J. DRUG DEL.SCt. TECH.,21 (6) 469-477 2011 Cellular uptake of liposomes monitored by confocal microscopy and flow cytometry E. Ducatr, B. Evrardr, O. Peulen2, G. Pielt* rLaboratory of Pharmaceutical Technology, CIRM, Department of Pharmacy, University of Liège, 836, Tour 4, Level 2, Avenuede I'Hôpital, I, 4000 Liège, Belgium rMetastasis Research Laboratory, GIGA-Cancer, University of Liège, Pathology building, B23,Level4,4000 Liège, Belgium *Correspondence: geraldine.piel @ulg.ac.be For several years, two advanced techniques, confocal laser scanning microscopy (CLSM) and flow cytometry, in particular fluorescence- .ittivated cell sorting (FACS) have been used more and more to study the cellular uptake of liposomal drug delivery systems. Thesetechniques :,rovide new potential to locali:e carriers in cells and quantifl the amount of liposomal uptake, leading to essentialinformation on the interaction .ent'een the formulation and the target cell. A better understanding of the underlying mechanismbehavior of liposomes in biological s-r'sterns is t\sential when adapting the liposomalformulation in order to improve carrier effectiveness.The present review describesthese wo techniques and their use in liposomalresearch. Keywords: Liposome - Cellular uptake - Confocal microscop\ - Flow cvtometry. Liposomes, spherical structurescomposed of one or several phospholipidbilayers, possess many attractivecharacteristics that stabilize and improve the pharmacological properties of drugs. Be- in,e biocompatible and biodegradable,liposomes cause either very mild or no antigenic, pyrogenic, allergicor toxic reactions. They can Èntrap hydrophilic drugs within theiraqueous compartment. lipophilic compoundsin their membraneor amphipathicdrugs. First generation liposomes have been shown to be easily eliminated from the blood- stream andto accumulate in the Kupffer cellsin the liver and in spleen macrophages [1]. Therefore, the strategy has been to graft polymers onto liposomal phospholipids.The mostwidely used polymers forthis application are polyethyleneglycols (PEGs). PEGs are synthetic and inert biocompatible polymers that allow the formation of a protective layeron the particle surface and provide protection against opsoniza- tion andcapture by the RES [2,3]. Moreover, the liposome surface can be modified with certain specific ligands (such as antibodies,sugars, peptides, etc.). This activetargeting allows control of their biological properties and theirbiological behavior [4-7]. To improve in situand/or intracellular delivery, several types of liposomes havebeen developed suchas pH, light or temperature-sensitive liposomes [8]. Fourmechanisms of liposome-cell interaction by which liposomes deliver their content in cells can occur: i) adsorption followed by extracellular release of liposome content; ii) endocytosis clathrin-(in) dependent; iii) lipid exchange by transferof lipophilic compounds from the liposomalbilayer to the cell membrane and iv) fusion with the intracellular membrane [9]. Mechanisms of liposome-cell interaction vary greatly as a func- tion of the type and the composition of the formulation but also as a function of the type of cell and cell cycld [0]. For example, Papahadjopoulos et al. observed that fusion could occur between the cellularand the liposomal membrane when liposomes areconstituted of purephosphatidylserine, but not when liposomes areconstituted of pure phosphatidylcholine [ ], l2]. On the other hand, Bergstrand er a/. observed that pH-sensitive liposomes containingI,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE) do not interactorfuse readily with endosomal model membranes [8]. For their experiments, they usedendosome-like liposomes composed of a mixture of phosphati- dylcholine, DOPE, sphingomyelin and cholesterol. In composition, these liposomes resemble those in endosomal membranes. Moreover, the presence of PEGs on the liposome surface can often hamper the interactionbetweenliposomesand cell membranes [3]. This also occurswhen cholesterol is present [11]. The study of the cellular uptakeof liposomes providesessential information about the formulation developed. The localization of the carrierand of its encapsulated materialin the cell can be evaluated as well as the quantity of liposomeinternalized. The type of liposome- cell interaction mechanism can also be investigated. Cellular uptake studies allow betterunderstanding of the mechanisms of interaction between liposomes and biological systems and allow the adaptation of liposomalformulation in order to enhance effectiveness. Herein, we focuson two techniques widely used for several years to evaluate andstudy theintracellularfate ofliposomes and theircontent: confocal laserscanning microscopy (CLSM) and flow cytometry (FCM). I. CONFOCAL LASER SCANNING MICROSCOPY Confocal laser scanning microscopy(CLSM) has beenrvidely used for a number of yearsby researchers in the field of liposomesto study the cellular uptakeof theselipid vesicles and of their encapsulated material. 1. CLSM principle - gainoverconventional f luorescence mlcroscopy CLSM allows the acquisitionof in-focus imagesfrom selected depths usingthe process of optical sectioning, which eliminates arte- factsoccurring during physical sectioning and fluorescent staining of tissue specimens fortraditional forms of microscopy. Optical sectioning is a non-invasive methodof image collection,using light to section the specimen. Two techniques of optical sectioning can be achieved: deconvolution and multiphotonimaging.Deconvolution occursafter image acquisition and usesnearest-neighbor algorithmsin order to extractinformation out of bluned images. Multiphoton imaging uses two long-wavelength photons thatmustarrivesimultaneously in order to excitethe fluorochrome. CLSM is built around a conventional light microscope, using alaseras a light source andsensitive photomultiplier tubedetectors (PMT) placed behinda pinholeto detect pointsof light from the specimen. PMT produce a signal directly proportional to the brightness of the light and then a computerbuilds imagesusing the informationoutput acquired from PMT (Figure 1) tl4l. 469