Liposome-labeling methods

Tc-Liposome-Labeling Method Using Hexamethylpropyleneamine Oxime-Glutathione... 

The BMEDA method shares some of the same features as the HMPAO method such as good in vitro and in vivo stability with a variety of preformed liposome formulations, and the need for coencapsulation of glutathione. In addition, an advantage of the BMEDA-labeling method is that it can also be used for labeling liposomes with therapeutic rhenium radionuclides (22). Currently, for the BMEDA method, there is no commercially available kit. Also the BMEDA chemical is not currently commercially available and must be synthesized. 

A planar BLM cannot be investigated by means of the molecular spectroscopical methods because of the small amount of substance in an individual BLM. This disadvantage is removed for liposomes as they can form quite concentrated suspensions. For example, in the application of electron spin resonance (ESR) a spin-labelled phospholipid is incorporated into the liposome membrane this substance can be a phospholipid with, for example, a 2,2,6,6-tetramethylpiperidyl-A-oxide (TEMPO) group ... 

Hwang SH, Maitani Y, Qi XR, Takayama K, Nagai T. Remote loading of diclofenac, insulin and fluorescein isothiocyanate labeled insulin into liposomes by pH and acetate gradient methods. Int J Pharm 1999 179 85-95. 

Phillips WT, Rudolph AS, Goins B, et al. A simple method for producing a technetium-99m-labeled liposome which is stable in vivo. Nucl Med Biol 1992 19 539. 

Recently, this method was adapted to label two commercially available liposomal formulations doxorubicin encapsulated in polyethylene glycol (PEG)-coated liposomes (Caelyx /Doxil ) (14) and daunorubicin encapsulated in small distearoyl-phosphatidyl-choline/cholesterol liposomes (Daunoxome ) (15). Although no DTPA was encapsulated in these liposomes, the labeling efficiency was typically between 70% and 80% and the radiolabeled preparations were stable in vivo during the time course of the experiment (four hours). Most likely, the lipophilic In-oxine avidly associates with the lipid bilayer and encapsulation of DTPA might not be necessary when the experimental observation period does not exceed four to six hours. 

One method for the labeling of liposomes with chelator, hexamethylpropyleneamine oxime (HMPAO) was developed by Phillips et al. (16). Lipophilic HMPAO enters the liposome where it interacts with glutathione and becomes converted to the hydrophilic form, which is trapped in the liposome. A detailed protocol for radiolabeling liposomes using Tc-HMPAO has been reported (3). In a typical experiment, 10 to 15mCi (370-555 MBq) of Tc in the form of sodium pertechnetate... 

The HMPAO-glutathione method has been used in a number of preclinical animal studies (19). An example is shown in Figure 1, where the biodistribution of Tc-HMPAO-labeled PEG liposomes is compared to... 

A new method for radiolabeling glutathione-containing liposomes with using a different chelator based on SNS/S pattern complexes has been recently reported (21). One particular chelator, A,A-bis(2-mercaptoethyl)-A, A diethyl-ethylenediamine (BMEDA), was shown to efficiently label the liposomes. 

More recently, a new chelation method based on the technetium chelator, HYNIC, was developed by Laverman et al. (36). HYNIC is well known for its use in labeling peptides and proteins with high efficiency and excellent stability (37). A-hydroxysuccinimidyl hydrazino nicotinate hydrochloride was conjugated to the free amino group of distearoylpho-sphatidyl-ethanolamine (DSPE) and subsequently incorporated in the lipid bilayer during the liposome preparation. 

This method is relatively inexpensive and easy to apply, although there is no commercially available kit. However, recently succinimidyl-HYNIC became commercially available (Solulink, Inc., San Diego, California, U.S.). Liposomes are labeled rapidly and with a labeling efficiency generally higher... 

Laverman P, Dams ET, Oyen WJ, et al. A novel method to label liposomes with 99mTc jjg hydrazino nicotinyl derivative. J Nucl Med 1999 40 192. 

Measurements of the quantities of glycolipids inserted into the membrane have also been reported by a technique based on the use of C-labeled lipid anchors. In this method, the carbohydrate (a-o-Man) was covalently coupled to the anchor at the surface of a pre-formed vesicle. Indeed, the liposome structure was shown to remain intact in the treatment. Nevertheless, the measurement of the incorporated mannose was performed after separation of bound and unbound material by centrifugation. The yields of coupling were shown to increase with the increase of the initial mannose/ C-anchor ratio, but non covalent insertions were displayed at high initial mannose concentrations. Therefore, the aforementioned method was not as accurate as could have been expected for the use of radioactive materials [142]. Radiolabeled phospholipids were also used for such determinations thus the amounts of glycosphingolipids incorporated into liposomes were quantified by the use of H-phospholipids whereas the amounts of glycolipids were determined by a sphingosine assay [143]. 

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