UV light-induced calcein release from gold nanoparticle-loaded liposomes

Our results indicate that calcein is released from gold nanoparticle-loaded liposomes by a light-triggered mechanism. At 37 °C without UV light, liposomes showed much lower release rates than with irradiation. When liposomes were exposed to 250 nm UV light, calcein release was simultaneously triggered (Figure 5). Photobleaching of calcein during the 20 min UV exposure was not significant.

Low Au-C6SH concentrations in the liposomes had only slight effect on liposomal bilayer integrity at 37°C. The half-live of calcein release without UV light was 2.75 h at 8.6 µg/ml, and 3.3 h at 17.2 µg/ml. At 43 g/ml and 86 g/ml Au-C6SH, the leakage half-lives were 1.1 h and 0.5 h, respectively. It is likely that high gold nanoparticle concentrations in the liposomal bilayer may interfere with the integrity of bilayer, thereby causing non-specific leakage of the encapsulated calcein. However, it was observed that all the liposomes with different Au-C6SH concentrations were able to induce calcein release with 250 nm UV light for 30 min (Figure 5A). It was also clearly seen that without gold particles, UV radiation of liposomes did not induce any calcein release from liposomes, whereas the liposomes with Au-C6SH, Au-MSA or Nanogold^® released calcein upon UV irradiation.

Hydrophilic Au-MSA nanoparticles were encapsulated in liposomes and, interestingly, were also able to induce UV light-triggered calcein release (Figure 5B). A low Au-MSA concentration (8.6 g/ml) did not compromise bilayer integrity at 37 ºC, where the leakage half-life was 2.8 h without UV light. Higher Au-MSA concentrations resulted in faster rates of calcein release upon UV irradiation, but also decreased the leakage half-lives (1 h at 43 g/ml, and 0.6 h at 86 g/ml) during the incubation without UV light. This suggests that there are interactions between the Au-MSA nanoparticles and the liposomal bilayer inner surface. These interactions may be concentration dependent, thus explaining calcein leakage at high MSA concentrations. It is also possible that calcein leakage at the highest Au-MSA concentration was due to altered liposome formation because of the large volume of Au-MSA solution, resulting in decreased organic phase/hydrophilic phase ratio (from 2.5 to

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1.9) in the liposome preparation. Also, Au-MSA particles themselves can escape from the liposomes. Because the heating intensity is inversely proportional to the square of the distance, it is likely that nanoparticles in the bulk phase will be too far from the liposomes to cause any perceivable heating. At some point, most of the Au-MSA particles will have escaped from the liposomes and they will no longer be efficiently heated, leaving some of the calcein still inside the liposomes.

Liposomes with 1000 pmol of commercial DPPE-Nanogold^® released calcein after UV irradiation (Figure 5C). These gold nanoparticles with core diameter of 1.4 nm are covalently attached to the ethanolamine headgroup of the lipid, and thus, they are presumably located on the outer and inner surfaces of the liposomes. Incubation of the samples at 37 °C without UV irradiation caused only minor calcein leakage compared to the Au-C6SH and Au-MPC liposomes. The rate of calcein release upon UV exposure was comparable to liposomes with 86 g/ml of Au-C6SH nanoparticles. Liposomes with 1000 pmol of DPPE without gold nanoparticles were used as a control. They did not show light-induced contents release.

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Figure 5. UV light-induced calcein release from liposomes at constant temperature (37°C) with different types of loaded nanoparticles: A) Au-C6SH, B) Au-MSA and C) Nanogold^®.

81 6.4 Discussion

Light-induced release of contents from gold nanoparticle-loaded liposomes was demonstrated. Liposomes remained intact at 37°C but released calcein upon UV light irradiation. Triggered release of calcein was more pronounced with hydrophobic Au-C6SH and DPPE-Nanogold^® than with hydrophilic Au-MSA nanoparticles (Figure 5) giving over a 100-fold increase in the release rate compared to plain liposomes (Figure 6). Gold nanoparticles work as localized heat sources by absorbing the energy of UV radiation and transferring it as heat to the surrounding environment. The permeability of the bilayer is increased by the separation of gel and fluid phases (Needham, Dewhirst 2001) and calcein can escape from the liposomes. Since the bilayer-associated Au-C6SH and Nanogold^® particles are in direct contact with the lipids, heat is conducted more efficiently to the lipid molecules, thereby inducing the phase transition and consequent calcein release. Light-induced heating by nanoparticles is presumably extremely localized and therefore Au-MSA particles located in the core of the liposome do little to heat the liposomes, whereas particles close or attached to the bilayer are much more efficient in this respect. Furthermore, it was recently reported that integration of gold nanoparticles to DPPC bilayers also increases the fluidity of the membranes above their gel-to-liquid crystalline transition temperature (Park et al. 2006).

Figure 6. The slope of calcein release curve (k) from UV light-irradiated Au-C6SH liposomes at various gold nanoparticle concentrations at 37 °C.

This study demonstrated for the first time the use of gold nanoparticles as energy collectors for triggering the selective contents release from gold nanoparticle containing liposomes. UV light was used here to demonstrate the proof of principle. The same approach

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can be further modified by using different lipid compositions or different light signals in terms of the wavelength, duration of the pulse, and the light intensity. Calcein leakage half-lives from gold nanoparticle-loaded liposomes were relatively low without UV irradiation. It is possible that there is a threshold concentration which is able to induce the contents release with UV irradiation. Decreasing the concentration to the threshold value would allow UV-triggered release while causing minimal non-specific leakage. The presence of serum proteins presumably increases unwanted leaking, so liposome composition needs to be further developed for in vivo applications. The blood circulation time could be extended by e.g.

incorporation of PEG coating to the liposomes. Heating of liposomes is highly selective, and no heating of bulk solution, as with “classical” heat sensitive liposomes, is needed. This is highly advantageous as it makes the delivery mechanism more compatible with living tissues, where one must be careful not to affect the structure and activity of the cell membranes. The approach could also be extended to other materials, like heat-sensitive polymers.

The liposomes with embedded gold nanoparticles could prove useful in controlled release of drugs in specific areas such as the eye and skin, which are easily accessible to light irradiation. Deeper tissues of the body could be reached by adjusting the wavelength of the triggering light. The method could also be adapted to a number of other interesting applications. Nanoparticle modified liposomes could be included in immobilized phases on lab-on-chip technologies and cell arrays, where controlled release of diagnostic substances could be released by laser signal. The temperature range could further be extended to useful regions other than body temperature by modifying the lipid composition, allowing sensor applications for non-biological purposes.

6.5 Conclusions

We demonstrated that the contents release from liposomes with embedded gold nanoparticles can be selectively induced with UV light irradiation, whereas liposomes devoid of gold nanoparticles remained unaffected.

Acknowledgements

This study was funded by the National Technology Agency of Finland (TEKES).

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7 PHOTOSENSITIVE LIPOSOMES WITH EMBEDDED GOLD NANOPARTICLES

In document External signal-activated liposomal drug delivery systems (sivua 78-86)