pH-Reversible Cationic RNase A Conjugates for Enhanced Cellular Delivery and Tumor Cell Killing

Abstract

Intracellularly-acting therapeutic proteins are considered promising alternatives for the treatment of various diseases. Major limitations of their application are low efficiency of intracellular delivery and possible reduction of protein activity during derivatization. Herein, we report pH-sensitive covalent modification of proteins with a histidine-rich cationic oligomer (689) for efficient intracellular transduction and traceless release of functional proteins. Enhanced Green fluorescent protein (EGFP), as model for the visualization of protein transduction, and RNase A, as therapeutic protein with antitumoral effect, were modified with the pH-sensitive bifunctional AzMMMan linker and varying amounts of cationic oligomer. The modification degree showed impact on the internalization and cellular distribution of EGFP as well as the biological effect of RNase A conjugates, which mediated considerable toxicity against cancer cells at optimal ratio. The presented conjugates demonstrate their qualification to achieve efficient intracellular delivery and controlled release without protein inactivation and potential prospective applications in protein-based therapies.

Biological Activities of Secretory RNases: Focus on Their Oligomerization to Design Antitumor Drugs

“…However, RNases can have distinct but not always successful outcomes in several cellular organelles, such as endosomes, lysosomes, ER and the trans-Golgi network (136). Thus, many studies have been performed to unveil the intracellular routing of extracellular RNases once internalized by endocytosis, and the relative results indicated that diverse RNases, such as ONC or BS-RNase as well as RNase A, RNase 1, or ANG follow different cellular pathways within one another, as described or proposed in several reports (32, 118, 120, 128, 130–132, 135).…”

“…More recently, an interesting strategy to intracellularly internalize RNases was proposed: three cationic amine-reactive linkers were attached to RNase A, and the stability of these conjugates was pH-dependent. Therefore, the endocytic vesicles' acidic environment led to the release of high cytosolic amounts of RNase A to make its concentration high enough to overcome the RI binding capacity and become cytotoxic (135).…”

Engineered Polymeric Materials for Biological Applications: Overcoming Challenges of the Bio–Nano Interface

“…However, demonstration that activity was retained was not explored. Delivery of functional enzymes has been demonstrated by Schepartz and Wagner [201,202]. Wagner, Lächelt and coworkers developed a three-arm cationic star succinoyl tetraethylenepentamine conjugated with a tetraethylene glycol linker by a pH-responsive aminated methyl maleic anhydride bond [201].…”

“…Delivery of functional enzymes has been demonstrated by Schepartz and Wagner [201,202]. Wagner, Lächelt and coworkers developed a three-arm cationic star succinoyl tetraethylenepentamine conjugated with a tetraethylene glycol linker by a pH-responsive aminated methyl maleic anhydride bond [201]. The protein star conjugate showed some diffuse fluorescence of enhanced green fluorescent protein (eGFP) at a 1 μM of protein, and was demonstrated to induce apoptosis through the similar delivery of RNase A star conjugate.…”

One‐Step Microfluidic Synthesis of Nanocomplex with Tunable Rigidity and Acid‐Switchable Surface Charge for Overcoming Drug Resistance

“…We have previously demonstrated that drug‐loaded rigid nanoparticles (NPs) show advantages with respect to improved cellular uptake and better cancer therapeutic efficiency than their soft counterparts . To improve the endo/lysosomal escape of nanovectors after internalization, the presence of cationic surface charge could facilitate disruption of the endo/lysosomal membrane . However, it is critical to shield the positive surface charge during blood circulation to avoid non‐specific interaction with serum proteins and negatively charged molecules presented on the cell membrane .…”