A. Motivation
Glioblastoma Multiforme (GBM) is classified by the World Health Organization as a highly malignant, grade IV astrocytoma affecting 2-3 per 100,000 individuals in Europe and the United States 1. It is the most aggressive and common brain tumor, and accounts for approximately 52% of all primary brain tumors 2. GBM tumors are usually surgically removed, however doing so is difficult, due to its topographically diffuse and rapidly invasive nature. Aside from surgical removal, chemotherapeutic drugs such as Temozolomide are currently being used as treatments 2,3. However, these therapeutics do not effectively reach the tumor sites, as drug delivery to GBM is especially difficult due to the highly selective blood brain barrier (BBB).
In 2015, the National Cancer Institute reported a 33% survival rate from surgery and chemotherapy, with survivors only living 15 months for newly-diagnosed GBM and 5-7 months for relapsed GBM 2,3. In order to improve Quality of Life (QoL) and increase survival rate, it is clear that new and innovative ways to deliver therapeutics is vital.
B. Glioblastoma Multiforme and the Blood Brain Barrier
The blood brain barrier (BBB) acts as a specialized barrier between systemic circulation (where the cancer fighting drugs start) and the brain (where the glioblastoma cells are). It is designed to stop potentially harmful compounds from reaching the brain while still allowing essential nutrients to pass through. Even though the BBB is compromised and “leaky” at primary tumour sites when the tumour is too big and diffuse, the BBB is still intact at sites of secondary tumors4. Therefore, in order to reach all of the glioblastoma multiforme tumors with chemotherapy drugs, we must take advantage of the mechanisms that make the BBB permeable to certain molecules. Most compounds that pass through the barrier do so via one of two methods, transcellular passive diffusion or carrier-mediated transport. In many cases, BBB cells are equipped with proteins that recognize certain ligands and transport compounds with that ligand through the cell interiors and across the BBB. For example, the Low-Density Lipoprotein (LDL) receptor recognizes proteins like APOE protein and permits the transcytosis of the molecule with APOE on it through the cell that the LDL receptor is on. In order to get drugs past the BBB, we have designed a drug delivery system (DDS) that takes advantage of the LDL receptor’s ability to permit certain molecules passage from circulation into the brain.
C. Liposomes as Drug Delivery Vehicles
Liposomes are artificially-made phospholipid bilayer spheres with a fluid-filled center that are often used as therapeutic carrying vessels. The phospholipid membranes are commonly modified with the addition of molecules like cholesterol, peptides, and ligands to increase stability and provide functionality. The composition of the membrane and the hydrophilicity of the chosen therapeutic determines how leaky the liposome is to a drug, and thus controls the speed at which therapeutics can be released5. For decades, liposomes have been recognized for their desirable drug carrying characteristics - namely, their ability to stay stable and bear drugs for extended periods of time in circulation, their ability to carry a wide range of therapeutics, and their capacity for triggered release5 Liposomes are especially useful for delivering therapeutic drugs that would be normally be rejected across the BBB - the drugs are enclosed inside the liposome, and the modified surface of the liposome has receptors that facilitate endocytosis of the entire system through the BBB4. As well, liposomes and other similar sized particles tend to accumulate in tumorous tissue much more than they do in normal tissue because of the enhanced permeability and retention effect, leading to more concentrated drug release at the cancerous tissue of glioblastoma.
Based on a recommendation by Saul, Annapragada and Bellamkonda6, we use 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC)- cholesterol as the components of our liposome. DSPC is a long chain saturated phospholipid that has been shown to have a significantly lower drug leak rate, and a longer half-life compared to unsaturated lipids7. Cholesterol is included in the liposome formulation since it is found to improve plasma stability, and increases the rigidity and packing density of the lipids, thus reducing permeability of water, small molecules, and other ions8.
D. DSPE-PEG as Liposome to Ligand Linkers
In order to attach our receptor ligands to the liposome surface, we need a linker. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Polyethylene Glycol (DSPE-PEG 2000) is a Food and Drug Administration (FDA) approved amphiphilic block copolymer9 that can anchor into the similarly hydrophobic tails of the DSPC-cholesterol liposome bilayer. When in solution, DSPE acts as a hydrophobic core, while PEG acts as a hydrophilic shell with exposed hydroxyl terminal groups that can be modified to attach various ligands such as APOE or EGF. In our design, we modify the distal end of PEG2000 with either an amine, maleimide or carbohydrazine group, depending on the ligand. Modified DSPE-PEG linkers have also demonstrated increased drug circulation time and delivery specificity9, which overall makes it an ideal lipid linker for our dual-ligand system.
E. EGF as an EGFR-targeted ligand
The Epidermal Growth Factor (EGF) ligand is an approximately 40 amino acid, 6.2 kDa protein growth factor that has a high binding affinity to EGF receptors (EGFR). EGFRs contain a ligand binding site for EGF heterodimerization, which activates the tyrosine-kinase domain for regulation of key cellular processes such as proliferation, survival, differentiation, homeostasis and tumorigenesis10. In glioblastomas, amplification of EGFRs have a frequency of around 50% which makes them an attractive ligand-targeting option 10. Although, EGFRvIII is the most common mutant of EGFR found in glioblastoma patients, our ligand targets wild-type EGFR due to experimental limitations (i.e., finding a cell line that specifically over expresses EGFRvIII).
According to Kim, S. et al 10, permeating the blood brain barrier effectively is still the biggest hindrance to curing glioblastoma. EGF-ligand carrying liposomes are taken up by the cell through receptor-mediated endocytosis11 which allows the liposome system to be digested in lysosomes.
To conjugate the EGF ligand onto the liposome, we form a thioether bond between an thiolated EGF (EGF-SH) and a maleimide group on the distal end of a DSPE-PEG-Maleimide liposome.
F. APOE ligand and receptor-mediated transcytosis
Apolipoprotein-E is a 34 kDa low density lipoprotein (LDL) ligand that directs delivery of triglycerides and cholesterol to cells by binding to low density lipoprotein receptors (LDLRs). On fully differentiated endothelial cells, such as the BBB, the LDLR captures LDL ligands by lateral diffusion in the caveolae pathway and delivers intact ligands via receptor-mediated transcytosis12. This is in contrast with non-differentiating, proliferating endothelial cells which undergo receptor-mediated endocytosis. Once the ligand is internalized, it is shuttled through multivesicular bodies, specifically the early and late endosomes, with subsequently decreasing pH ( approximately pH 6.0 to pH 5.0). The receptor and the internalized ligand is then ejected to the abluminal (brain side) side. In our project, ‘smuggling’ the liposome via APOE binding to LDLR ensures that the system remains intact for further EGFR targeting on the glioblastoma. One study approximates that this transcytosis mechanism happens fairly quickly (within 15-20 mins)13.
However, there are limitations to targeting LDLRs, since these receptors are not unique to the BBB, and their surface expressions may vary from patient to patient. They are also widely found on the surface of hepatocytes, and upregulation of LDLRs are dependent on cholesterol levels. Despite these limitations, targeting LDLR has proven to be promising due to the receptor-mediated transcytosis mechanism of APOE binding. APOE binds to the LDLR on an arginine or lysine group within amino acid residues 135-15114.
G. pH-sensitive Hydrazone (HZ) bond as an APOE cleave
Hydrazone is a bond that has been previously shown to be stable at physiological pH (pH 7.0- 7.4), and acid labile at low pH levels (pH 5.0-6.0)15. Hydrazone bond linkages have been used to conjugate drugs to certain ligands or antibodies for rapid release of drug under acidic conditions such as the tumor microenvironment or endosomal compartments16. For this reason, we form a hydrazone bond between a carbohydrazine group on the distal end of a DSPE-PEG lipid, and a periodate oxidized Apolipoprotein E.
To synthesize this, we adapt a protocol outlined by Biswas, Dodwadkar, Sawant and Torchillin17. We begin by the addition of a sulfhydryl group (SH) to DSPE-PEG-Amine to form DSPE-PEG-SH, then a thioether bond is formed when reacted with a maleimide-hydrazine cross-linker, and the result is the formation of DSPE-PEG-CO(NH2)2 (CO(NH2)2 represents the carbohydrazine group). Since hydrazone bonds form strongly with reactive aldehyde groups, we can oxidize sialic acid groups on the APOE. For our specific human plasma derived APOE sequence (gene id: 348, UniProtKB-P02649), a sialic acid moiety is present on the Serine 308 (Ser 308) amino acid site. Serum derived APOE exists as multiple sialylated glycoforms, with monosialo forms dominating18.
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