NG degradation during endocytosis.…”

Cytochrome c in cancer therapy and prognosis

“…One of these groups consists in enclosing cyt c inside pockets formed by large molecular structures that can target cancer cells and release cyt c into the cytoplasm, inducing their apoptosis ( Figure 3 B). Those large structures where cyt c is encapsulated include ferritin (an iron-storage protein) [ 51 ], polypeptide micelles [ 52 ], and nanogels [ 53 , 54 ]. To ensure their specific targeting to cancer cells, some of these pocket structures were modified on their surface with ligands that bind specific molecules on the surface of cancer cells, triggering their endocytosis ( Table 3 ).…”

“…The polypeptide micelle delivery system is sensitive to oxygen scarcity within tumors, releasing its content faster under hypoxic conditions [ 52 ]. Additional nanoparticle-based systems are nanogels sensitive to the reductive chemical environment in the cytoplasm of cancer cells [ 53 , 54 ]. Reductive environment causes the reduction in disulfide bonds in the nanogels and their consequent disruption, which releases cyt c ( Figure 3 C).…”