Recombinant stimulus-responsive biomaterials to target hypoxic/ischemic perinatal brain injury

  • Author / Creator
    Alshememry, Abdullah
  • Hypoxic-ischemic brain damage (HIBD) is a major concern during the neonatal period, and can result in chronic neurological complications arising from damage to the term newborn brain. Delivering drugs at the site of brain injury is thought to be crucial to the advancement of therapy for these patients. Herein is described a two prong approach for the development of nanoparticles for delivering drugs at the site of injury, namely, temperature sensitive elastin-like polypeptide based nanoparticles and identification of peptides that can target the penumbra and ischemic core observed in HIBD. It is thought that this approach will lead to the development of a platform system that can facilitate the release of drugs at the site of injury with multiple triggers so as to prevent off-target toxic side-effects.A novel family of leucine-containing, short, marginally soluble elastin-like polypeptides (ELPs) were developed. In order to successfully express and purify these ELPs, a novel purification method needed to be developed. The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of ELP constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified ex¬pression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC). This protocol has been used to express multiple leucine-containing elastin-like polypeptides, with final yields 10-20 times greater than reported yields for comparable constructs. Due to the relative hydrophobicity of the tested constructs, even compared with commonly employed ELPs, conventional methods would not have been able to purify these peptides.In order to better understand the effect of drug loading on the size and self-assembly and disassembly behaviour of ELPs particles, multiple ELPs with varying concentrations and guest amino acid hydrophobicity were tested. The results revealed that the nature and hydrophobicity of the guest amino acid position and the ratio of ELP:drug have a significant effect on the particle size and the assembly/disassembly behaviour, and the drug entrapment efficiency was increased as the hydrophobicity of the guest amino acid increased. Moreover, less ELP when mixed with the drug seemed to increase the entrapment efficiency as well. Therefore, a certain balance needs to be maintained between the ELP:drug ratio and the choice of the guest amino acid as together they will have an effect on the phase transition behaviour of ELPs and its drug entrapment capability.Peptides that can specifically interact to the injury within the brain are promising agents for a variety of applications, including site-specific drug delivery that reduces toxic side effects and diagnostics platforms for the injured brain. To this end, in vivo phage display technology was employed where several unique peptides that home preferentially to ischemic tissue were identified. Peptides preferentially homing for the vasculature of penumbra (PHP1), ischemic core (CHP1) and healthy brain tissue (HHP1 and HHP2) were the most frequently occurring sequences after three rounds of biopanning. These identified vasculature-homing peptides may have significant clinical applications as targeting moieties to be used in diagnostic and ligand-mediated targeted therapy for hypoxic-ischemic perinatal brain injury.

  • Subjects / Keywords
  • Graduation date
    Spring 2019
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
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