Reviews of siRNA Nanocarriers for Blood-Brain Barrier(BBB) Penetration

What is the Blood-Brain Barrier(BBB)?

The blood-brain barrier (BBB) is a highly selective semi-permeable boundary formed by endothelial cells, which regulates the transfer of solutes and chemicals between the circulatory system and the central nervous system (CNS), thereby protecting the brain from the influence of harmful or unnecessary substances in the blood. The BBB is a natural protective membrane that prevents the CNS from being harmed by toxins and pathogens in the bloodstream. It is composed of endothelial cells lining the capillary walls, astrocytic end-feet enveloping the capillaries, and pericytes embedded in the capillary basement membrane. This system allows for the passive diffusion of some small molecules, as well as the selective and active transport of various nutrients, ions, organic anions, and macromolecules (such as glucose and amino acids) essential for neuronal function. However, the existence of the blood-brain barrier complicates the treatment of CNS diseases because most chemical and biological drugs are prevented from entering the brain.

A schematic view of the Bood Brain Barrier and its components including bood vessel, endothelial cells and astrocytic feet.

Barriers for siRNA Penetrating the Blood-Brain Barrier(BBB)

Difficult to Penetrate the Blood-Brain Barrier

As the global population continues to age and external factors such as work pressure increase, the incidence of central nervous system (CNS) diseases continues to rise. Among them, the high mortality rate of stroke has surpassed that of coronary heart disease, becoming the second leading cause of death worldwide. In addition, Alzheimer's disease also imposes a significant economic and health burden. Due to the presence of the blood-brain barrier (BBB), many diseases lack effective treatment methods and drugs, making the treatment of CNS diseases a major challenge in today's medical and pharmaceutical fields.

Issues for siRNA Delivery

Small interfering RNA (siRNA) is a double-stranded RNA molecule consisting of 21 to 25 nucleotides in length, which can complementarily bind to the target gene sequence and induce the degradation of the corresponding mRNA, thereby preventing mRNA translation into protein and achieving the purpose of treating diseases. Therefore, theoretically, siRNA, if designed with a rational sequence, can silence any disease-related gene expression, including those that contribute to CNS diseases. siRNA possesses high specificity and efficiency toward disease-causing targets, as well as rapid and simple candidate drug development, giving siRNA a unique advantage in the treatment of CNS diseases. However, naked siRNA also faces some challenges, such as being small in size, which makes it prone to kidney filtration, and having poor stability, as it can be hydrolyzed by nucleases in the bloodstream and degraded by immune cells. Although siRNA itself exhibits strong target specificity when silencing genes, it lacks targeting specificity during the delivery process, which may result in off-target effects and unpredictable adverse reactions. Moreover, siRNA carries a negative charge, making it difficult to penetrate the blood-brain barrier and target cells, thus unable to fully exert its gene silencing effect. Even if siRNA enters the cells, it cannot escape from endosomes and lysosomes. Therefore, siRNA requires an appropriate carrier to overcome its inherent limitations.

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Nanocarriers in siRNA Delivery for Penetrating the BBB

Lipid-based Nanoparticles

• Liposomes used for BBB penetration research mainly undergo functionalization of the liposome surface with biologically active ligands such as peptides, antibodies, or small molecules, enabling liposomes to possess targeted BBB penetration capabilities.
• Solid Lipid Nanoparticles (SLNs) are a new generation of submicron drug delivery systems. These nanoparticles are primarily composed of solid-state natural or synthetic lipids such as phospholipids, triglycerides, etc., providing good biocompatibility and low cytotoxicity. This carrier can encapsulate or entrap drugs within lipid cores to form solid lipid particles for drug delivery systems, which can significantly improve BBB penetration efficiency after surface modification.
• Nanoemulsions are heterogeneous dispersions stabilized by surfactants consisting of "water-in-oil" (O/W) or "oil-in-water" (W/O) compositions. The surface of these nanoparticles can also be modified with biologically active ligands to facilitate BBB penetration through receptor-mediated endocytosis.
• Lipid nanoparticles have long been used as drug carriers for liver targeting. In brain delivery, similar to the liver, the penetration into the brain is achieved by the adsorption of endogenous apolipoprotein E (ApoE) by cationic lipids in LNP.

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Chitosan Nanoparticles

Chitosan is a natural biopolymer with excellent biocompatibility, high cellular uptake, and large drug loading capacity. Amino, hydroxyl, and N-acetyl amino groups on the molecular chain can form intermolecular hydrogen bonds with siRNA chains, making it widely used in the delivery of nucleic acid drugs such as siRNA. Positively charged chitosan modified with positive charge will electrostatically bind to anionic sialic acid residues on the BBB, thereby promoting nanoparticle penetration. Additionally, PEGylation of chitosan followed by coating with polysorbate 80 can also promote drug penetration through the BBB.

Polyethyleneimine Nanoparticles

PEI is a cationic polymer and one of the most studied gene carrier materials. It has good solubility and pH buffering capacity in the endosome/lysosome pathway, and can electrostatically bind to siRNA with negative charges to form nanocomposites. Furthermore, modification with RVG targeting peptide significantly enhances the ability of these nanoparticles to target and penetrate the BBB.

Dendrimer Nanoparticles

Dendrimers consist of three parts: an internal core, internal branching functional groups, and external surface groups, highly branched and radially symmetrical with precise three-dimensional spatial structures. The internal cavity and internal branching functional groups can be used to carry gene drug molecules as carriers for targeted transport, while the externally surface groups can be targeted modified to improve stability and enhance active targeting efficiency. The cationic polyamidoamine dendrimer (PAMAM) carrying active amino groups and its branching structure provide convenience for encapsulating and compressing siRNA. Researchers have connected RGD peptide, tLyp-1 peptide, and T7 peptide to PAMAM for BBB penetration and brain tumor treatment. Both in vitro and in vivo experiments have demonstrated the good targeting penetration ability of this carrier through the BBB and glioma.

Nanogels

Nanogels with a three-dimensional network structure have many advantages in drug delivery, such as high drug loading, avoiding enzymatic degradation of drugs, and reducing the risk of phagocytosis by the reticuloendothelial system. Specific modifications can not only increase the circulation time of the nano-carrier in the blood, enhance the BBB penetration, and promote efficient cellular uptake but also facilitate the release of siRNA in cells, ensuring the effectiveness of siRNA therapy.

Nano-Micelles

Polymer micelles are thermodynamically stable colloidal solutions self-assembled by synthetic amphiphilic block copolymers in water. Micelles, as drug carriers, have many advantages such as increasing the solubility of poorly soluble drugs, enhanced permeability and retention (EPR) effect, and improving the bioavailability of drugs. Targeting can be enhanced by modifying the hydrophilic shell with ligands such as folic acid, peptides, antibodies, etc., and the PEG part of the hydrophilic segment has a long circulation effect.

Exosomes

The method of loading siRNA onto exosomes is not like traditional cationic carriers, but rather achieved through methods such as incubation, electroporation, or transfection reagents. Exosomes as siRNA drug carriers also have many research reports on their application in the brain. For example, RVG modified exosomes secreted by dendritic cells can penetrate the BBB.

siRNA Conjugates

The appearance of early siRNA conjugates was to increase the stability of siRNA, that is, to covalently bind different compound molecules to the sense or antisense chains of siRNA through chemical synthesis methods without affecting the efficacy of siRNA. Nowadays, peptide-siRNA conjugates have begun to be used for brain delivery, mainly focusing on cell-penetrating peptides (CPPs)-siRNA and ligand-siRNA.

How to Design BBB-Penetrating siRNA Nanocarriers

• Designing high-quality and efficient nanocarrier systems requires meeting the following requirements as much as possible:
• Safety of carrier materials: The human brain is a special organ with high requirements for the safety of carriers. Therefore, good biodegradability and biocompatibility are necessary conditions.
• No aggregation and dissociation in the blood, maintaining a relatively long circulation time.
• Excellent drug loading and release capabilities.
• Non-immunogenicity.
• Presence of coupling groups for easy attachment of other functional groups such as ligands.
• Effective and safe penetration of the blood-brain barrier (BBB) without complete disruption.
• Good targeting ability and endosomal/lysosomal escape ability.
• In addition, the surface properties of nanocarriers, such as particle size, surface charge, density of modified ligands, and affinity of ligands, will also affect the distribution of nanocarriers in the body and the efficiency of BBB penetration.