ABSTRACT
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.
Subject(s)
Bacteriophages , Blood-Brain Barrier , Biological Transport , Blood-Brain Barrier/metabolism , Drug Delivery Systems , Endothelial Cells/metabolism , Humans , Peptides/metabolism , TechnologyABSTRACT
Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids.
Subject(s)
Pichia/chemistry , Protein Conformation , Viral Core Proteins , Capsid/chemistry , Capsid/ultrastructure , Humans , Particle Size , Viral Core Proteins/chemistry , Viral Core Proteins/isolation & purification , Viral Core Proteins/metabolism , Viral Core Proteins/ultrastructureABSTRACT
Little is known about the in vivo assembly pathway or structure of the hepatitis C virus nucleocapsid. In this work the intermediates of HCcAg multimerization in Pichia pastoris cells and the nucleic acid binding properties of structured nucleocapsid-like particles (NLPs) were studied. Extensive cross-linking was observed for HCcAg after glutaraldehyde treatment. Data suggest that HCcAg exists in dimeric forms probably representing P21-P21, P21-P23, and P23-P23 dimers. In addition, the presence of HCcAg species that might represent trimers and multimers was observed. After sucrose equilibrium density gradient purification and nuclease digestion, NLPs were shown to contain both RNA and DNA molecules. Finally, the analysis by electron microscopy indicated that native NLPs were resistant to nuclease treatment. These results indicated that HCcAg assembles through dimers, trimers, and multimers' intermediates into capsids in P. pastoris cells. Assembly of NLPs in its natural environment might confer stability to these particles by adopting a compact structure.
