Cell-Penetrating and Lysosomal Escape Peptides: BR2, GALA

The efficient intracellular delivery of therapeutic molecules (e.g., nucleic acids, proteins, or drugs) is one of the major challenges of modern biomedicine. The cell membrane acts as a natural barrier, limiting the free entry of these macromolecules or charged substances. In addition, even when molecules enter the cell by endocytosis, they are often trapped in the endosomal/lysosomal compartment and degraded, unable to reach their targets of action in the cytoplasm or nucleus. Overcoming these barriers is critical for gene therapy, protein replacement therapy and targeted drug delivery. Cell-penetrating peptides (CPPs) and lysosomal escape peptides (e.g., BR2, GALA) show great potential for application as key tools to break through these delivery bottlenecks. In this thesis, we will delve into the mechanisms of CPPs and lysosomal escape mechanisms, focusing on analyzing the structure and function of BR2 and GALA peptides and their applications in delivery systems, especially gene therapy and nanomedicines.

Understanding Cell-Penetrating Peptides (CPPs) and Lysosomal Escape Mechanisms

In order to effectively utilize peptide tools such as BR2 and GALA, it is first necessary to gain a deeper understanding of the basic concepts of cell-penetrating peptides as well as the centrality of lysosomal escape in the overall intracellular delivery process. Together, these two aspects form the basis for overcoming barriers to intracellular delivery.

What are Cell-Penetrating Peptides?

Cell-penetrating peptides (CPPs), also known as protein transduction domains, are a class of short peptides typically consisting of 5-30 amino acids. They possess unique physicochemical properties (e.g., positive charge due to the abundance of basic amino acids, amphiphilicity) that enable them to efficiently cross the phospholipid bilayer barrier without significantly disrupting the integrity of the cell membrane, and transport their own or covalently/non-covalently bound biologically active "cargoes" (e.g., DNA, siRNA, proteins, nanoparticles, small-molecule drugs) to the cell interior. (e.g. DNA, siRNA, proteins, nanoparticles, small molecule drugs) to the cell interior. The core value of CPPs lies in their powerful transmembrane delivery ability, which provides a key entry point for therapeutic molecules that would otherwise be difficult to enter the cell.

Importance of Lysosomal Escape in Intracellular Drug Delivery

Successful crossing of the cell membrane is only the first step in the delivery process. The vast majority of exogenous substances that enter the cell through endocytosis are encapsulated in endocytic vesicles. These vesicles gradually mature and go through the stages of early endosomes, late endosomes, and eventually fuse with lysosomes. Lysosomes contain high concentrations of various hydrolytic enzymes (proteases, nucleases, lipases, etc.) and an acidic environment (pH ≈ 4.5-5.0), and their main function is to degrade foreign substances and the cell's own waste products. Therefore, for many therapeutic molecules (especially nucleic acids and functional proteins), if they cannot escape in time before the endosomes mature into lysosomes, they will be enzymatically inactivated, leading to therapeutic failure. Lysosomal escape strategies aim to design systems that can respond to changes in the environment of endosomes/lysosomes (e.g., pH reduction, activation of specific enzymes), disrupt the membrane of endocytosed vesicles, and release the cargoes into the cytoplasm to significantly enhance their bioavailability and therapeutic efficacy, which is an indispensable core component of intracellular delivery technologies.

Functional Overview of BR2 and GALA Peptides

BR2 and GALA, as two representative classes of peptides, provide efficient solutions for two key aspects of intracellular delivery, cell membrane penetration and endosomal/lysosomal escape, respectively. An in-depth understanding of their respective structural features and mechanisms of action is fundamental to the design of optimized delivery systems.

BR2 Peptide: Structure and Cell Membrane Penetration

BR2 peptide (Sequence: RAGLQFPVGRLLRRLLRRLLR) is derived from the C-terminal fragment of bee venom peptide (Melittin). It retains the strong cationicity and amphiphilicity of Melittin, but significantly reduces its cell membrane cleavage toxicity through key amino acid substitutions (e.g., replacing the N-terminal hemolytic site of Melittin with alanine), while retaining its highly efficient membrane-penetrating ability.The core function of BR2 lies in its highly efficient membrane-penetrating activity. Its arginine (Arg, R)-rich structure confers a strong positive charge and facilitates interaction with negatively charged cell membranes. Its α-helical amphiphilic structure facilitates the insertion into the phospholipid bilayer and efficiently mediates the entry of itself and its attached or complexed cargoes into the cell, mainly by facilitating endocytosis (e.g., megacytosis) as well as a certain degree of direct penetration of the membrane.BR2 has attracted much attention in the design of drug delivery carriers because of its high efficiency and low toxicity.

GALA Peptide: Facilitating Endosomal Escape for Enhanced Delivery

Unlike BR2, which focuses on initial membrane penetration, the core point of action of the GALA peptide (WEAALAEALAEALAEALAEHLAEALAEALEALEALAA) is to address the delivery bottleneck after the endocytosis pathway - endosomal/lysosomal escape. It is a well-designed pH-sensitive fusion peptide. Its core function is to respond to the acidic environment in endosomes/lysosomes, triggering conformational changes and membrane fusion/disruption effects. At physiological pH (~7.4), GALA exists mainly in a randomly coiled form with high hydrophilicity and weak interaction with membranes. However, when the ambient pH is lowered to the endosomal/lysosomal range (~5.0-6.0), its glutamate (Glu, E) and histidine (His, H) residues undergo protonation, which results in a dramatic increase in the peptide's hydrophobicity and induces it to fold into a stable amphiphilic α-helical structure. This hydrophobic helix is able to efficiently insert itself into the lipid bilayer of the endosome membrane, destabilizing the membrane through a mechanism similar to that of viral fusion proteins, forming pores or inducing membrane fusion, which ultimately leads to the release of the endosome contents, including therapeutic cargoes, into the cytoplasm.GALA is a key component in enhancing the efficiency of endocytosis-based pathway delivery systems.

Applications in Gene Therapy and Nanomedicine

Integrating the functional advantages of peptides such as BR2 and GALA into delivery systems has shown great potential, especially in the field of gene therapy and nanomedicines. These applications aim to address the challenge of full delivery of therapeutic molecules (e.g., nucleic acids, drugs) from extracellular to intracellular targets.

Using CPPs for Efficient Gene Delivery

Efficient and safe delivery of therapeutic genes (e.g., plasmid DNA, siRNA, mRNA, CRISPR-Cas9 components) to the cytoplasm or nucleus of the target cell is the key to successful gene therapy.CPPs, especially membrane-penetrating peptides with high efficiency and low toxicity like BR2, provide a powerful tool to address this challenge. Strategies include:

1. direct covalent coupling: chemically linking CPPs (e.g., BR2) directly to nucleic acid molecules to form complexes;
2. non-covalent complexation: utilizing the positive charge of CPPs to form nanocomplexes with the negative charge of nucleic acids through electrostatic interaction;
3. heterogeneous carrier construction: integrating CPPs onto the surface of liposomes, polymer nanoparticles, or inorganic nanocarriers or into inorganic nanocarriers, to form a multifunctional delivery systems ("CPP-decorated nanoparticles").

These strategies significantly improve the efficiency of nucleic acids across cell membrane barriers, overcoming many of the limitations of conventional viral vectors and cationic liposomes/polymers (e.g., immunogenicity, low transfection efficiency, and cytotoxicity). BR2 has been demonstrated to be able to efficiently deliver siRNAs, plasmid DNAs, and so on, and to exhibit enhanced gene silencing or expression effects in in vitro and in vivo models.

Designing Multifunctional Delivery Systems with Lysosomal Escape Peptides

A single delivery strategy is often difficult to overcome all the barriers in complex intracellular delivery pathways. Integrating efficient cell-penetrating elements (e.g., BR2) and endosomal/lysosomal escape elements (e.g., GALA) into the same delivery platform is a core strategy for building multifunctional and efficient delivery systems. This synergistic design idea is particularly important in the field of nanomedicine:

1. Co-modification of nanocarriers: modification of liposomes, polymer micelles or inorganic nanoparticles with both BR2 (responsible for efficient cellular uptake) and GALA (responsible for endosomal escape) on the surface of liposomes, polymer micelles or inorganic nanoparticles.
2. Fusion peptide construction: fusion of the membrane-penetrating sequences of BR2 and the pH-sensitive sequences of GALA is designed to form a single polypeptide that has dual functionality.
3. Modular assembly: BR2 and GALA are integrated as separate functional modules in peptide- or polymer-based nanocomplexes.

These smart systems integrating membrane-penetrating and lysosomal escape functions have been widely used to deliver anticancer drugs (to increase intracellular drug concentration), protein/peptide drugs (to prevent enzymatic inactivation), and gene therapy vectors as described above (to protect nucleic acids and facilitate their entry into the cytoplasm). For example, liposomes co-modified with BR2 and GALA on the surface exhibited significantly higher cellular uptake efficiency, endosomal escape ability, and ultimately anti-tumor effects than single modified or unmodified vectors when delivering siRNA or chemotherapeutic drugs.
In summary, cell-penetrating peptides (e.g., BR2) and lysosomal escape peptides (e.g., GALA) represent two key molecular tools to overcome the obstacles of intracellular delivery. BR2, with its highly efficient and low-toxicity membrane-penetrating ability, opens up a gateway for biomolecules to enter into the cell, while GALA utilizes its pH-responsive membrane-disrupting property to rescue the therapeutic molecules trapped in the endosomes/lysosomes. A deep understanding of their mechanisms of action and skillful functional integration of the two, especially synergizing them in the design of nanocarriers, is a key pathway to develop the next generation of efficient, targeted, and smart intracellular delivery systems. These technologies have extremely broad application prospects and significant translational value in cutting-edge biomedical fields such as gene therapy, tumor-targeted therapy, and protein replacement therapy. Continuously optimizing the structure and function of these peptides and exploring their combination with novel nanomaterials will be an important direction for future research.

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