biodegradable polymeric micelles (10–100 nm in diameter) are recognized as one of the most promising nanovectors for poorly water-soluble drugs. Several cytotoxic anticancer drugs (e.g. doxorubicin, paclitaxel and cis- platin) formulated in such Leflunomide nanovectors are presently in clinical trials with encouraging results ( Nishiyama and Kataoka, 2006; Matsumura, 2008 ). Self-assembled from amphiphilic copolymers, polymeric micelles can solubilize hydrophobic drugs by incor- porating the drug molecules in the vicinity of the hydrophobic core, and remain intact upon dilution in a large volume of blood ( Gaucher et al., 2005 ). Restricted from the normal vasculature (1–2 nm fenestrations in most healthy tissues), long-circulating nanovectors can extravasate and accumulate at the tumor site was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. d – -Tocopheryl polyethy- lene glycol 1000 succinate (TPGS) was a gift from Eastman Co. (Kingsport, TN). 17-AAG was purchased from LC Laboratories (Woburn, MA). All other chemical reagents used were of analytical or reagent grade. 2.2. Preparation of 17-AAG-incorporating micelles Drug-loaded micelles were prepared by a dry film method ( Musacchio et al., 2009 ).
Briefly, varying amounts of PEG-DSPE, TPGS and 17-AAG were dissolved in chloroform in a round-bottom flask. Chloroform was removed under vacuum, resulting in the for- mation of a homogenous film. The drug-polymer film was hydrated in 10 mM HEPES-buffered saline (pH 7.4, HBS), and sonicated at the room temperature for 10 min. The resulting mixture was centrifuged at 12,000 × g for 10 min to yield clear micelle purchase Leflunomide disper- sion. 2.3. Quantification of 17-AAG by HPLC analysis The content of 17-AAG loaded in micelles was quantified by reverse phase HPLC based on a previously reported method ( Egorin et al., 2001 ), which consisted of a Waters (Milford, MA) 2795 pump with an autosampler, a Waters 996 photodiode array detector and a C8 column (5 m, i.d. 4.6 × 150 mm). The isocratic mobile phase was comprised of 75% (v/v) methanol and 25% sodium phosphate buffer (25 mM, pH 3.0) with 10 mM triethylamine at a flow rate via the enhanced permeability and retention (EPR) effect, owing of 1.2 ml/min. -Naphthoflavone (Sigma, St. Louis, MO) was used to the leaky vasculature (usually >100 nm fenestrations) and the as an internal standard at a concentration of 2 M.
The detection impaired lymphatic drainage in solid tumors ( Matsumura and wavelength for 17-AAG and -naphthoflavone were 333 nm and Maeda, 1986 ). The conjugates of polyethylene glycol (PEG) and diacyllipids, such as order Leflunomide PEG-distearoylphosphatidylethanolamine (PEG-DSPE), form stable micelles in aqueous environment because of strong hydrophobic interaction between double acyl chains of phospholipid residues ( Gao et al., 2002 ). PEG-DSPE micelles are spheroidal and have a narrow size distribution of 7–35 nm, depending on the molecular size (750–5000 Da) of the PEG block. With a critical micelle concentration (CMC) in 10 − 6 to 10 − 5 M range, PEG-DSPE micelles retain their size even after 48-h incu- bation in plasma ( Lukyanov et al., 2002 ). A number of sparingly water-soluble anticancer drugs, such as paclitaxel, tamoxifen and doxorubicin, can be incorporated into PEG-DSPE micelles ( Gao et al., 2002; Musacchio et al., 2009; Tang et al., 2007 ).
The small and uniform particle size of PEG-DSPE micelles offers a distinct advan- tage in terms of targeted drug delivery to the tumor tissue via the EPR effect. The administration of doxorubicin-loaded PEG-DSPE micelles lead to extensive drug nutrients accumulation and penetration in the tumor, greatly enhancing the anticancer efficacy of the drug in tumor-bearing mice ( Tang et al., 2007 ). In the present work, we report on the design of PEG-DSPE micel- lar nanocarriers for 17-AAG without the inclusion of any organic solvent. We found that by modulating the PEG-DSPE concentra- tion and incorporating d – -tocopheryl polyethylene g