Characteristics, Targets, and Peptides for Crossing the Blood-Brain Barrier

This article focuses on the latest developments in brain penetrating peptides and peptide-drug conjugates (PDCs) as delivery vehicles for transporting small molecule drugs across the BBB into the brain parenchyma. The paper summarizes peptides expressed on brain endothelial cells and their specific receptors, brain transport mechanisms, and delivery efficiency to the brain.

BBB as a Major Obstacle in CNS Drug Delivery

The brain is a highly complex and important organ that contains various types of brain cells, each with different functions. Neurons are electrically excitable cells used for transmitting information, endothelial cells form brain blood vessels, astrocytes support neuronal functions, microglia are the brain's resident macrophages responsible for immune surveillance, and other brain macrophages, like perivascular macrophages, are located on the outer surface of blood vessels, carrying out activities at the interface between the brain parenchyma and circulation. The human brain contains a blood vessel network over 400 miles long, including cerebral arteries, arterioles, and capillaries, with nearly every neuron being supplied by its own brain capillary.

There are three main obstacles to drug delivery in the brain. First, the BBB is a physiological barrier between the blood vessels and the brain parenchyma. Second, the blood-cerebrospinal fluid barrier (BCSFB) is a barrier between blood circulation and cerebrospinal fluid circulation. The BCSFB is formed by the following structures: the choroid plexus epithelial cells tightly linked to the blood-CSF. Third, the avascular arachnoid barrier lies beneath the dura mater and completely seals off the CNS, playing an important role in CNS drug transport to the brain. Among these barriers, the BBB is the most important, as it strictly controls the direct microenvironment of brain cells.

Properties of the Blood-Brain Barrier

The blood-brain barrier tightly regulates brain access, allowing only essential ions, nutrients, and hormones while blocking harmful substances. It consists of endothelial cells, astrocytes, pericytes, basement membranes, microglia, and neurons. Unlike peripheral endothelial cells, brain endothelial cells lack fenestrations and have tight junctions that form a continuous barrier, limiting the paracellular flow of hydrophilic molecules. These tight junctions contribute to a high trans-endothelial electrical resistance (TEER >1000 Ωcm²) and reduce endothelial cell phagocytosis. The BBB is dynamic, with neurovascular unit (NVU) signaling regulating permeability. Specialized endothelial transport proteins control metabolite exchange, while microglia modulate immune responses. Peripheral immune cells, such as T lymphocytes, can enter the brain during CNS diseases. Age-related changes shift plasma protein transport from receptor-mediated transcytosis to non-specific uptake. The BBB functions as a physical, transport, metabolic, and immune barrier, ensuring CNS homeostasis.

Transport Mechanisms Across the Blood-Brain Barrier

Due to the "compactness" of the BBB, bypass cellular transport of molecules is minimized; Drugs and essential molecules enter the brain primarily through passive diffusion, carrier-mediated transport, receptor-mediated transcell swallowing, adsorption-mediated transcell swallowing, or through cell-mediated transcell swallowing. Transcell swallowing is a process in which molecules are transferred from one side of the cell to the inside of the cell and then to the other side of the cell through membrane cysts. Brain endothelial cells exhibit relatively low vesicle trafficking rates relative to peripheral endothelial cells.

Passive Diffusion

Passive diffusion is when a compound moves in the direction of a concentration gradient without energy and without saturation. Simple diffusion is the primary mechanism of entry for most lipophilic small-molecule CNS drugs. The maximum cell-capillary distance is 20 microns, and small molecules can penetrate in half a second. The diffusion of substances into the brain can be divided into paracellular diffusion and intracellular diffusion. Paracellular diffusion is limited by tight junctions between brain endothelial cells. Intracellular diffusion mainly depends on the permeability of the molecule. Small lipophilic substances such as alcohol and steroid hormones can undergo intracellular penetration through unsaturated diffusion. The highly fat-soluble drug dexamethasone can easily cross the blood-brain barrier and have a sedative effect. In general, small molecular weight (<500 6="" weak="" hydrogen="" bonds="" less="" than="" lipophilicity="" logp="">2), and the absence of free spin bonds and a polar surface area (PSA) of < 60 to 70 square angstroms are all favorable for crossing the blood-brain barrier by diffusion. When measuring drug permeability through passive diffusion, all of these parameters are considered in combination and not individually. For native peptides, passive diffusion is very limited unless the peptide has an amphiphilic structure or is made lipophilic by a synthetic method.

Vector-Mediated Endocytosis

Essential nutrients such as glucose, amino acids, and nucleotides cross the blood-brain barrier through highly selective and stereospecific transporters through carrier-mediated transcellular transport (CMT) mechanisms. The transporter recognizes the substrate on the luminal side, and the substrate-transporter binding system then triggers a conformational change in the carrier protein from the outward to the inward state, resulting in the substrate being transported through the concentration gradient to the outer luminal side of the membrane. If the compound needs to move against the concentration gradient, ATP may provide energy to facilitate this process. There are several transport systems in the brain endothelial cell membrane for the transport of nutrients and endogenous compounds, such as the GLUT1 glucose transporter for the transport of glucose and certain hexose, the acidic amino acid transport system for the transport of glutamate and aspartic acid, and the LAT1 large neutral amino acid transporter for the transport of phenylalanine and other neutral amino acids. LAT1 also transports drugs such as L-dopamine, gabapentin, or melphalan, which have similar molecular structures and sizes to endogenous substrates.

Receptor-Mediated Transcytosis

Brain capillary endothelial cells have a receptor-mediated transcytosis (RMT) mechanism as a means of specific and selective uptake of certain macromolecules. In general, ligands located on the luminal side of brain endothelial cells in the circulation bind to their specific receptors, and the receptor-ligand complex crosses the intracellular compartment through vesicle trafficking mechanisms, usually through the endosome/lysosomal system. The receptor-ligand complex is then dissociated, and the ligand is released from the complex into the extracellular space of the non-luminal side of the endothelial cell, and the receptor is recycled to the luminal side of the endothelial cell. Certain macromolecules in the blood cross the blood-brain barrier through well-characterized endogenous receptors, such as insulin, transferrin, and leptin. There is limited understanding of how different central nervous system diseases affect receptor expression and their regulation. Receptor-mediated transcytosis has the potential to be exploited as a strategy for the delivery of therapeutic molecules to the brain.

Adsorption-Mediated Endocytosis

Adsorption-mediated transcellular swallowing (AMT) is a non-specific type of transcellular swallowing that is triggered by electrostatic interactions between the positively charged moiety of certain macromolecules and the membrane of brain endothelial cells containing anionic heparin proteoglycans. The process of AMT involves the invagination of electrostatic complexes followed by the formation of endosomes, transport from the luminal side to the non-luminal side, and the release of the AMT substrate. In AMT-facilitated central nervous system drug delivery strategies, polycationic proteins, such as protamine or cell-penetrating peptides containing cationic sequences, such as SynB peptides, are commonly used. Interestingly, the type 1 protein of the SARS-CoV-2 virus responsible for the COVID-19 outbreak can cross the blood-brain barrier in mice through a mechanism similar to AMT. Potential drawbacks associated with cationic proteins or peptides are their random distribution and therefore lack of selectivity for the brain, and potential toxicity associated with endothelial damage.

Cell-Mediated Transcytosis

Cell-mediated transfect swallowing is a recently discovered drug transport pathway across the BBB. Monocytes are considered a "Trojan horse" and are used by pathogens as a means of transportation into the central nervous system. Some pathogens, such as the human immunodeficiency virus (HIV), enter the brain through this transport pathway, in which HIV-infected monocytes and/or macrophages cross the blood-brain barrier for immune surveillance during normal migration, or alter vascular permeability due to the production of pro-inflammatory mediators. Many neurological diseases, such as Alzheimer's disease, Parkinson's disease, brain tumors, and AIDS-related dementia, have an inflammatory component. During the inflammatory process, leukocytes (monocytes and neutrophils) are widely recruited. These cells migrate to the site of inflammation through a process known as extravasation and chemotaxis, which is unique. Cell-mediated transcell swallowing has been developed as an exciting strategy for achieving therapeutic drug delivery at the blood-brain barrier. Drug carriers for the treatment of brain tumors based on immune cells, neural stem cells, and mesenchymal stem cells have been explored.

Active Efflux Transport

The capillary endothelial cells of the blood-brain barrier express drug efflux transporters, which limit the ability of many drugs to enter the brain. These efflux transporters are able to pump drugs out of the cells, creating additional restrictions on access to the blood-brain barrier by excluding them from the brain endothelial cells. Most of these efflux transporters belong to the ATP-binding cassette superfamily of proteins, including P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and breast cancer resistance protein. In summary, the blood-brain barrier tightly regulates the inflow and outflow of essential substances and information molecules (e.g., peptides) and serves as a communication interface between the central nervous system and the blood.

Discovery of Peptides for Crossing the BBB

Peptides for Brain Delivery

Research into brain-penetrating molecular carriers has led to the discovery of blood-brain barrier shuttle peptides, which offer advantages such as ease of synthesis, low immunogenicity, and cost-effectiveness compared to Trojan horse antibodies. These peptides, often linear or cyclic (≤50 amino acids), facilitate non-invasive transport across the BBB via receptor-mediated uptake or direct membrane penetration. Many derive from endogenous neurotropic proteins, neurotoxins, and viruses, or are identified through phage display technology (PDT).

PDT, pioneered by G.P. Smith in 1985, enables high-throughput screening of peptides binding to biological targets. It involves inserting foreign protein genes into bacteriophage capsid proteins, expressing peptide conjugates on the phage surface, and selecting high-affinity binders through iterative bio-panning. This method has identified numerous BBB shuttle peptides, including PepC7 (CTSTSAPYC) with a 41-fold higher translocation efficiency, and LRPep2 (HPWCCGLRLDLR), which binds LDL receptors on brain endothelium. Other notable peptides identified via PDT include Angiopep-2, Apamin, SynB1, and GSH, demonstrating the potential of BBB shuttle peptides in CNS drug delivery.

Peptides Derived from Neurotrophin and Viruses

Some BBB shuttle peptides originate from endogenous neurotrophins and neurotropic viruses, identified through sequence alignment, synthetic screening, or structural studies like NMR. For example, ApoE interacts with the LRP-1 receptor for BBB transcytosis, while the HIV-1 TAT peptide and dengue virus-derived PepH3 facilitate brain penetration.

Optimization of BBB shuttle peptides involves chemical modifications to enhance stability, permeability, and receptor binding. Strategies include integrating D-enantiomers for protease resistance and modifying hydrophobicity. For instance, (PhPro)₄ improves solubility and BBB permeability, while trans-racemic THRre enhances transport. The fusion peptide gHoPe2, combining CPP pVEC and gHo, demonstrates improved tumor targeting.

Common Targets of Action of Brain Penetrating Peptides

Transferrin Receptors

The transferrin receptor (TfR), a key RMT pathway receptor in the BBB, regulates iron uptake and homeostasis. Though found in brain endothelial cells, choroid plexus, and neurons, TfR is mainly expressed in hepatocytes and erythrocytes. TfR-mediated transcytosis enables iron-bound transferrin transport into the brain, similar to insulin transport.

Cancer cells overexpress TfR, making transferrin a vehicle for drug delivery. The OX-26 antibody facilitates nanoparticle transport across the BBB, enhancing drug concentration. Bispecific antibodies targeting TfR and β-secretase improve brain uptake. BBB shuttle peptides like THR, CRT, and HAI also target TfR.

Low-Density Lipoprotein Receptors

Low-density lipoprotein receptors (LDLRs) mediate endocytosis of cholesterol-rich low-density lipoprotein (LDL) and have been extensively studied for transporting proteins and therapeutics across the blood-brain barrier to the central nervous system (CNS). LDLR is most prominently expressed in bronchial epithelial cells and adrenal and cortical tissues. It is also highly expressed in the blood-brain barrier, as well as in various tumor cells such as glioma cells. Apolipoprotein B (ApoB) and apolipoprotein E (ApoE) peptides derived from LDLR-binding sites have been shown to be effective for LDLR-targeted therapies in central nervous system diseases, although competition with endogenous LDL and disruption of cholesterol homeostasis in the brain have been a concern. Peptide-22, developed by phage display screen, has an affinity for LDLR and is not in competition with endogenous LDL, enabling efficient and rapid transfer to the central nervous system.

LDL Receptor-Associated Proteins 1 and 2 (LRP-1 and LRP-2)

LRP-1 and LRP-2, multifunctional endocytic receptors in the LDLR family, mediate ligand internalization and degradation, playing roles in metabolism and cell signaling. They interact with over 40 molecules, including apolipoprotein E, α2-macroglobulin, and amyloid precursor proteins, making them key targets for brain drug delivery via RMT.

LRP-1 (CD91) is widely expressed in the CNS and regulates amyloid clearance and BBB permeability. LRP-2 (megalin) is abundant in the kidneys and brain and upregulated in Alzheimer's disease. Nanoparticles coated with polysorbate 80 utilize LRP-1 transcytosis for enhanced BBB penetration.

Leptin Receptors

Leptin is a 16 kDa hormone produced primarily by white peripheral adipocytes and plays an important role in regulating food intake and maintaining energy balance. Leptin is also involved in regulating learning and memory processes. Leptin receptors have been found to be present in glial cells, astrocytes, choroid plexus, and cerebral capillary endothelial cells. Leptin is released from fat cells into the bloodstream and is taken up by interacting with leptin receptors expressed in the brain parenchyma. Brain endothelial cells affect physiological and behavioral aspects such as eating, thermogenesis, and activity. In obese patients, leptin receptors may be saturated due to elevated leptin levels. A leptin-derived peptide sequence g21 (12-32 amino acids) fragment has been used to bind to the surface of polylactic acid-glycolic acid copolymer (PLGA) nanoparticles that cross the blood-brain barrier after intravenous injection and reach the brain 2 hours later, accounting for 0.16% of the injected dose. Another 30-amino acid peptide derived from leptin (Leptin30) was developed as a brain-targeting ligand for nanoparticles.

Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAchR) belong to a ligand-gated ion channel superfamily that includes γ-aminobutyric acid type A and C receptors, glycine receptors, and serotonin receptors. nAchR mediates synaptic transmission between nerve and muscle cells. nAchR responds to both the neurotransmitter acetylcholine and the addictive drug nicotine. nAchR is mainly expressed in the central nervous system, peripheral nervous system, and muscles. Neurons nAchR consists of five transmembrane subunits arranged around a central water-filled pore. A 505-amino acid neurotoxic rabies virus glycoprotein (RVG) has been reported to interact with nAchR and is used to deliver various nanoparticles, RNA, and DNA fragments. RVG-derived 29-amino acid peptide RVG29 binds to PAMAM (polyamide amine) nanoparticles and shows higher blood-brain barrier crossing efficiency than conventional nanoparticles in vitro blood-brain barrier models. Another report suggests that a 16-amino acid D-CDX peptide facilitates brain-targeted drug delivery through nAchR-mediated endocytosis.

BBB Penetrating Peptides for PDCS

Despite advances in antibodies or endogenous protein ligands against transporters expressed on the BBB, some high-affinity antibodies hinder the delivery of cargo due to their high affinity for the receptor and the release of cargo that is ineffective in the brain parenchyma. In addition, the high cost of protein production and complex structure limit the wide application of these blood-brain barrier shuttle proteins. Small-sized peptides are gaining traction due to their ability to exhibit excellent target specificity, low toxicity and immunogenicity, and minimal synthesis and manufacturing challenges. Various blood-brain barrier shuttle peptides have been studied to assess their ability to cross the blood-brain barrier. Some blood-brain barrier shuttle peptides cross the blood-brain barrier by using a receptor-dependent mechanism that utilizes the selective RMT pathway, while some synthetic or protein-derived cell-penetrating peptides (CPPs) have shown promise as brain delivery vehicles through receptor-independent mechanisms, primarily through the AMT pathway. CPPs are typically made up of 5 to 30 amino acids and are broadly divided into two categories: highly cationic and amphiphilic. The positive charge and amphiphilic nature of CPPs are key features that allow CPPs to cross biological membranes and drive bioactive cargo into cells. Some CPPs exhibit blood-brain barrier penetration. Some commonly used blood-brain barrier shuttle peptides derived from PDT, CPP, or native proteins are highlighted below for the construction of PDCs.

Table.1 Peptide Transporters and Their Drug Conjugates.

Peptides Targeting LRP-1, LRP-2, and LDLR

An 18-residue apolipoprotein E peptide and a 39-residue apolipoprotein B peptide derived from endogenous neurotropies apolipoprotein E and apolipoprotein B proteins exhibit hemorrhagic brain barrier penetration by interacting with low-density lipoprotein receptors in the brain. The latest phage displays biological screen also identified several blood-brain barrier shuttle peptides, including Peptide-22, VH4127, LRPep2, and AEP peptides targeting LDL receptors, and L57 peptides targeting LRP-1 receptors.

Angiopep-2

Angiopep-2 is a 19-amino acid peptide (TFFYGGSRGKRNNFKTEEY, molecular weight 2.3 kDa) developed by sequence alignment with human proteins such as bikunin, secreted amyloid precursor protein, and Kunitz inhibitor-1 precursor protein. All three proteins contain a Kunitz domain known for its affinity for the LRP receptor. Using the in vitro blood-brain barrier bovine brain capillary endothelial cell Transwell assay, Angiopep-2 demonstrates a transcytosis capacity more than 50 times higher than that of transferrin and lactoferrin. In addition, the near-infrared fluorescent dye Cy5.5-labeled Angiopep-2 was shown to be able to rapidly enter the brain parenchyma through in vivo imaging and fluorescence analysis of brain sections. Cy5.5-Angiopep-2 is located in the brain parenchyma.

At 1 h after intravenous injection, fluorescently labeled Angiopep-2 was observed to be close to the nucleus of brain cells, while Cy5.5-Angiopep-7 (negative control) did not cross the cerebral capillaries. By in vivo imaging, the accumulation of fluorescently labeled Angiopep-2 in the meninges and parenchyma of the brains of living mice was observed after 5 days of intravenous administration. In vivo, mouse brain imaging showed a significantly higher accumulation of Cy5.5-Angiopep-2 in the brain than Cy5.5-Angiopep-7 over the time frame of 30 minutes to 24 hours after intravenous administration. The AUC value of Cy5.5-Angiopep-2 fluorescence was 9.5-fold higher than that of Cy5.5-Angiopep-7, indicating the selective uptake of Angiopep-2 to the brain. Initial applications of Angiopep-2 focused on the chemical synthesis of Angiopep-2 small molecule drug conjugates, including conjugates of paclitaxel, etoposide, doxorubicin, morphine, morphine-6-β-glucuronate, caffeic acid, and neurotransmitters. The subsequent utilization of Angiopep-2 is the modification of carriers (e.g., nanoparticles, micelles, carbon nanotubes, and antibodies) to deliver drugs to the brain, which supports the versatility of the blood-brain barrier shuttle protein Angiopep-2 in delivering small and large molecules to various central nervous system diseases.

Table.2 Peptide Modification Services at Creative Peptides.

Peptides Against TfR

A 12-residue THR peptide (THRPPMWSPVWP) and a 7-residue HAI peptide (HAIYPRH) were found to be binding peptides for human transferrin receptors by phage display biological screening process. The TfR peptide fused with green fluorescent protein (GFP) can be internalized by transferrin receptor-expressing cells. The blood-brain barrier shuttle THR peptide has been conjugated to gold nanoparticles (AuNPs) to increase nanoparticle penetration in the rat brain. THR peptides bind to adeno-associated virus AAV8 virions and significantly enhance their ability to cross the blood-brain barrier and transduce neuronal cells. Blood-brain barrier shuttle HAI peptide-conjugated liposomes improve delivery and treatment outcomes in a mouse model of glioma. The HAI peptide is able to deliver AuNPs to the rat brain and shows better transport capacity than the THR peptide. Another blood-brain barrier shuttle peptide, CRT (cyclic CRTIGPSVC peptide), selectively interacts with transferrin receptors, inducing alloconismal changes that "mimic" iron functionally. CRT-directed viral particles show improved blood-brain barrier permeability in normal mouse brains, and CRT-conjugated nanoparticles show enhanced brain distribution in glioma parenchyma in mouse models.

TAT-Derived Peptides

The TAT (transduction domain of human immunodeficiency virus type 1 [HIV-1]) protein is an 86-amino acid transcription factor involved in the replication cycle of the HIV-1 virus, capable of entering cells and transferring to the nucleus through adsorption endocytosis. The 11-residue TAT peptide derived from the α-helix domain of the TAT protein was the first peptide to be identified as a cationic CPP. TAT peptides have been shown to increase the pervasive tissue penetration of TAT-β-galactosidase conjugates beyond capillaries, including the brain. Various PAT-conjugated nanoparticles have shown improved brain delivery. PDCs synthesized by conjugation of dual TAT and Angiopep-2 peptide with paclitaxel showed improved drug translocation to the brain in a mouse model of intracranial glioma.

Transportan 10

Transportan 10 (TP10) is a 21-residue amphiphilic CPP (AGYLLGKINLKALAALAKKIL) with high membrane translocation capacity. TP10 has been used as a delivery vehicle for peptide nucleic acids, peptides, and proteins.

TP10-dopamine conjugates, linked via small-sized PEGs, can penetrate brain tissue, exhibit high affinity for dopamine D1 and D2 receptors, and demonstrate anti-Parkinson's disease activity in animal models. Additionally, a TP10-vancomycin conjugate effectively crosses the blood-brain barrier, achieving a brain tissue concentration 200 times higher than free vancomycin.

SynB

SynB peptides are derived from the natural antimicrobial peptide protegrin and belong to the cationic CPP family. The 18-residue SynB1 (RGGRLSYSRRRFSTGR) and 10-residue SynB3 (RRLSYSRRRF) peptide have been shown to enhance drug trafficking across the blood-brain barrier through adsorption-mediated endocytosis. L-SynB1, L-SynB3, and their enantiomer D-SynB3 are conjugated to doxorubicin, and the conjugate significantly increases the brain uptake of doxorubicin by approximately 30-fold. SynB1 also enhances the effects of benzicillin or morphine-6-glucuronate in the brain without compromising the integrity of the blood-brain barrier. The SynB1 peptide is linked to dalargin via a disulfide linker, a conjugate that significantly improves dalargin delivery and analgesic activity in the brain.

Penetratin

Penetratin is a 16-residue cationic CPP (also known as pAntp-(43-58)) derived from Drosophila antennal foot protein (amino acids 43-58). Penetratin exhibits cell-penetrating properties through adsorption-mediated endocytosis. Penetratin has been used as an intracellular delivery vehicle for various cargoes such as GFP, oligonucleotides, and liposomes. Compared to polylysine (CPP with high arginine content) functionalized nanoparticles, Penetratin-functionalized polymer nanoparticles showed significantly enhanced brain uptake and reduced accumulation in non-target tissues. Transferrin-Penetratin-modified liposomes improve brain delivery of plasmid ApoE2 and plasmid β-galactosidase. Doxorubicin-D-Penetratin conjugate showed 6-fold higher brain uptake of doxorubicin.

Cytoplasmic Transduction Peptides

Cytoplasmic transduction peptide (CTP) is an 11-residue cationic CPP (YGRRARRRRR) that is designed to cross the cell membrane and preferentially aggregate in the cytoplasm. CTP-conjugated Smac/DIABLO peptide and CTP-fused β-galactosidase are able to efficiently enter the cytoplasmic compartment of the cell. The CTP-coupled neurotransmitter γ-aminobutyric acid (GABA) is able to penetrate the blood-brain barrier and increase GABA levels in rat and mouse plasma.

Arginine-Glycine-Aspartate Peptide

The RGD (arginine-glycine-aspartate) peptide targets αvβ3 integrins and has been widely used as a brain drug delivery vehicle due to its overexpression in neovascularization, especially in brain tumors. RGD peptide-modified polymers, radiolabeled peptides, micelles, and liposomes have been reported to be useful in the diagnosis and treatment of glioblastoma. These RGD nanoparticles are thought to mediate indirect endocytosis by internalizing into leukocytes, which are recruited into the brain in response to inflammation. The RGD-PEG4-Suc-PD0325901 (a MEK1/2 inhibitor) conjugate has shown superior therapeutic efficacy in the treatment of xenografts in U87MG glioblastoma mice, suggesting that the drug has the potential to enable brain delivery.

Glutathione

Glutathione (L-γ-glutamyl-L-cysteine-glycine, GSH) is the most abundant (up to 10 mM intracellularly and 5 μM circulating in circulation) small molecular weight thiol in living cells, and it has a range of functions from detoxification to protection from oxidative damage. Endogenous GSH was identified as a blood-brain barrier shuttle peptide, which can be demonstrated by the saturation process of 35S-labeled GSH in the rat brain after carotid artery injection. The most successful example of GSH being used as a blood-brain barrier shuttling peptide for drug delivery to the brain is G-Technology. Sodium-dependent glutathione-PEG liposomes have been shown to be able to deliver antiviral drugs to the brain. GSH is also covalently linked to the N-methyl-D-aspartate receptor antagonist memantine (MEM) for drug delivery to the brain.

Polyarginine Peptides

Polyarginine peptides, such as R8, R11, and R18, are arginine-rich CPPs that protonate under physiological conditions and interact with the negatively charged carboxy, sulfate, and phosphate groups of phospholipid membranes, resulting in a distortion of the membrane structure and initiating migration of hydrophilic water pores in the membrane, thereby triggering receptor-independent intracytosis. Polyarginine CPPs can facilitate the transport of molecular cargo, including nucleic acids, active proteins, quantum dots, and nanoparticles, across membranes. Octaarginine (R8), D-R8, and R11 have been used as blood-brain barrier shuttle peptides for drug delivery to the brain. R8 and D-R8 are shown to enhance insulin uptake and accumulation deep in the rat brain after intravenous combined administration of a mixture of D-R8 and insulin. The R11 peptide can cross the normal intact blood-brain barrier and reach the cortex, striatum, and thalamus. R11-mediated brain delivery was greatly enhanced in ischemic mouse brains up to 8 hours after systemic administration. R18 and type D antigen D-R18 show neuroprotective properties in different rodent stroke models by reducing the severity of ischemic brain injury and improving functional outcomes. These findings suggest that polyarginine peptides are promising carriers for the delivery of therapeutics across the blood-brain barrier with potential neuroprotective effects.

Summary

In this review, we investigated blood-brain barrier shuttle peptides and brain-penetrating PDCs as non-invasive drug delivery systems for transporting chemotherapy drugs and other molecular cargo across the restrictive blood-brain barrier. Early blood-brain barrier shuttle peptides, such as cell-penetrating peptides, mainly used poorly selective adsorption-mediated transcytosis to improve trafficking across the blood-brain barrier, while novel peptides discovered through phage display technology or from native neurotrophin used receptor-mediated molecular mechanisms of transcytosis to cross the blood-brain barrier.