Ammonium sulfate gradient loading of weakly basic amphiphilic small molecules.

摘要

The purpose of my research project is to understand how trans-membrane ammonium sulfate gradients in liposomes produce efficient remote loading of weakly basic amphiphilic small molecules. To understand the effect of pre-loaded ammonium ions, I have developed the following mathematical model to describe loading: 1F=1+do rugH+ 0NIH +4 0Where INH4 +0 is the mol of ammonium ions in the liposome compartment prior to loading, odrugH +0 is the mol of protonated drug in the external compartment prior to loading, and F is the fraction of protonated drug incorporated into liposomes by loading. The model provides a way to estimate the optimal loading efficiency using the ammonium sulfate gradient technique, and should apply to all weak bases that are soluble as sulfate salts. When INH4 +0 is experimentally estimated, a plot of odrugH+ 0 vs 1/F for a series of loading experiments using different amounts of drug should yield a straight-line plot, whose slope is 1/INH4 +0 and intercept is 1. This model was proved to be valid for loading of egg PC/cholesterol liposomes preparations with hydromorphone, oxymorphone, lidocaine, and ketamine.Osmotic gradients that lead to liposome swelling can cause suboptimal loading, because ammonium ions leak from the liposomes through swelling and rupture. For hydromorphone and oxymorphone, this occurs for large uni-lamellar liposomes, not for multi-lamellar liposomes. For more hydrophobic drugs such as lidocaine and ketamine, this occurs also for multi-lamellar liposomes. Ketamine hydrophobicity leads to suboptimal loading of LUV even when no osmotic gradient is present.Hydromorphone loading of DPPC/Cholesterol liposomes also follows the model. For DPPC/cholesterol liposomes, loading is optimal if allowed to proceed for I hour at 55°C, or for 6 hours at 25°C. DPPC/cholesterol LUV are resistant to osmotic pressure when loaded below the Tm, and thus provide a technique to reach optimal loading in the presence of external osmotic pressure. Following this observation, the ammonium sulfate content of DPPC/cholesterol liposomes was increased to 1.5 M, and loading efficiency for 40 mg hydromorphone was raised to over 67%.