Augmenting Hypothermia Therapy for Hypoxic-Ischemic Encephalopathy in Neonates: Localizing Drug Delivery using pH and Temperature Responsive Nanoparticles

  • Author / Creator
    Narayanamurthy, Rukhmani
  • The obstruction of blood flow to regions of an infant brain leads to a reduction of oxygen and essential nutrients, causing what is known as hypoxic-ischemic (HI) damage to that region. Presently the only treatment available for affected neonates is hypothermia: head or whole-body cooling. Despite its limited effectiveness, hypothermia delays disease progression, thereby providing an opportunity for a supplement therapy to be beneficial within that time frame. It has long been the goal to combine hypothermia with pharmacology to improve the overall neuroprotective potential. Many drugs have shown promise in treating the HI damaged neonate brain, however, the systemic dosage used to achieve therapeutic concentrations in the brain lead to significant side-effects that have prevented their use in clinic.

    Self-assembling peptides can be engineered to respond to biological cues and deliver drugs in an on-demand manner. Although some classical pH sensitive polymer systems have been studied in the past, we focus on using relatively biocompatible elastin-like polypeptides (ELPs) as a self-assembling peptide that can be engineered to respond to pH and temperature to allow for localized release of drug. ELPs are biopolymers composed of repeating units of the pentapeptide sequence Val-Pro-Gly-X-Gly (VPGXG), where the variable residue X can be manipulated to trigger the phase transition at desired conditions. Once in nanoparticle form, appropriate drugs can be incorporated into the ELPs such that hypothermia and/or the pH changes that arise due to disrupted metabolism in the damaged tissue, would induce nanoparticle dissolution. Subsequently, the drug would be released at the site of injury in a safe and controlled manner.

    ELPs were synthesized from genetically engineered plasmids in recombinant Escherichia coli and purified from the bacterial lysate by inverse temperature cycling (ITC). Dexamethasone (DEX) was selected as the candidate anti-inflammatory drug for neonatal brain injury. Dynamic light scattering was used to characterize the temperature-dependent assembly of ELP nanoparticles containing DEX followed by the pH-dependent disassembly. The drug loading or encapsulation efficiency and the stability of the nanoparticles over time were determined using high-performance liquid chromatography. Next, the ELP-DEX nanoparticles were injected in a neonatal rat model of brain injury and focal brain cooling was applied. To test the ability of the ELP nanoparticles to deliver DEX to the injured brain, HPLC was used to detect and measure the amount of DEX in the brain tissue.

    On screening several valine, leucine, and histidine containing-ELPs in the guest position, we identified the construct (AG)40-(VH4)24 as the one that could undergo nanoparticle formation with DEX encapsulation under physiologically relevant pH 7.4 and temperature 37°C. This construct demonstrated assembly between 35 – 40 oC to form particles of size ~300 nm, and disassembly between 35 – 33 oC to <10 nm, at 0.25 mg/mL and pH 7.4. When the pH of the solution was lowered to 6.4 during cooling, the disassembly temperature decreased by 1oC and at pH 6 complete dissociation was observed, with presence of ~10 nm particles across all temperatures below 45°C. This shows the pH-responsiveness and sensitivity of the ELP construct (AG)40-(VH4)24 within a narrow pH range between 7.4 and 6, is an important trigger owing the local acidosis (~pH < 6.75) that develops at the site of brain injury. The drug loading or encapsulation efficiency of the particles was determined to be 1.3 ± 0.6 % at the same concentration of 0.25 mg/mL.

    Selective brain hypothermia was induced using a focal cooling device. Our method of cooling was successful in establishing and maintaining a temperature differential of 3.2ºC between the head and body of the animal throughout the experiment. It also offered the same extent of neuroprotection as in whole-body hypothermia and significantly reduced the amount of tissue damage when compared to normothermic animals (p = 0.0457). A single injection of DEX containing-(AG)40-(VH4)24 particles resulted in ~0.4 µg of DEX/g of brain tissue thus validating the ability of ELP nanoparticles to release DEX in the brain.

    The findings suggest that this drug delivery system holds immense potential to enhance the beneficial effect of hypothermia in a clinical setting, thus improving the quality of life in infants with brain injury. The proposed nanoparticles could then be used to understand the effects of multiple drugs under investigation for the treatment of other brain disorders.

  • Subjects / Keywords
  • Graduation date
    Fall 2022
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.