Conjugtation of EGF to Liposome
The conjugation of EGF to liposomes was successful, which was verified by cell-culture experiments. The experiments showed that liposomes treated with EGF were endocytosed by U251 cells, whereas liposomes not treated with EGF were not. This confirms that EGF facilitates endocytosis, and was successfully conjugated onto the liposomes. DLS results confirm that liposomes were not damaged during the conjugation process. Due to the fluorescent component of the liposomal samples, we were unable to determine the amount of EGF conjugated onto the liposomes.
Synthesis of DSPE-PEG-CONHNH2
Thiolation of DSPE-PEG(2000) and MPBH crosslinker addition was successfully performed. This was verified using H-NMR analysis which identified the aromatic protons present in DSPE-PEG(2000)-CO(NH2)2. Hydrazone bond content was quantified using a TNBSA assay. Successful conjugation of APOE to the system was also verified using a human APOE sandwich ELISA. Finally, the product was able to be conjugated to APOE for use in the drug delivery system.
Cell Culture Experiments
Through the experiment’s results, the positive effect of EGF on their endocytosis into cancerous cells is observed. Additionally, the needed concentration and time for effective endocytosis of liposomes into cells were identified as 100 µM and 2 hours. A positive effect from ApoE was not observed in our experiments, however this could be a result of many factors:
- Low number of conjugated ApoE ligands on the liposomes
- Poor conjugation of ApoE ligands on the liposomes
- Low amount of time for liposomes to settle and reach the bottom of the transwells
- Low concentration of liposomes used for these tests
- Low expression of LDL receptors on the b.END3 cells used These factors can be assessed by conducting the following experiments:
- Titrating the amount of ApoE ligands conjugated to liposomes and testing all batches with cells simultaneously
- Characterizing the ApoE ligands on the liposomes using ELISA assays and improving conjugation process if necessary
- Titrating the incubation time and only having one incubation time per condition (as the problem with the old liposomes does not persist with the new ones)
- Titrating the concentration of liposomes added
- Characterizing the membrane proteins of the cells using western blot After assessing these factors, liposomes with ApoE and hydrazone should be put through the endothelial cell layer and the cleavage of ApoE should be characterized by conducting an ELISA assay on samples from below the transwells. The next step would include conducting an experiment with the full system, where the transcytosis and endocytosis of liposomes+ApoE-HZ is compared with liposomes+ApoE+EGF and with liposomes+EGF that are simply added to a monolayer of U251 cells. This would show the positive effect of having the ApoE cleave off during the endocytosis of liposomes into cancerous cells. In addition, it would also compare the endocytosis of liposomes with EGF and ApoE cleaved off to liposomes with EGF.
Additional steps can be taken to improve the synthesis yield of the final product by including thiolation reagent in excess and using gel filtration chromatography to minimize loss of product during intermediate purification steps.
Optimization of the conjugation may be useful to investigate to also increase the yield of the final product. Such parameters for optimization may include changing the oxidation buffer, temperature, duration and performing a titration of the volumes of DSPE-PEG(2000)-CO(NH2)2 and oxidized APOE during the conjugation steps.
Further mass spectrometry experiments may be conducted to assess the success of the acid labile bond to cleave in the endosomal compartment environment. This may include testing the cleavage at various pHs and duration of incubation to determine the most efficient pH and incubation time for cleavage.