Project Goals


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.


Project Design

Discussion