Christopher G. Morgan

Active Uptake of Drugs into Photosensitive Liposomes and Rapid Release on UV Photolysis

Abstract Liposomes containing high concentrations of the anticancer drug doxorubicin, prepared by active-loading techniques, have been intensively investigated as potential agents for chemotherapy. The present study investigates the possibility of active uptake and photoinduced release of such solutes from liposomes incorporating a photoisomerizable lipid. The active loading of acridine orange and doxorubicin was investigated using liposomes containing entrapped ammonium sulfate. The liposomes were prepared with dipalmitoyl-l-α-phosphatidyl choline (DPPC) and a photochromic lipid, (1,2-(4′-n-butylphenyl)azo-4′-(γ-phenylbutyroyl))-glycero-3-phosphocholine (Bis-Azo PC), which isomerizes on exposure to near-UV light with resulting changes in membrane permeability to solutes. The rate of loading of the vesicles below the phase transition temperature of DPPC was investigated as a function of Bis-Azo PC and cholesterol concentrations in the liposome. The rate of doxorubicin uptake was found to be greatly decreased in the presence of cholesterol, while below 30°C the rate of acridine orange uptake was increased in the presence of cholesterol. On exposure to a single UV laser pulse, actively loaded acridine orange was rapidly released from liposomes containing Bis-Azo PC at a rate similar to that found for the indicator dye calcein. However while cholesterol had previously been shown to greatly enhance the rate of photoinduced calcein leakage, it had no significant effect on the rate of acridine orange release. After active loading into DPPC vesicles containing Bis-Azo PC, doxorubicin was also released after exposure to a single laser pulse, but at a rate slower than for acridine orange and calcein. The difference in behavior between these systems is ascribed to the interactions of acridine orange and doxorubicin with the liposome bilayer. Photoinduced release of pharmacologically active materials from sensitized liposomes might provide a useful adjunct or alternative to conventional photodynamic therapy.

Direct modulation of the effective sensitivity of a CCD detector: a new approach to time-resolved fluorescence imaging

In this paper a novel approach to frequency‐domain fluorescence lifetime imaging (FLIM) is described. In a CCD camera a single pixel is defined by a charge pattern on a group of electrodes. By modulation of the pattern of voltages defining the pixel structure it is possible to modulate the sensitivity of the CCD at radio frequency. The modulation enhances the noise performance of the CCD, in contrast to the deterioration in performance seen when an intensifier stage is similarly modulated. The new technology has potential applications to a wide range of assays as well as in conventional FLIM applications. Unlike intensifier‐based systems, the directly modulated CCD is physically small, inexpensive, robust and offers superior resolution and noise performance.

Measurement of nanosecond time-resolved fluorescence with a directly gated interline CCD camera.

CCD cameras coupled optically to gated image intensifiers have been used for fast time‐resolved measurements for some years. Image intensifiers have disadvantages, however, and for some applications it would be better if the image sensor could be gated directly at high speed. Control of the ‘charge drain’ function on an interline‐transfer CCD allows the sensor to be switched rapidly from an insensitive state. The temporal and spatial properties of the charge drain are explored in the present paper and it is shown that nanosecond time resolution with acceptable spatial uniformity can be achieved for a small commercial sensor. A fluorescence lifetime imaging system is demonstrated, based on a repetitively pulsed laser excitation source synchronized to the CCD control circuitry via a programmable delay unit.

Prospects for confocal imaging based on nanosecond fluorescence decay time

A fluorescence microscope has been developed to give contrast on the basis of fluorescence decay time. Two different detection schemes are used, one based on an imaging single photon detector equipped with an external photon correlator and the other using an RF‐modulated intensified CCD camera. Examples of the new contrast are shown, and the possible routes to development of a confocal version of the microscope are discussed.

The use of a phospholipid analogue of diphenyl-1,3,5-hexatriene to study melittin-induced fusion of small unilamellar phospholipid vesicles

A phospholipid analogue incorporating the diphenyl-1,3,5-hexatriene (DPH) chromophore has been synthesized. The compound has been shown to have similar fluorescence properties to DPH itself but, unlike DPH, is unable to exchange freely through solution when incorporated as probe in a subset of phospholipid vesicles of given composition. The non-exchangeability of this probe has been exploited to study the fusion of phospholipid vesicles to form larger structures. The peptide melittin was used to initiate fusion, and it was shown that vesicles which had been induced to fuse by heating in the presence of melittin would not fuse with subsequently added vesicles.

Fast solute release from photosensitive liposomes: an alternative to ‘caged’ reagents for use in biological systems

The kinetics of release of soluble marker trapped in liposomes of gel phase phospholipid containing a photoisomerisable phospholipid analogue have been investigated. Marker release is triggered by UV laser flash photolysis at 355 nm. A markedly temperature‐dependent release rate is seen, and above 25°C millisecond release kinetics can be achieved. These results suggest that such liposomes might find application as an alternative to conventional ‘caged’ reagents for photo‐triggered reagent release in biological research.

The formation and Langmuir-Blodgett deposition of monolayers of novel photochromic azobenzene-containing phospholipid molecules

Abstract A group of novel phospholipid molecules has been synthesised, in which one or both acyl chains has been replaced with an azobenzene-containing acid. These lipids photochromic, isomerising from the trans form in which the azobenzene unit is almost linear to a cis form with a bent chain. The influence of this isomerisation on the properties of monolayers of these lipids on an aqueous subphase has been investigated using the Langmuir trough. The lipids are shown to form compact monolayers when in the trans form, but to have greater molecular areas after isomerisation. All lipids could be transferred to a glass substrate by Langmuir-Blodgett deposition. Evidence suggestive of phase equilibria is seen for surface monolayers of one of the lipids, which also shows evidence of a temperature-dependent phase transition by light scattering when dispersed as vesicles in water. Marked hysteresis is seen between cycles of compression and expansion for surface monolayers of azo-lipids before and after photoisomerisation.

Photosensitive liposomes as ‘cages’ for laser‐triggered solute delivery: the effect of bilayer cholesterol on kinetics of solute release

Liposomes containing acyl chains incorporating azobenzene chromophores have been investigated as potential ‘caging’ agents for fast solute release. On photolysis, trapped marker dye can be released from gel‐phase liposomes within milliseconds. Solute release is markedly sensitive to the presence of cholesterol in the bilayer. Phospholipids bearing one saturated acyl chain and an azobenzene‐substituted chain are ineffective as sensitisers unless cholesterol is present, while doubly substituted phospholipids sensitise release in its absence. Cholesterol markedly affects the temperature profile of solute release depending on the host phospholipid chain length. Solute release is not seen for lipid hosts with unsaturated acyl chains.

Photobleaching. A novel fluorescence method for diffusion studies in lipid systems

(1) The fluorescent molecular 12(9-anthroyloxy)-stearic acid dimerises on irradiation with light of 366 nm wavelength. (2) The dimer is nonfluorescent and can be reconverted to the parent compound by irradiation at 254 nm. (3) Kinetic analysis suggests that the dimerisation proceeds by a diffusion-limited second order mechanism in many solvents. (4) Anomalously high rates seen in other systems can be attributed to localised high concentration regions (clusters) of the fluorescent molecule. (5) The analysis has been extended to oriented lipid bilayers and the results suggest that below the gel-liquid crystalline transition temperature the 12(9-anthroyloxy)-stearic acid is excluded by the lipid matrix and forms regions of localised high concentration. (6) In fluid lipid the results suggest an isotropic distribution of the probe. Calculated diffusion coefficients correspond to those found by other techniques.

Liposome fusion and lipid exchange on ultraviolet irradiation of liposomes containing a photochromic phospholipid.

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