Metabolic Pathways and Liposome Drug Delivery

Metabolic and Biochemical Processes

BMI Calculation: BMI = Kg/M². Interpretation: <25 = Normal, 25-30 = Overweight, >30 = Obese. Ethanolamine (Ethan-NH₃), Choline = N(CH₃)₃

Leptin and Adipose Tissue

Leptin is released from adipose tissue when mass is high. It travels through the blood to the arcuate nucleus, binds to neuronal cells, and activates the JAK-STAT pathway. This increases gene expression of POMC, which produces alpha-MSH (a neurotransmitter). Alpha-MSH reaches neurons connected to adipose tissue, releasing norepinephrine. Norepinephrine binds to beta-adrenergic receptors on adipose tissue, promoting an increase (via the G-protein coupled receptor pathway and activation of PKA). This leads to upregulation of UCP1 expression and TAG breakdown. Fatty acids from TAG breakdown enter the mitochondrial matrix, generating a proton gradient uncoupled to ATP synthase. Protons flow through UCP1, ultimately depleting TAG content and decreasing mass.

Leptin Supplementation

1. Leptin supplements (even by injection) are generally unhelpful because most humans are already producing sufficient leptin. Increased body weight is often due to other factors.
2. Orally ingested leptin is broken down by stomach HCl or proteases in the digestive tract.

Type 2 Diabetes Progression

Early Stage: Pancreatic beta cells release more insulin until they are exhausted.

Intermediate Stage: Metabolic syndrome (syndx), high LDL, glucose, blood pressure, and obesity.

Progression: TAG capacity is reached, tissues transform to a pro-inflammatory state, secreting MCP1 which attracts macrophages. TNF alpha is produced, promoting TAG breakdown and release of fatty acids. Fatty acids taken to muscle tissue form ectopic lipid deposits, inhibiting Glut4 (which normally brings in glucose).

Type 2 Diabetes Treatments

• Weight Loss: Reduces TAG.
• Exercise and Metformin: Activate AMPK, increasing the AMP to ATP ratio.
• Sulfonylureas: Increase insulin secretion from pancreatic beta cells.
• Thiazolidinediones: Stimulate PPARgamma, increasing glucose uptake and TAG storage via PEPCK.
• GLP-1 Agonists: Similar effect to sulfonylureas.
• Gastric Bypass: Bypasses the majority of the stomach and duodenum.

Other Notes

Amphotericin B (AmphB): Antifungal and treatment for visceral leishmaniasis (enlarged liver, spleen, fever, night sweats, cough, weakness).

Fatty Acids: Stearic acid (C18:0), Oleic acid (C18:1-cisΔ9), Palmitic acid (C16:0). Lipid transition temperatures: DOPC=-17, DPPC=41, DSPC=55. Cholesterol broadens Tc.

Lipid Structures

Micelles: A > B. Bilayer: A = B. H₂ phase (inverted hexagonal phase): A < B - helps in destabilizing membranes (e.g., DOPE, a helper lipid).

P-serine: Provides a negative charge on liposomes, typically we want neutral.

Liposome Properties and Targeting

PEG: Prevents aggregation of lipids and opsonins (cb3-liver) from binding to the liposome, and stops clearance of liposomes from the bloodstream. Targeting ligands (small molecules or antibodies) can be coupled to the PEG-lipid for tissue-specific targeting. For example, PSMA-targeting PEG-lipid targets “prostate-specific membrane antigen” on prostate cancer cells. High rate of lipid exchange = membrane destabilization.

HDL: Can cause leakage via exchange of apolipoprotein A1. At tumor sites, it enters via leaky vasculature and releases its payload.

Liposome Preparation and Drug Loading

Rotovap: Evaporates organic solvent, leaving lipid in a round-bottom flask (RBF). MLV to SUV/LUV: sonication or extrusion (polycarbonate membrane). Use dynamic light scattering to check size.

Loading into liposomes:

1. Weakly basic drugs: Hydrate with aqueous buffer, then extrude. This gives an internal pH of 4. Use a dialysis bag to change the outer pH to 7.4, leading to a neutral drug that diffuses in and is then protonated.
2. Ionophore: Use nigericin to move H+ in and K+ out.
3. Ammonium Sulfate: Hydrate with ammonium sulfate, resulting in a high pH (8) inside, then dialyze with 7.4.

Liposome Characterization

EGGPC: C18:0, C18cis9. HSPC: 2C18:0. Ciprofloxacin cannot crystallize, so it is not good for drug retention (fix with freezing and thawing). Determine [drug] non-exchange using 3H-CHE with a scintillation counter. Doxorubicin is fluorescent at 590nm. D/L = [doxo]/[lip. in liposome]. Study pharmacokinetics of doxorubicin (how much drug is released) using AUC. Vd (volume of distribution): high = moves into peripheral tissue, low = stays in blood. Clearance of drug = rate (mg/h) / [plasma] (L). Tumor accumulation = amount of drug (ug) / mass of tumor (g) - due to doxorubicin fluorescence and 3H-CHE (liposome), then calculate TA.