Fluorescence imaging revolutionises drug delivery

Drug delivery systems are revolutionising pharmaceutical industry with new concepts like targeted, intracellular and intracellular-organelle targeted drug delivery systems. At a time like this, an extensive characterisation of these is essential. A study by Rakesh Singh, Komal Gupte and Aditya Pattani

Fluorescence imaging has become an invaluable tool in biomedical research and its power has been extended by recent developments, thus, improving the likelihood of identifying molecules with advantageous properties early on in the discovery cycle. While conventional techniques like transmission electron microscopy provide the highest level of morphologic/spatial resolution, it requires a significant level of technical expertise and time resource to gain informative data. As a result, it is unable to capture dynamic real-time events.

In contrast, fluorescence imaging technology has the capability for real-time quantitative measurements that allow integration of anatomic, functional, and statistical data. Its contributions focus on studies of intracellular trafficking, epithelial function and viral gene delivery systems. This illustrates the capabilities of different modes of fluorescence imaging in addressing questions in cell biology and physiology research and also highlights their applications in pharmaceutical research.

There are three major types of fluorescence imaging. Fluorescence correlation spectroscopy (FCS) is an analysis method that can measure the dynamics of molecular processes from observations of spontaneous microscopic fluctuations in molecular concentration. In wide-field fluorescence microscopy, wide-field microscopes collect emitted fluorescence from the specimen across a large depth of field, including light from outside the focal plane. As a result, the axial resolution or the ability to distinguish between two separate point sources of light through the depth of the sample of the standard wide-field instrument will approximately be not much less than 1Å (Angstrom), even when using an oil immersion objective of high numerical aperture.

However, confocal laser scanning microscopy (CLSM) is by far the most important of the emerging techniques. Here a laser beam passes through the sample and the resultant emerging light is mathematically broken down into 'plane by plane' fluorescence. This means that the images that are out of focus are suppressed and clear images in individual planes are seen, thus enabling clear and unambiguous delineation of data. CLSM uses various techniques like fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), photoactivation, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging, and total internal reflection fluorescence microscopy.

Application in drug delivery

When it comes to application in novel drug delivery systems, fluorescence microscopy provides important information regarding interactions between nanoparticulate drugs carriers such as liposomes and micelles with target cells, as well as their intracellular fate. The enhanced antibody-mediated targeting of drug-loaded immuno micelles confirmed by fluorescence microscopy resulted in an increased killing of cancer cells compared to free drug or drug-loaded non-targeted micelles. The technology was also used to prove the endosomal escape of properly assembled polymeric micelles containing various additives destabilising the endosomal membrane. When loaded with the anti-cancer drug such micelles demonstrate increased cytotoxicity.

Fluorescence microscopy was also applied to investigate the capture of cell-penetrating TAT peptide-modified liposomes by various cells and stability and intracellular trafficking of captured TAT-liposomes inside cells. It was also used to confirm the successful transfection of cells with TAT-liposomes bearing the plasmid encoding for Green Fluorescent Protein (GFP).

CLSM technique was found to be effective in examining the behaviour of an enhancer in Transdermal Drug Delivery Systems (TDDS) of the drug-in-adhesive type. It was possible to obtain images indicating a homogeneous incorporation of the lauric acid fluorescent probe in the adhesive, to measure diffusion inside the TDDS and release out of the TDDS. The release characteristics measured for the probe were similar to the characteristics for lauric acid measured using conventional methods. And the diffusion inside the TDDS showed good correspondence to the theoretically calculated profiles. Though CLSM technique is considered to be the most efficient, a major difficulty in this area is that the drug delivery scientist needs to be skilled in physics, optics and cell biology, as well as in the chemistry and physics of nanomaterials. However, it is noteworthy that many of the informative studies have exploited experimental approaches coupling CLSM with other imaging (or related) techniques such as that of FRET.

Nevertheless, CLSM has become a prime tool for the analysis of molecular events due to its extreme sensitivity and high spatial resolution. In particular, its application at the cellular level is rapidly developing and should provide a large amount of important and new information. The future is sure to see fluorescence imaging, especially CLSM, which plays a decisive role in drug delivery research.

(The authors are associated with H(S)NCB's College of Pharmacy)