Timothy Troutman and Marek Romanowski PhD Candidate Biomedical Engineering GIDP Biomedical Optics (BIOMED) Topical Meeting and Tabletop Exhibit St. Petersburg, Florida March 16 to 19, 2008 "Biodegradable Nanoshells for Optical Contrast and Controlled Release"

Liposome-supported arrays of gold nanodots exhibit plasmon resonance, making them useful as contrast agents in optical imaging techniques and degradable to components of clearable size. Encapsulated volume can be released with high energy incident light.

1. Introduction
The use of exogenous contrast agents for in vivo imaging is precluded by the fact that such agents must be cleared from the body in a timely manner. Size and material limitations prevent the clinical use of many presently developed scattering-based optical contrast agents. The generation of plasmon resonance in the near infrared imaging window requires the use of particle sizes that are not readily cleared from the body. One solution to such a problem is to fabricate plasmon resonant structures that can be degraded so that following use, the breakdown of the nanoparticle yields products within the range of sizes readily cleared by the kidney. A means of accomplishing this involves the generation of nanoparticles with a metastable core, but also the metallic components necessary for generating plasmon resonance must be discrete particles of a size on the order of angstroms or single nanometers rather than continuous shells. (Figure 1) We have developed such a composite that is based upon a liposome template with ionic gold reduced to form nanodots that are coordinated to charged surfaces on the hydrophilic perimeter of the liposome. Individual metal atom coordination to functional groups has been characterized. This individual atom, once coordinated to a single charged phospholipid can serve as a nucleation site for further reduction. These biodegradable nanoshells are shown to be susceptible to several types of degradation including dissolution by surfactant, and enzyme induced breakdown. In addition, the structure of the coated liposome can be disturbed to release its contents with incident high energy light.

Fig. 1 Assembly and degradation of biodegradable nanoshells.

2. Model
As in the case of other plasmon resonant particles, the resonance peak of gold-coated liposomes can be tuned in the near infrared. The nature of this tunability is different from that of any previously described structure, particularly that of silica-based nanoshells. With increased gold concentration, the resonance peak shifts further into the infrared; a process that can be described by a model derived from Kerker’s concentric spheres approximation, and the effective medium theory. This model indicates that with increased density of gold particles in the outer layer of the liposome, the dielectric constant of the shell-layer will change, resulting in a shift of the plasmon resonance maxima.

3. Characterization
Electron Microscopy was used to image the intact liposomes to determine whether reduced gold nanodots were resolvable with high magnification field emission electron microscopy. Individual nanodots of gold were not observable at a magnification of 800,000x and a pixel resolution of 0.125 nm. Energy Dispersive X-ray Spectroscopy (EDS) maps indicated that gold was indeed present on the surface of the liposomes.

Dynamic light scattering was used to determine the actual size of extruded liposomes and their degradation products. Liposomes extruded through a 50 nm pore size polycarbonate membrane were found to have an average diameter of 63 nm, and degradation products following dissolution by Triton X-100 containing surfactant, lipids and nanodots had an average size of 5.7 nm.

These new materials exhibit scattering characteristics that generate signal in Optical Coherence Tomography (OCT) similar to that observed in other types of plasmon resonant particles [5,6]. For characterization, we used a time-domain OCT system with a center wavelength of 890 nm and bandwidth of 150 nm. In the images generated by this system the biodegradable nanoshells were observed to yield an intensity increase of 7.2 dB vs. uncoated liposomes at the same lipid concentration, and 9.3 dB vs. PBS when the plasmon resonance maxima was matched to the source output wavelength. (Figure 2)

Fig. 2 OCT image of capillary tubes filled with non-coated liposomes, biodegradable nanoshells, triton treated biodegradable nanoshells, PLA2 treated biodegradable nanoshells, and phosphate-buffered saline (from left to right).

4. Degradation and Controlled Release
Degradation of the biodegradable nanoshells was mediated through two means, the first was dissolution with the surfactant Triton X-100, the other was a more physiological-based assay in which phospholipase A2 (PLA2) was used to directly cleave the acyl tails of the phospholipids comprising the liposomal scaffold. Degradation was monitored by observing the loss of plasmon resonance. We hypothesized that the resonance is dependent on the integrity of the liposome. This was verified thorough the findings of dynamic light scattering and the concomitant loss of plasmon resonance with incubation with both Triton and PLA2. (Figure 3)

Fig. 2 OCT image of capillary tubes filled with non-coated liposomes, biodegradable nanoshells, triton treated biodegradable nanoshells, PLA2 treated biodegradable nanoshells, and phosphate-buffered saline (from left to right).

An additional benefit of the use of liposomes as the basis of these plasmon resonant structures is their ability to carry a payload. Liposomes have frequently been the subject of controlled release techniques.Their composition is similar to that of the bilayer membrane of cells making them conducive to biological applications and their metastable nature leads to several means of biologically-induced breakdown and release. In addition to the imaging function of these nanoparticles, the ability to encapsulate and retain a payload in an aqueous environment is particularly useful. This may enable the use of multi-modal imaging using a single contrast agent, or as we propose here, the light-induced delivery of contents. This could open the door to a new type of phototherapy. Presently, we report the release of contents with a high-power pulsed femtosecond laser more commonly used to induce multi-photon excited luminescence. With an energy input slightly higher than that used when imaging the agent, we are capable of bursting the liposome and releasing its contents.

>5. Conclusion
The biodegradable nanoshells we have investigated and revealed here can fulfill the present role of similar plasmon resonant materials for imaging applications while meeting the need of biodegradation necessary for clinical in vivo use as well as adding new functionality. This composite represents a major step toward the development of scattering-based contrast agents that are useful for clinical imaging and may still be readily cleared from the body. The possibility of targeting such agents is the next step toward making a device that can be used to locate target cells for identification as well as to administer a local dose of encapsulated medication or dye while preventing systemic exposure.