Calcein release

Fig. 7. Ultrasound-triggered release of calcein and air from acoustically active liposomes composed of EggPC DPPE DPPG CH at molar ratio of 69 8 8 15 including 4% DHPC. Mean SD, n=6. Internal control non-acoustically active liposomes were evaluated for calcein release. Reproduced with permission from (3)...

Fig. 3.5 Total calcein release from 1,2-O-SPC target liposomes 20 min after addition of 10 pm PA and lyso-PPC separately or simultaneously in an equimolar mixture. Concentration of the target liposomes was 20 pm in a HEPES buffer (pH = 7.5), temperature = 39°C.

Temperature has a dramatic and highly non-trivial effect on SPLA2 activation in the region of the main phase transition of saturated phospholipid bilayers [17, 19] (Fig. 3.2). As noted above, this is caused by dramatic lateral structural changes in the lipid bilayer [28]. It is possible to take advantage of this physical effect as a thermally activated release trigger mechanism in the biophysical drug-delivery model system, as illustrated by the data displayed in Fig. 3.6. As the temperature approaches the main phase transition temperature at 41 C of the DPPC Upid bilayer, the rate of calcein release is dramatically enhanced as quantified by the time of 50% calcein release (insert in Fig. 3.6). 

Fig. 3.6 Temperature-dependent SPLA2 (A. pisciuorus piscimms) controlled permeation of calcein across 1,2-di-O-SPC target liposomes as a function of time at different temperatures. As the temperature is raised, calcein release is enhanced due to an increased activity of the enzyme induced by structural...

Ollila et al. (2002) studied the interaction between flavonoids and membranes composed of di-palmitoylphosphatidylcholine by means of nonco-valent immobilized artificial membrane chromatography and flavonoid-induced calcein release from fluid egg phosphatidylcholine vesicles. Flavonoids with more hydroxyl groups showed longer retention delays in the immobilized artificial membrane studies, suggesting stronger interactions between the flavonoids, which are rich in hydroxyl groups, and the dipalmitoylphosphatidylcholine membrane interface. Both polar and nonpolar forces were shown to have a significant impact on the flavonoid-biomembrane interactions. 

Abstract When the vesicle suspension of HCO-10 was heated to 40-45 °C and then cooled to room temperature, larger vesicles were produced, which not only encapsulated an increased fraction of the solutes but also was narrow in size distribution. The improvement of their size uniformity was dependent on the rate of cooling. During the heating and cooling of HCO-10 suspensions, the transformation process of the vesicles was investigated by observing the freeze fracture electron microscopic appearances and the temperature dependence of the calcein release from those vesicles. It was found that... 

Key words HCO-10 vesicles - heating-cooling cycle - high entrapment efHciency - narrow size distribution - calcein release... 

The calcein release from the vesicles was measured as follows the vesicle suspensions of HCO-10 were prepared in 100 mM calcein/20mM Tris buffer (pH 7.4, 388 mOsm), in which the fluorescence intensity of calcein is self-quenched. The vesicles were separated from un-trapped calcein by gel-permeation chromatograph using a Sephadex G-75 gel (0.5 x10 cm column) equilibrated with an isotonic buffer, glucose/20mM Tris buffer (pH 7.4). The osmolarity of buffers was monitored with an Osmometer (Semi-micro Osmometer, Knauer). The separated vesicles, suspended in a cold isotonic buffer, were rapidly diluted (1 200) into the well-stirred isotonic buffer equilibrated to the experimental temperature. The fluorescence emission intensity of a sample was recorded continuously subsequent to this dilution (zero time) on a RF-5000 fluorescence spectrophotometer, Shimadzu (excitation A = 490 nm, emission X = 520 nm) equipped with temperature control accessories and a magnetic stirrer [11]. 

Fig. 5 Temperature dependence of the calcein release from HCO-10 vesicles...

Our laboratory mostly works with PEG-cholesterol. It is easily obtained in one step by addition of cholesteryl chloroformate and amino-methoxy-PEG (31). Introduction of a linker between the cholesterol and the PEG part would induce higher membrane fluidity and reduce more efficiently protein interactions as compared to PEG-cholesterol. A diaminobutane spacer was shown to improve significantly the sustained release of calcein from lipoplexes incubated in 30% serum (32). The spacer effect on bicatenar PEG-lipid has not been intensively studied because it can be expected that it would induce less effect on PEG-dioleoyl than on PEG-cholesterol, the lipidic anchor being predominant in the bilayer stabilization (Fig. 2). 

Membrane permeabilization activity of peptides is currently measured by the use of artificial membrane bilayers, such as liposomes or erythrocytes. The hposome leakage assay can be performed by using spectrofluorimetry with a concentration-dependent quenching of a dye (calcein, carboxyfluorescein) encapsulated in liposomes. Disruption of hposomes in the presence of peptide-inducing leakage will lead to an increase in the fluorescence intensity of the liposome solution. Erythrocyte lysis assay is based on the absorption of hemoglobin, which can be measured once released into the extracellular medium upon erythrocyte lysis in the presence of peptide. 

FIGURE 13.5 (a) Visualization of a typical multilamellar ethosome containing 2% PL, 30% ethanol, and water by TEM (b) Entrapment of fluorescent probes by phopholipid vesicles as visualized by CSLM. Liposomes (a-c) or ethosomes (d-f) were prepared with one of three fluorescent probes rhodamine red (a, d), D-289 (b, e), or calceine (c, f). White represents the highest concentration of probe. (Reproduced from Touitou, E. et al., J. Control. Release, 65, 403, 2000. With permission from Elsevier.)... 

Tokudome, Y., and K. Sugibayashi. 2003. The synergic effects of various electrolytes and electroporation on the in vitro skin permeation of calcein. J Control Release 92 (l-2) 93. 

Bidleman et al. (2JD) used a palladium chloride-calcein chelate spray for organothiophosphorus compounds and detected 10 ng per spot. The mechanism is based on the release of the ligand because of the affinity of palladium ions for sulfur atoms. The technique does not necessitate bromine vapours and is therefore an improvement in terms of selectivity. Unfortunately, the reaction is slow and the fluorescence increases with time. 

Add lOpL 10% triton X-100 to lyse the liposomes, allowing the release of entrapped calcein. Measure the background fluoresence at zero encapsulated volume. Measure the fluorescence (E jq) after lysing liposomes. 

Calculate the amount of released calcein upon ultrasound application. 

Co combines with calcein to form a chelate that does not have fluoresence in the neutral pH range. However, in the presence of EDTA, a stronger Co chelator, calcein is released and can be readily detected at very low concentrations. Therefore, the inclusion of EDTA or other strong chelators in these measurements will cause interfere to the quenching effect of Co. ... 

In order to evaluate the membrane integrity of the different ARSL types, the release of vesicle-encapsulated 5,6-carboxyfluorescein (9) or calcein (10, 11), has been measured. 

The leakage of small water-soluble dyes encapsulated in the aqueous interior of liposomes during their preparation is often used as a method to study their membrane integrity during incubation under various conditions (temperature, pH, presence of serum proteins, etc.). In the case of ARSL s, the release of CF or calcein has been used as a measure of the vesicle membrane integrity, during incubation of ARSL in buffer or in presence of serum proteins [80% FCS] at 37°C under mild agitation. Calcein (or CF) is encapsulated in the vesicles in a quenched concentration (100 mM), and, therefore, its release from the membrane can be calculated without separation of free and liposomal dye, as reported before (23). In brief, 20 pL of the incubated ARSL dispersion are drawn out from each incubation tube and diluted with 4 mL of PBS, pH 7.40. The fluorescence intensity of the samples is then measured (EM 490 nm, EX 520 nm, slit-slit 10-10), before and after the addition of Triton X-100 at a final... 

For calcein (or any drug that is sensitive to traces of heavy metals), 10 mM EDTA (ethylenedinitrilotetraacetic acid disodium salt) should be included within the buffer in order to complexate traces of cobalt ions, which are released from the stainless steel material of the homogenizer during processing. Alternatively, a valve made of ceramic may be used. 

Incubate the cuvettes at 37°C for 10 min and measure the cal-cein fluorescence intensity again. Subtract 1 fi om the intensity value evaluated after 10 min incubation of liposomes to obtain the fluorescence of the released calcein at different pH (7 )... 

---