Novel Blood-Brain Barrier Shuttle Peptides Discovered through the Phage Display Method.

Delivery of therapeutic agents into the brain is a major challenge in central nervous system drug development. The blood-brain barrier (BBB) prevents access of biotherapeutics to their targets in the central nervous system and, therefore, prohibits the effective treatment of many neurological disorders. To find blood-brain barrier shuttle peptides that could target therapeutics to the brain, we applied a phage display technology on a primary endothelial rat cellular model. Two identified peptides from a 12 mer phage library, GLHTSATNLYLH and VAARTGEIYVPW, were selected and their permeability was validated using the in vitro BBB model. The permeability of peptides through the BBB was measured by ultra-performance liquid chromatography-tandem mass spectrometry coupled to a triple-quadrupole mass spectrometer (UHPLC-MS/MS). We showed higher permeability for both peptides compared to N-C reversed-sequence peptides through in vitro BBB: for peptide GLHTSATNLYLH 3.3 × 10-7 cm/s and for peptide VAARTGEIYVPW 1.5 × 10-6 cm/s. The results indicate that the peptides identified by the in vitro phage display technology could serve as transporters for the administration of biopharmaceuticals into the brain. Our results also demonstrated the importance of proper BBB model for the discovery of shuttle peptides through phage display libraries.

Phage display as a tool to discover blood-brain barrier (BBB)-shuttle peptides: panning against a human BBB cellular model.

Most potential drugs for the treatment of central nervous system disorders do not cross the blood-brain barrier (BBB). Much research effort has been devoted to the discovery of new BBB-shuttle peptides-most of which have been identified by phage display. Here we report for the first time on the use of phage display against a human BBB cellular model which mimics the characteristics of the BBB. From the panning experiment of a 12-mer library, the SGVYKVAYDWQH (SGV) peptide sequence was selected and its permeability validated in the aforementioned model. Furthermore, internalization studies suggested that SGV internalizes through a clathrin-mediated mechanism and that it increases the uptake of a cargo in endothelial cells. These results highlight the usefulness of in vitro BBB models for the discovery of BBB-shuttle peptides through phage display libraries.

Signature Effects of Vector-Guided Systemic Nano Bioconjugate Delivery Across Blood-Brain Barrier of Normal, Alzheimer's, and Tumor Mouse Models.

The ability to cross the blood-brain barrier (BBB) is critical for targeted therapy of the central nerve system (CNS). Six peptide vectors were covalently attached to a 50 kDa poly(ß-l-malic acid)-trileucine polymer forming P/LLL(40%)/vector conjugates. The vectors were Angiopep-2 (AP2), B6, Miniap-4 (M4), and d-configurated peptides D1, D3, and ACI-89, with specificity for transcytosis receptors low-density lipoprotein receptor-related protein-1 (LRP-1), transferrin receptor (TfR), bee venom-derived ion channel, and Aß/LRP-1 related transcytosis complex, respectively. The BBB-permeation efficacies were substantially increased ("boosted") in vector conjugates of P/LLL(40%). We have found that the copolymer group binds at the endothelial membrane and, by an allosterically membrane rearrangement, exposes the sites for vector-receptor complex formation. The specificity of vectors is indicated by competition experiments with nonconjugated vectors. P/LLL(40%) does not function as an inhibitor, suggesting that the copolymer binding site is eliminated after binding of the vector-nanoconjugate. The two-step mechanism, binding to endothelial membrane and allosteric exposure of transcytosis receptors, is supposed to be an integral feature of nanoconjugate-transcytosis pathways. In vivo brain delivery signatures of the nanoconjugates were recapitulated in mouse brains of normal, tumor (glioblastoma), and Alzheimer's disease (AD) models. BBB permeation of the tumor was most efficient, followed by normal and then AD-like brain. In tumor-bearing and normal brains, AP2 was the top performing vector; however, in AD models, D3 and D1 peptides were superior ones. The TfR vector B6 was equally efficient in normal and AD-model brains. Cross-permeation efficacies are manifested through modulated vector coligation and dosage escalation such as supra-linear dose dependence and crossover transcytosis activities.

Brain penetrating peptides and peptide-drug conjugates to overcome the blood-brain barrier and target CNS diseases.

Nearly one in six people worldwide suffer from disorders of the central nervous system (CNS). There is an urgent need for effective strategies to improve the success rates in CNS drug discovery and development. The lack of effective technologies for delivering drugs and genes to the brain due to the blood-brain barrier (BBB), a structural barrier that effectively blocks most neurotherapeutic agents from reaching the brain, has posed a formidable hurdle for CNS drug development. Brain-homing and brain-penetrating molecular transport vectors, such as brain permeable peptides or BBB shuttle peptides, have shown promise in overcoming the BBB and ferrying the drug molecules to the brain. The BBB shuttle peptides are discovered by phage display technology or derived from natural neurotropic proteins or certain viruses and harness the receptor-mediated transcytosis molecular machinery for crossing the BBB. Brain permeable peptide-drug conjugates (PDCs), composed of BBB shuttle peptides, linkers, and drug molecules, have emerged as a promising CNS drug delivery system by taking advantage of the endogenous transcytosis mechanism and tricking the brain into allowing these bioactive molecules to pass the BBB. Here, we examine the latest development of brain-penetrating peptide shuttles and brain-permeable PDCs as molecular vectors to deliver small molecule drug payloads across the BBB to reach brain parenchyma. Emerging knowledge of the contribution of the peptides and their specific receptors expressed on the brain endothelial cells, choice of drug payloads, the design of PDCs, brain entry mechanisms, and delivery efficiency to the brain are highlighted. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease.

Application of the Phage Display Technology for the Development of Peptide- mediated Drug Delivery Systems through the Blood-Brain Barrier.

The main obstacle to biopharmaceutical delivery in therapeutic concentration into the brain for treating neurological disorders is the presence of the Blood-Brain Barrier (BBB). The physiological process of Receptor-Mediated Transcytosis (RMT) to transport cargo through the brain endothelial cells toward brain parenchyma has prompted researchers to search for non-natural ligands that can be used to transport drugs across the BBB. Conjugation of drugs to RMT ligands would be an effective strategy for its delivery to the central nervous system. An attractive approach to identify novel transcytosing ligands is the screening by phage display combinatorial libraries. The main technology strength lies in the large variety of exogenous peptides or proteins displayed on the phage's surface. Here, we provide a mini-review of phage display technology using in vitro and in vivo BBB models for the development of peptide-mediated drug delivery systems.

Peptide-mediated drug delivery across the blood-brain barrier for targeting brain tumors.

INTRODUCTION: Transportation of the nutrients and other substances from the blood to the brain is selectively controlled by the brain capillary endothelial cells that form a restrictive barrier, so-called blood-brain barrier (BBB). Currently, there is no unimpeachable approach to overcome the BBB obstructiveness because the existing options are either invasive or ineffective. AREAS COVERED: This review delineates the biological impacts of BBB on brain drug delivery and targeting. The nanoscaled multifunctional shuttles armed with the targeting entities (e.g., antibodies and peptides) are discussed. Important insights are remarked into the combinatorial screening methodologies used for the identification of de novo peptides capable of crossing BBB and targeting the brain. EXPERT OPINION: Depending on the physicochemical properties of small molecules and macromolecules, they may cross the BBB and get into the brain either through passive diffusion or active/facilitated transportation and transcytosis in a very selectively controlled manner. Phage-derived shuttle peptides can specifically be selected against BBB endocytic machinery and used in engineering novel peptide-drug conjugates (PDCs). Nanoscaled multitargeting delivery systems encompassing PDCs can overcome the BBB obstructiveness and deliver drugs specifically to diseased cells in the brain with trivial side effects.