Anionic Cloaking Revolutionizes Intracellular Protein Delivery: Expanding the Therapeutic Potential of Protein-Based Therapies

The study introduces a pioneering method aimed at addressing a persistent challenge in biomedical research: the effective delivery of protein-based therapeutics into the interior of cells, known as the intracellular space. While proteins offer immense potential as therapeutic agents due to their specificity and diverse biological functions, their large size and hydrophilic nature pose significant obstacles to crossing the hydrophobic plasma membrane of cells. This limitation has confined most protein therapies to act on targets located outside of cells, severely constraining their therapeutic utility.

In response to this challenge, the researchers present a transformative approach centered around the concept of "anionic cloaking" coupled with lipid nanoparticle (LNP) formulations. Anionic cloaking involves chemically modifying the surface charge of proteins using lysine-reactive sulfonated compounds, effectively creating a reversible "cloak" that imparts an anionic character to the protein surface. This modification enables electrostatic complexation with cationic lipid nanoparticles, which have been widely studied for their ability to deliver nucleic acids into cells. By exploiting the electrostatic interactions between the anionic cloaked proteins and the cationic LNPs, the researchers aim to facilitate the efficient internalization of protein cargos into the cytosol of target cells.

The key findings and innovations of this study can be summarized as follows:

• Bioconjugation-Based Strategy: The researchers demonstrate the feasibility of modifying protein surfaces with anionic cloaks through bioconjugation chemistry. This strategy offers a versatile means of altering the charge characteristics of diverse protein cargos, irrespective of their molecular weight or surface properties
• Reversible Modification: Crucially, the anionic cloaking is designed to be reversible, allowing for the traceless delivery of protein cargos into the cytosol following endocytic uptake and escape from intracellular vesicles. The cleavage of the anionic cloak within the reducing environment of the cytosol ensures the restoration of native protein structure and function.
• Broad Applicability: The efficacy and generalizability of the anionic cloaking approach are demonstrated across a range of protein cargos, including therapeutic enzymes and antibodies. This broad applicability underscores the potential of the method to facilitate the intracellular delivery of various protein-based therapeutics for diverse biomedical applications.
• Enhanced Cellular Uptake: The study highlights the enhanced cellular uptake efficiency achieved through the combination of anionic cloaking with clinically validated lipid nanoparticle formulations. This synergistic effect suggests improved delivery efficacy compared to existing methods, promising greater therapeutic impact.
• Preservation of Protein Function: Functional assays confirm that the delivered protein cargos retain their biological activity post-delivery, indicating minimal interference from the modification process. This preservation of protein function is essential for ensuring the therapeutic efficacy of the delivered proteins in the intracellular environment.
• Simplicity and Versatility: Unlike existing strategies that often rely on genetic manipulation or specific protein domains, the anionic cloaking approach offers a straightforward and adaptable solution applicable to a wide range of proteins without prior sequence knowledge. This simplicity and versatility enhance the translational potential of the method for clinical applications.

A significant advancement in the field of intracellular protein delivery, offering a promising solution to a longstanding challenge in biomedical research. The innovative combination of anionic cloaking with lipid nanoparticle formulations holds great potential for enabling the development of more effective protein-based therapeutics for a variety of human diseases. Further research and refinement of this approach could lead to transformative advances in precision medicine and targeted therapy.