Targeting Folate Receptor with Folate Linked to Extremities of Poly(ethylene glycol)-Grafted Liposomes: In Vitro Studies

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Abstract
Conjugates of three components, folic acid−poly(ethylene glycol)−distearoylphosphatidylethanolamine (FA−PEG−DSPE), derived from PEG with molecular masses of 2000 and 3350 Da were synthesized by a carbodiimide-mediated coupling of FA to H2N-PEG−DSPE. The conjugates were characterized by 1H NMR, MALDI-TOF, and HPLC analysis of enzymatic cleavage with carboxypeptidase G. As a prototype of a folate receptor (FR)-targeted system, the conjugates were formulated at 0.5 mol % phospholipid in hydrogenated phosphatidylcholine/cholesterol liposomes with or without additional methoxyPEG2000−DSPE. In vitro binding studies were performed with sublines of M109 (murine lung carcinoma) and KB (human epidermal carcinoma) cells each containing high and low densities of FR. FA−PEG−DSPE significantly enhanced liposome binding to tumor cells. The best binding was observed when FA−PEG liposomes contained no additional mPEG−lipid. In fact, our experiments showed that the presence of mPEG on liposomal surfaces significantly inhibited FA−PEG−liposome binding to FR. Increasing the molecular mass of the PEG tether from 2000 to 3350 Da improved the FR binding, particularly in the case of mPEG-coated liposomes. The FA−PEG liposomes bound to M109-HiFR cells very avidly as demonstrated by the inability of free FA (used in a 700-fold excess either at the beginning or at the end of the incubation) to prevent the cell binding. This is in contrast to the 5−10-fold lower cell binding activity of mPEG−FA compared to that of free FA, and likely to be related to the multivalent nature of the liposome-bound FA. Only 22% of FA−PEG3350 and 32% of FA−PEG3350/mPEG cell-associated liposomes could be removed by exposure to pH 3, conditions that dissociate FA−FR, suggesting that more than two-thirds of the bound liposomes were internalized during incubation for 24 h at 37 °C. FA-targeted liposomes also show enhanced nonspecific binding to extracellular tissue culture components, a phenomenon especially relevant in short incubation time experiments.