Encapsulation and delivery of Ivermectin as a potential new anti-cancer formulation using recombinant human heavy-chain Apoferritin
Publication Date
March 15, 2026
Creator
Aspinall, Christian
Abstract
Many drugs that have been used for decades show anti-cancer activity. However, their low bioavailability limits their effectiveness as anti-cancer treatments.
Ivermectin (IVM) possesses several anti-cancer properties, such as inhibiting angiogenesis, potentiating the ubiquitination of PAK1, inhibiting the JAK/STAT signaling pathways, and affecting the WNT-TCF pathway, which vary depending on the specific type of cancer cells. Due to its low solubility in water-based buffers and its high protein-binding affinity of ~93 % for albumin in the blood, it is not used in clinical settings to treat cancer. IVM is widely used as an anti-parasitic agent orally and topically in humans and animals. This drug can induce autophagy, which is a self-repair mechanism of cells, resulting in reduced proliferation or cell death, depending on the dosage.
Apoferritin (Apo) is a self-assembling protein nanocage that is able to encapsulate drugs within its structure and act as a delivery system, entering cells via Transferrin receptor 1 (TfR1), for these drugs. In order to improve the Pharmacokinetic-Pharmacodynamic properties of IVM, we have investigated its encapsulation in Apo and did subsequent in vitro studies in cancerous and healthy cells.
IVM was encapsulated into Apo by different variations of encapsulation methods, which employed Sodium dodecylsulfate (SDS) as a protein denaturant. The Apo+IVM formulation was purified via acetone precipitation. The effect of acetone on the Apo cage was investigated via native PAGE and circular dichroism. Thereafter, the Apo+IVM formulation was characterised by Size exclusion (SEC)-HPLC.
Afterwards, the Apo+IVM formulation was tested in cell viability assays on MCF-7 (breast cancer) cells and MRC-5 (fibroblast) cells as a biological control.
A significant decrease of 49 %, p<0.05, in cell viability was observed when treating MCF-7 cells with Apo+IVM at a protein treatment concentration of 23.8 nM. In contrast, the biological control did not show a comparable significant reduction in cell viability. This difference can be attributed to the overexpression of TfR1 receptors in MCF-7 cells compared to MRC-5.
In the case of lower protein treatment concentrations, the differences in the reduction of cell viability between MCF-7 and MRC-5 were negligible. Even at 2.4 nM, a reduction of 20 % in cell viability/proliferation compared to its filtrate control was seen. The last two MTT assays indicate that with a higher loading of IVM inside Apo, the reduction in cell viability is similar. Thereby, a lower IVM loading inside Apo would be more selective in reducing cell viability in cancer cells but not in the biological control. Furthermore, MALDI-TOF-MS was used to investigate the appearance of the two protein bands at the position of the full nanocage, where only one band should appear. This was either due to self-cleavage of the HIS-TEV-Tag or contamination with an enzyme cleaving Apo HIS-TEV-Tag.
It has been shown that IVM induces a G1/S cell cycle arrest typical for inducers of autophagy. This work holds promise to encapsulate hydrophobic molecules within Apo, thereby being able to employ hydrophobic drugs against cancer.
Item Type
ethesis
Thesis Type
MRes
Supervisors
Thomas, Neil
University of Nottingham
Bradshaw, Tracey
University of Nottingham
Subjects (LC)
Associated Schools / Departments
UoN Repository URI
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Christian Aspinall-20704468-Final MRes Thesis.pdf
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