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 degrees C. FA-targeted liposomes also show enhanced nonspecific binding to extracellular tissue culture components, a phenomenon especially relevant in short incubation time experiments.

No abstract

Like most low molecular weight drugs, carboplatin has a short blood circulation time, which reduces tumor uptake and intracellular DNA binding. Drugs conjugated to PEG carriers benefit from prolonged blood circulation, but suffer from reduced cell permeability. In this work we attempted to develop long-circulating PEGylated carboplatin analogues with improved cell permeation abilities, by conjugating the platinum moiety to folate-targeted PEG carriers capable of utilizing the folate receptor-mediated endocytosis (FRME). Two bifunctional FA-PEG conjugates, FA-PEG-Pt and FA-PEG-FITC, were prepared, and their cell uptake, DNA binding, and cytotoxicity were studied by fluorescent microscopy, FACS, and platinum analysis. Folate-targeted PEG conjugates enter the cells efficiently by the FRME pathway but form relatively few DNA adducts and have higher IC(50) values than carboplatin and their nontargeted analogues. Nontargeted PEG-Pt conjugates have a lower cellular uptake but produce higher levels of DNA binding and improved cytotoxicity. Carboplatin, used as a control, has the fastest cellular uptake, but after 16 h of postincubation a large percentage of the drug is excreted from the cells. The findings of this study suggest that folate-targeted conjugates such as FA-PEG-Pt, may not be an optimal prodrug for the carboplatin family compounds, because the conjugates or the active moieties are neutralized or blocked during the FRME process and do not manage to effectively reach the nuclear DNA.

Anthracyclines entrapped in small-sized, sterically stabilized liposomes have the advantage of long circulation time, reduced systemic toxicity, increased uptake into systemic tumors, and gradual release of their payload. To date, there is no information on the behavior of these liposomes in brain tumors. The objective of this study was to compare the biodistribution and clinical efficacy of free doxorubicin (F-DOX) and stealth liposome-encapsulated DOX (SL-DOX) in a secondary brain tumor model. Nine days after tumor inoculation Fischer rats with a right parietal malignant sarcoma received an intravenous dose of 6 mg/kg of either F-DOX or SL-DOX for evaluation of drug biodistribution. For therapeutic trials a single dose of 8 mg/kg was given 6 or 11 days after tumor induction, or alternatively, weekly doses (5 mg/kg) were given on Days 6, 13, and 20. Liposome-encapsulated DOX was slowly cleared from plasma with a t1/2 of 35 hours. Free-DOX maximum tumor drug levels reached a mean value of 0.8 microgram/g and were identical in the adjacent brain and contralateral hemisphere. In contrast, SL-DOX tumor levels were 14-fold higher at their peak levels at 48 hours, declining to ninefold increased levels at 120 hours. A gradual increase in drug levels in the brain adjacent to tumor was noted between 72 and 120 hours (up to 4 micrograms/g). High-performance liquid chromatography analysis identified a small amount of aglycone metabolites within the tumor mass from 96 hours and beyond, after SL-DOX injection. Cerebrospinal fluid levels were barely detectable in tumor-bearing rats treated with F-DOX up to 120 hours after drug injection (< or = 0.05 microgram/ml), whereas the levels found after SL-DOX were 10- to 30-fold higher. An F-DOX single-dose treatment given 6 days after tumor inoculation increased the rats' life span (ILS) by 135% over controls (p < 0.05) but was not effective if given on Day 11. In contrast, SL-DOX treatment resulted in an ILS of 168% (p < 0.0003) with no difference when given after 6 or 11 days. Treatment with three weekly doses of SL-DOX produced an ILS of 189% compared to 126% by F-DOX (p < 0.0002). The authors conclude that the use of long-circulating liposomes as cytotoxic drug carriers in brain tumor results in enhanced drug exposure and improved therapeutic activity, with equal effectiveness against early small- and large-sized brain tumors.

No abstract