Release date:2022/5/27 10:51:55

Achieving drug delivery across the blood-brain barrier is a major challenge when developing drugs to treat central nervous system (CNS) diseases, especially when using biologics such as monoclonal antibodies and enzyme replacement therapies.

The Blood-Brain Barrier (BBB) is a protective barrier between the blood vessels (capillaries) and the cells and other components that make up brain tissue, providing a defense mechanism against foreign pathogens and toxins in the bloodstream, etc. The development of therapies for central nervous system (CNS) diseases is complicated by the presence of the BBB. While the BBB maintains CNS homeostasis by tightly controlling specific nutrients and limiting the passage of harmful xenobiotic molecules, the BBB also prevents drugs and macromolecular therapies such as biologics from entering the brain, thereby greatly reducing their efficacy.

Possible Strategies To Cross The Blood-Brain Barrier

Over the past two decades, biologics such as monoclonal antibodies have become increasingly popular in the field of drug development. In fact, among the top 10 selling drugs for 2021 (excluded COVID-19 related drugs), five are biologics (see Figure 1), yet none of these drugs are for CNS diseases. For large molecule biologics such as monoclonal antibodies, it can be difficult to across the BBB to enter the brain for treatment. Some studies have demonstrated that after non-intestinal administration of therapeutic antibodies, their levels in the brain are only 0.01-0.1% of the levels in plasma.

Figure 1: Top 10 Selling Drugs of 2021, sources:

Thus, the BBB poses a barrier to the treatment of CNS diseases and is currently an important area for drug delivery technology development. Here, we focus on listing and summarizing potential drug delivery approaches ( except invasive technologies) to across the BBB.

1. Receptor-mediated transcytosis (RMT)

On the BBB, several endogenous substance transport processes exist for normal substance transport, such as adsorptive-mediated transcytosis, carrier-mediated transcytosis, and receptor-mediated transcytosis (RMT). Among them, the use of RMT pathway for drug development has been extensively studied.

Biological Transport Mechanisms Across the Blood-Brain Barrier
Figure 2. Biological Transport Mechanisms Across the Blood-Brain Barrier, image source: References 1

Therefore, finding the right receptor is the first step, and an excellent receptor needs to meet the conditions of high expression on vascular endothelial cells in the brain and low expression on the peripheral vascular system. However, until now, no proteins or pathways have been identified that meet these criteria, and most studies in the past decades have focused on targets that are commonly expressed on BBB cells, such as transferrin receptor (TfR), insulin receptor, low-density lipoprotein receptor (LDLR) family members, melanotransferrin (MTf) and CD98 heavy chain (CD98hc, also known as SLC3A2).

Moreover, based on these receptors, scientists have developed a variety of antibody therapies that can bind to such targets (modifying the antibody to have a region that binds to the above receptors) and protein replacement therapies (where the above receptor-targeted region is combined with a replacement protein to build a fusion protein), and the modified therapeutic drugs can bind to specific receptors on the surface of the BBB. After binding, the complex molecules cross the vascular endothelium and enter the brain to exert therapeutic effects through receptor-mediated transcytosis. This is considered to be one of the most established strategies for brain-targeted drug delivery and has been validated in several clinical trials (Table 1).

Table 1. Clinical Trials of Brain Drug Delivery Using the RMT Pathway

2. Neurotropic virus

Neurotropic viruses are a class of viruses that have an affinity for neural tissue and enter the brain through three main mechanisms, including direct transcytosis across the BBB, CNS entry via immune cell migration across the BBB, and retrograde transport of rabies virus from peripheral nerves to the CNS. (Figure 3).

Ways in which viruses enter the central nervous system
Figure 3. Ways in which viruses enter the central nervous system, source: reference 1

The use of neurotropic virus-based vectors is currently limited to gene therapy and often relies on invasive delivery, such as intrathecal injections.

A variety of viral vectors as well as virus-like vectors, such as adeno-associated virus (AAV), lentivirus, herpes simplex virus (HSV) and virus-like particles (VLP), have emerged as major delivery vehicles for gene therapy. These vectors have also demonstrated the ability to cross the BBB for therapeutic purposes in numerous CNS disease models in preclinical studies and are well tolerated. Therefore, they could be potential carriers to cross the BBB for gene therapy in the brain. Several clinical trials are also currently underway (Table 2).

Clinical Trials of Brain Drug Delivery Using Neurotropic Viruses
Table 2. Clinical Trials of Brain Drug Delivery Using Neurotropic Viruses

The use of neurotropic viruses for brain drug delivery is still in its infancy and there are many considerations to be explored. First, these viruses may have the ability to target not only the brain, but also other sites such as the liver, heart, and skeletal muscle, so the rate of infection in the brain may still be low (one study reported a 2% infection rate for AAV9 in non-human primate species), and targeting ability needs to be improved. In addition, the lack of targeting ability then raises another issue, namely the possibility of requiring high doses of viral vectors to cross the patient's BBB, which not only poses a higher risk of toxicity, but also poses a challenge for production and manufacturing. Secondly, the virus is immunogenic, which also poses an autoimmune risk. Finally, the payload carried by the virus has a size limitation, for example, the loading capacity of AAV is limited to 4.7 kb. Therefore, more research and further modifications are needed to enhance the delivery efficiency and targeting ability to improve the therapeutic effect and avoid side effects.

3. Nanoparticle system

Nanoparticle systems contain a variety of carriers, such as liposomes, polymeric nanoparticles and solid lipid nanoparticles (SLNP), which can often be classified according to size (typically 10-300 nm in diameter), chemical composition and physical shape. Nanoparticles with a diameter of 50-200 nm have been studied in many studies for stroke, Alzheimer's disease or Parkinson's disease. Nanoparticles have been investigated as drug delivery platforms for decades, and the first nano-formulation for cancer therapy was approved more than 20 years ago. However, there are currently only a few approved nanoparticle-based therapies and technologies, such as the use of LNP for delivery of mRNA COVID-19 vaccines, but no CNS therapy-related applications are currently available.

In early therapeutic studies of brain tumors, even non-targeted nanoparticles have shown good potential for clinical applications. However, the BBB of brain tumor patients is usually damaged to a certain extent which increases the brain exposure, and the ability of non-targeted nanoparticles to cross the intact BBB has been demonstrated to be very limited. Therefore, the use of nanoparticles for the treatment of other CNS diseases needs to improve their brain-targeting ability.

Currently, some progress has been made in the development and design of strategies for nanoparticles targeting the brain (Figure 5). First, the effective brain targeting of glutathione (GSH)-polyethylene glycol (PEG) liposomes (in which GSH is used to target GSH transporter proteins in the BBB) has been demonstrated in several preclinical studies. In a phase I/II trial (NCT01386580), intravenous administration of GSH-PEG liposomes loaded with doxorubicin as a therapeutic agent in 28 brain cancer patients was found to be safe and well tolerated with initial indications of antitumor activity. Intravenous administration of ribavirin, an antiviral drug encapsulated in GSH-PEG liposomes, resulted in a 4-fold increase in free ribavirin levels in brain microdialysis fluid. In addition, fluorescently labeled poly(lactic-co-glycolic acid) (PLGA) nanoparticles surface loaded with PEG and B6 (a peptide as a transferrin substitute), polyester-PEG copolymer nanoparticles surface loaded with Seq12 (a peptide that promotes BBB penetration) and SLNP surface loaded with polysorbate 80 were also validated in Alzheimer's disease, brain tumors and animal models, respectively.

In the development of nanoparticles for drug development targeting the CNS, there are still some issues that need to be further explored and validated. First, the intrinsic properties of nanoparticles, such as size, surface charge, stability, tissue distribution and pharmacokinetics, can affect the uptake of nanoparticles into phagocytes, thus preventing entry into the CNS. And it has been shown that only a small fraction of nanoparticles can reach the brain intact after injection, and most of them are removed from circulation and destroyed during circulation by the mononuclear phagocyte system. In addition, from a production and manufacturing perspective, the long-term stability of nanoparticles is also critical to ensure that therapeutic nanoparticle formulations have a sufficiently long shelf life. In conclusion, there is still a long way to go for the application of nanoparticles in CNS diseases.

Types of nanoparticles, surface functionality and modification methods
Figure 4. Types of nanoparticles, surface functionality and modification methods

4. Exosomes

Exosomes are a subtype of extracellular vesicles (EV) formed by an endosomal route and are typically 30–150 nm in diameter. Exosomes are widespread in vivo and have important functions involved in the transport of substances in vivo (Figure 5), a property that also confers their potential in disease therapy and is of particular interest in the field of drug delivery vehicles.

Functions of exosomes
Figure 5. Functions of exosomes

The ability of unmodified and untargeted modified exosomes to target the brain is also low. It has been shown that only 0.5% of non-targeted exosomes can reach the brain for therapy in animal models, but have demonstrated therapeutic effects on disease. Surface-modified exosomes can dramatically enhance brain uptake, with common modifications being the fusion of RVG peptides (the α7 subunit of the nicotinic acetylcholine receptor that targets the brain) to Lamp2b proteins expressed on exosomes to achieve brain targeting.

Exosomes also have an important property - homing property. Exosomes derived from neuronal cells tend to return to the brain after a cycle in vivo, which provides an additional idea for brain targeting of exosomes.

Aruna Bio, a company dedicated to the development of a neuroexosome therapeutic platform, utilizes the proprietary non-transformed neural stem cell-derived exosome AB126 as a therapeutic drug or delivery vehicle to cross the blood-brain barrier for the treatment of central nervous system disorders (Figure 6).

NEUREX TM Pipeline diagram
Figure 6. NEUREX TM Pipeline diagram

In addition to the above properties, it has the advantages of low immunogenicity, low toxicity, a wide range of deliverable substance types, high stability and ease of modification, and is undergoing proof-of-concept and clinical development in several research teams and companies (Figure 8).

Of course, there are some pressing issues in the field of exosome drug delivery that need to be addressed. First, the exosome industry is still in its infancy, the technology is not yet mature, and there is a lack of unanimously recognized industry standards and quality control systems. Secondly, there are still challenges in the production and manufacturing of exosomes on a large scale, mainly in terms of difficulty in ensuring uniformity and high purity, and difficulty in judging biological activity and specific potency. Finally, since exosomes are natural products secreted by cells, they naturally contain some intrinsically secreted substances within them, and therefore the safety of their use in clinical practice has yet to be confirmed.

Advantages of exosomes as drug delivery vehicles
Figure 6. Advantages of exosomes as drug delivery vehicles 


There is a great unmet clinical need in the treatment of central nervous system disorders, and one of the major reasons is the difficulty in crossing the blood-brain barrier, especially in the field of biopharmaceuticals, such as large-molecule antibodies, RNA therapy, and gene therapy. The emergence of drug modifications based on the basic principle of receptor-mediated transcytosis, neurotropic virus-mediated transport, nanoparticles and exosomes all provide solutions for crossing the blood-brain barrier and are currently being evaluated in several clinical trials, with the expectation that clinical products supported by these technologies will eventually be available and go to market for the benefit of patients.

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