1. Conjugate EGF onto Liposome
✔ Maleimide assay
Confirm number of maleimide groups on DSPE-PEG-MAL liposomes
✔ Thiol Assay
Confirm that EGF is successfully thiolated for thioether bond formation with DSPE-PEG-MAL liposomes
✔ Dynamic light scattering (DLS)
Measure size distribution of liposomes
✘ Protein Assay
Confirm that EGF is successfully conjugated and determine the its concentration on the liposome
✘ Fluorescence Assay
Normalize the results of the protein assay by determining if the ratio of liposomal fluorescence and EGF protein concentration is similar
2. Synthesize DSPE-PEG-HZ-APOE
✔ Thiol Assay
Confirm that DSPE-PEG-Amine is successfully thiolated for attachment to maleimide-hydrazine crosslinker
✔ Hydrazine (TNBSA) Assay
Confirm that hydrazine groups are present on the synthesized DSPE-PEG-CONHNH2 (carbohydrazine).
✔ Nuclear Magnetic Resonance (H-NMR)
Determine if DSPE-PEG-SH and DSPE-PEG-CONHNH2 are correctly synthesized
✘ APOE ELISA (Enzyme linked immunosorbent assay)
Confirm and determine the concentration of APOE on the synthesized DSPE-PEG-HZ-APOE ligands
3. Determine functionality of Hydrazone bond on APOE ligand
✔ APOE ELISA (Enzyme linked immunosorbent assay)
Quantify the amount of APOE before and after acid cleave
✘ Matrix assisted laser desorption/ionization- mass spectrometry (MALDI-MS)
Determine that pH-cleavable hydrazone bond is functional and cleaves DSPE-PEG-HZ-APOE into two fragments at acidic pH’s
4. Determine EGF-ligand uptake by overexpressed EGFRs
✔ High-content screening analysis (Cellomics)
Determine if EGF ligand on liposomes have a positive effect on glioblastoma (U251 cell line) cellular uptake
5. Conjugate APOE ligand onto liposome and determine its functionality
✘ Fluorescence permeability assay for transcytosis through cell culture permeable supports
Determine if APOE ligand on liposomes have a positive effect on brain endothelial (bEND3 cell line) cellular transcytosis
✔ Dynamic light scattering (DLS) Measure size distribution of liposomes
✘ APOE ELISA (Enzyme linked immunosorbent assay)
Determine the specific concentration of APOE that has transcytosed
6. Investigate cellular uptake of “Trojan Bull DDS” at its endpoint (U251 cells)
✘ Fluorescence Permeability assay for transcytosis through cell culture permeable supports
Determine if cleavable hydrazone bond (HZ) has a positive effect on glioblastoma cellular uptake
✘ APOE ELISA
Determine the specific concentration of APOE that has transcytosed through the permeable supports
✔ Dynamic light scattering (DLS)
Measure size distribution of liposomes
✔ High-content screening analysis (Cellomics)
Determine if DSPE-PEG-HZ-APOE liposomes can transcytose and have a positive effect on glioblastoma cellular uptake
7. Model protein-protein interactions of ligand to receptor
✔ Z-dock
Determine if there is a significant difference in the ligand-receptor interactions between modified and unmodified EGF/APOE ligands through scoring functions and ligand-receptor complexes.
Determine whether the predicted models reflect binding studies conducted in literature for EGF/APOE.
Determine a representative ligand-receptor complex for the modified EGF/APOE ligands and their corresponding receptor.
✔ Chimera
Determine and generate the approximate 3D structures of the modified ligands and their corresponding receptors.
Determine and conduct appropriate pre-docking processing modifications.
✔ CHEMDRAW
Determine approximate 3D structures for the modifications to EGF and APOE.
✔ Swiss-Model
Determine an appropriate model for APOE using homology modelling.
✘ ZRank
Determine a re-ranked list of docked complexes from the ZDOCK output.
✘ RosettaDock
Determine a ranked list of docked complexes after applying structural refinement through RosettaDock.
