Since the folate receptor is known to be over-expressed by various tumors, numerous studies have investigated the use of incorporating folate as a targeting ligand into liposomal delivery vehicles loaded with chemotherapeutics. There are two problems, however, associated with this approach. First, the incorporation of polyethylene glycol (PEG) is essential to allow prolonged circulation of liposomes, but the addition of this polymer often hinders cell-liposome interactions, and drug uptake at the target site may be negatively affected as a result. In addition, even when PEG is included in liposomal formulations to inhibit uptake by the reticuloendothelial system (RES), the attachment of folate to the exterior surface of liposomes has been shown to result in accelerated clearance of targeted formulations from the bloodstream.

These studies involve the incorporation of a cysteine cleavable PEG conjugate into liposomal formulations to allow for enhanced control over liposome-cell interactions. When the liposomes are in circulation, the cleavable PEG chains inhibit binding of plasma proteins and effectively "mask" the folate targeting ligands reducing RES clearance. After extravasating into the target site, the systemic administration of a safe cleaving agent results in detachment of PEG chains and exposure of the targeting ligand to promote liposome-cell associations due to receptor binding. In this manner, folate ligands are only exposed at the site of action where they are needed and do not interfere with prolonged circulation of liposomal formulations.

Figure 1. Schematic depicting FR-targeted nanocarrier options. A) When attached to PEG chains longer than those incorporated for RES evasion, folate targeting ligands are readily recognized by the RES resulting in accelerated clearance. These liposomes are often removed from circulation before extravasation to the target site is achieved. B) Longer PEG chains conceal folate from the RES but hinder receptor-mediated uptake by target cells over-expressing the folate receptor. C) Long cysteine-cleavable PEG-phospholipid conjugates mask folate during circulation to enable passive targeting to tumor but may be removed through the administration of cysteine at a later time after a majority of the nanocarriers have extravasated to the target site. Resultant exposure of folate enables targeting to cells over-expressing the folate receptor.