Aqueous phase, intraliposome

The octanol/buffer represents a partition coefficient between two bulk phases it is less affected by the structure of the analyte and therefore it cannot be used to predict the exact value of liposome membrane-to-buffer Xp, which is also affected by the geometry of the analyte (41 4). However, it is accepted and established that the octanol-to-buffer can help to predict transmembrane passive diffusion (40). In the case of liposomes such as Doxil, in which the internal aqueous phase (intraliposome aqueous phase) is different from the external liposome aqueous medium due to large differences in the composition and pH of these two aqueous phases, there are two different liposome membrane-to-aqueous phase partition coefficients this is referred to as asymmetry in the membrane-to-aqueous media partition coefficient. 

Namely, the intraliposome aqueous phase becomes acidic. 

The role of the ammonium salt anion is not the loading of the amphipathic weak base per se, but rather to control the stability of loading and the profile and rate of release of the amphipathic weak base from the liposome to the external aqueous phase. Two major factors that differentiate the different anions are, firstly, their ability to induce precipitation/crystallization/ gelation in the intraliposome aqueous phase (1,12), and secondly, their effect on the membrane/buffer and octanol/buffer partition coefficient of the amphipathic weak base (1). Regarding the precipitation, the higher the amount of precipitated amphipathic weak base, the more stable is the loading and the slower is its release rate (10-12,18,33,35) and (Wasserman et al.). There are also some risks involved in the precipitation which in some cases reduce the mechanical stability of the liposomes and change liposome shape (36). 

The extraliposomal ammonium ion concentration [(NH4)jJ yj ] was measured as ammonia with the ammonia electrode at pH 13.5. Under these conditions, all ammonium ions are converted to ammonia and no leakage of intraliposomal ammonium ion occurred during the measurement. For measurements of total ammonium ion plus ammonia present in both intraliposome aqueous phase and external medium ammonia [(NH4)jJ g4i y +(NH4)Jpg j ] the liposomes were sonicated under acidic conditions (pH 1.5-2.0) using the Transonic 460/H bath sonicator in sealed vials for 45 minutes. Then, in order to convert ammonium ion to ammonia, NaOH was added to bring the pH to... 

Use of the pH-sensitive membrane-impermeable flurophore pyranine based on the ratiometric method, which determines directly level of dissociation of pyranine from the ratio between the charged (nnprotonated) pyranine and total pyranine in the intraliposome aqneons phase Addition of impermeable DPX, which acts as a quencher to pyranine fluorescence, into the liposome external medium ensures lack of contribution of extraliposome medium pyranine fluorescence (18,22). This method is considered invasive as the pyranine has to be added in the hydration medium prior to liposome preparation and cannot be used for pH determination of intraliposome aqueous phase... 

In the ratiometric method, the fluorescence intensity of the liposomes containing pyranine (F) and in the presence of the quencher DPX was determined at 520 nm upon excitation at two wavelengths 460 nm (of the charged unprotonated pyranine) and 415 nm (of the pH-independent isosbestic wavelength that describe the total pyranine concentration). The ratio of is described as F. The ratiometric measurement is used to determine the intraliposome aqueous phase pH (18,22). Then nigericin (or nonactine) at final concentration of 5pM was added to disrupt the pH and/or ammonium ion gradient that induce complete gradient collapse and the measurement at the above two excitations was repeated, and indeed it demonstrated a shift of the intraliposome aqueous pH to be identical to the extraliposome medium pH (10). 

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