Conjugation proteins to liposomes

Addition of Protein-Palmitate Conjugate to Liposomal Membranes... 

Numerous experimental therapeutics have shown potency in vitro however, when they are tested in vivo, they often lack significant efficacy. This is often attributed to unfavorable pharmacokinetic properties and systemic toxicity, which limit the maximum tolerated dose. These limitations can be overcome by use of drug carriers. Two general types of carrier systems have been designed drug conjugation to macromolecular carriers, such as polymers and proteins and drug encapsulation in nanocarriers, such as liposomes, polymersomes and micelles. 

Characterizing the resultant complex for the amount of protein per liposome is somewhat more difficult than in other protein conjugation applications. The protein-liposome composition is highly dependent on the size of each liposomal particle, the amount of protein charged to the reaction, and the mole quantity of reactive lipid present in the bilayer construction. An approach to solving this problem is presented by Hutchinson et al. (1989). In analyzing at least 17 different protein-liposome preparations, the ratio of proteindipid content (pg protein/pg lipid) in most of the complexes ranged from a low of about 4 to as much as 675. In some instances, however, up to 6,000 molecules of a particular protein could be incorporated into each liposome. 

Add protein-palmitate conjugate to the formed liposomes in a ratio of 20 1 (w/w). Add concentrated deoxycholate to give a final concentration of 0.7 percent. Mix thoroughly using a vortex mixer. 

Add the protein or peptide to be conjugated to the liposome suspension. The protein may be dissolved first in PBS, pH 7.2, and an aliquot added to the reaction lipid mixture. The amount of protein to be added can vary considerably, depending on the abundance of the protein and the desired final density required. Reacting from 1 mg protein per ml liposome suspension up to about 20 mg protein/ml can be done. 

Beyer, U., Rothen-Rutishauser, B., Unger, C., Wunderli-Allenspach, H., Kratz, F., Differences in the intracellular distribution of acid-sensitive doxorubicin-protein conjugates in comparison to free and liposomal formulated doxorubicin as shown by confocal microscopy. Pharm Res 18, 29-38 (2001). 

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. 

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