Pyranine is a pH sensitive probe that was used in this work to test the stability of the pH gradient in LMVs. The maximum absorption wavelengths for the acid (protonated) and the base (unprotonated) forms of pyranine are 405 nm and 450 nm, respectively51 . The fluorescence intensity of pyranine excited at 450 nm is high at pH 7-8 but near background at acidic pH, while the inverse is true for the fluorescence produced by 405 nm excitation52 (link). Ratiometric measurements using an excitation ratio of 450/405 nm are for that reason frequently used to provide information about the pH of a determined solution. This is an advantageous method since it not depends on pyranine concentration and is directly related with pyranine ionization degree.
LMVs/LUVs with 3 mM total lipid concentration were prepared using Hepes or Citrate Phosphate buffer (pH 7.4 or pH 5.0, respectively), as above described. These vesicles contained 0.5 mM pyranine encapsulated35 (link). The following (POPC/SM/Chol ternary mixtures were used: 59.7:26.3:14 (Xlo = 26) and 34:32.7:33.3 (Xlo = 0.83). Liposomes encapsulating pyranine were recovered (after separation through a Sephadex G-25 column) mainly in fractions 3 and 4 (1 mL each). Liposome final concentration was determined by lipid phosphorous analysis50 (link), for the samples prepared in Hepes buffer. The liposomes were then diluted to approximately 0.2 mM lipid concentration in 96 well opaque plates and fluorescence measurements were performed at 24 °C, in a microplate reader (Spectramax Gemini EM), using 405 and 450 nm as the excitation wavelengths and 510 nm as emission wavelength. The auto mix option of the microplate reader was selected to mix the samples 5 seconds before the first read and 3 seconds between reads. To evaluate the stability of the pH gradient in LMVs, the fluorescence measurements were performed during ap. 5 hours. After this time, triton X100 (0.1% (v/v)) was added to the samples in order to obtain the fluorescence intensity induced by an immediate burst of the vesicles. Some of the wells with LMVs (without the addition of triton X100) were left overnight and measurements were also performed next day, to observe if the pH gradient remained stable. No significant changes were observed (data not shown). Samples with acidic and neutral pH both inside and outside the vesicles were prepared to obtain the fluorescence intensity of pyranine at only acidic and neutral conditions (control samples). The stability of the LMVs was evaluated in the absence and presence of Sph (pre-incorporation of 10 mol% Sph and external addition of 10 mol% Sph). In the studies where Sph is externally added, Sph was dissolved in a small volume of absolute ethanol (ethanol was kept below to 1% v/v to prevent vesicle destabilization) and added to lipid vesicle suspensions (the auto mix option of the microplate reader was selected to mix the samples 5 seconds before the first read and 3 seconds between reads). Control experiments were also performed by adding the same volume of ethanol, without Sph.
LMVs/LUVs with 3 mM total lipid concentration were prepared using Hepes or Citrate Phosphate buffer (pH 7.4 or pH 5.0, respectively), as above described. These vesicles contained 0.5 mM pyranine encapsulated35 (link). The following (POPC/SM/Chol ternary mixtures were used: 59.7:26.3:14 (Xlo = 26) and 34:32.7:33.3 (Xlo = 0.83). Liposomes encapsulating pyranine were recovered (after separation through a Sephadex G-25 column) mainly in fractions 3 and 4 (1 mL each). Liposome final concentration was determined by lipid phosphorous analysis50 (link), for the samples prepared in Hepes buffer. The liposomes were then diluted to approximately 0.2 mM lipid concentration in 96 well opaque plates and fluorescence measurements were performed at 24 °C, in a microplate reader (Spectramax Gemini EM), using 405 and 450 nm as the excitation wavelengths and 510 nm as emission wavelength. The auto mix option of the microplate reader was selected to mix the samples 5 seconds before the first read and 3 seconds between reads. To evaluate the stability of the pH gradient in LMVs, the fluorescence measurements were performed during ap. 5 hours. After this time, triton X100 (0.1% (v/v)) was added to the samples in order to obtain the fluorescence intensity induced by an immediate burst of the vesicles. Some of the wells with LMVs (without the addition of triton X100) were left overnight and measurements were also performed next day, to observe if the pH gradient remained stable. No significant changes were observed (data not shown). Samples with acidic and neutral pH both inside and outside the vesicles were prepared to obtain the fluorescence intensity of pyranine at only acidic and neutral conditions (control samples). The stability of the LMVs was evaluated in the absence and presence of Sph (pre-incorporation of 10 mol% Sph and external addition of 10 mol% Sph). In the studies where Sph is externally added, Sph was dissolved in a small volume of absolute ethanol (ethanol was kept below to 1% v/v to prevent vesicle destabilization) and added to lipid vesicle suspensions (the auto mix option of the microplate reader was selected to mix the samples 5 seconds before the first read and 3 seconds between reads). Control experiments were also performed by adding the same volume of ethanol, without Sph.
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