Screening of Polymer-Based Drug Delivery Vehicles Targeting Folate Receptors in Triple-Negative Breast Cancer.

PURPOSE: To compare cellular uptake and cytotoxicity of fluorescein (FL)-labeled polyethylene glycols (PEGs) carrying 2 folate groups (targeted delivery vehicles [TDVs]) to non-PEGylated molecules with 1 or 2 folate groups. MATERIALS AND METHODS: Three PEGylated TDVs and 2 non-PEGylated folic acid (FA)-fluorescein (FL) conjugates (FA-FL and FA-FL-FA) were synthesized. Two triple-negative breast cancer cell lines (MDA-MB-231and MDA-MB-468) were cultured to 70% confluency and incubated for 2 h in a folate-depleted medium. Folate receptor (FR) expression was confirmed by immunocytochemistry. Cellular uptake and cytotoxicity of compounds were measured by flow cytometry. Intracellular localization was confirmed using confocal microscopy. RESULTS: MDA-MB-231 demonstrated 40% more FR staining than MD-MB-468. Intracellular localization of the 2 non-PEGylated molecules (FA-FL and FA-FL-FA) and the 3 PEGylated TDVs was confirmed with confocal microscopy. Cellular uptake was independent of concentration for FA-FL, but there was 26.8% more cytotoxicity at 30 µg/mL compared with no treatment (P ≤ .05). Uptake was > 90% for FA-FL-FA at 10 µg/mL and 30 µg/mL without significant cytotoxicity (P ≤ .005). Cellular uptake was > 80% for all TDVs. The molecule containing monodispersed PEG with Mn = 1,000 g/mol had the highest uptake in both cell lines without cytotoxicity. Maximum toxicity was demonstrated by the molecule containing PEG2,000 only at the highest dose of 30 µg/mL (8.66% ± 3.94% cytotoxicity; cut-off was 20%). CONCLUSIONS: The molecule containing monodispersed PEG with Mn = 1,000 g/mol and 2 FA targeting groups demonstrated better targetability and cellular uptake as a TDV.

PEGylation of Fluorescein by Enzyme-Catalyzed "Click" Michael Addition.

This paper reports the first "Click" Michael addition catalyzed by Candida antarctica lipase B (CALB) between fluorescein o-acrylate and thiol-functionalized poly(ethylene glycol)s (HS-PEG-SH, Mn = 1200 g mol-1 , D = 1.14, and Mn = 2200 g mol-1 , D = 1.09). The progress of the reactions is monitored with 1 H-NMR spectroscopy. In the absence of CALB, the reaction does not go to completion even after 18 h but completes in less than 2 min when CALB is added. Similarly, the reaction with HS-PEG-SH having Mn = 2200 g mol-1 and D = 1.09 completes in less than 2 min by CALB catalysis. The structures of the products are also confirmed by 13 C-NMR. This enzyme-catalyzed "Click" Michael addition is found to be a powerful tool to synthesize fluorescein-based polymeric conjugates for a wide variety of applications.

Enzyme-independent catabolism of cysteine with pyridoxal-5'-phosphate.

Pyridoxal-5'-phosphate (PLP) is a versatile cofactor that assists in different types of enzymatic reactions. PLP has also been reported to react with substrates and catalyze some of these reactions independent of enzymes. One such catalytic reaction is the breakdown of cysteine to produce hydrogen sulfide (H2S) in the presence of multivalent metal ions. However, the enzyme-independent catalytic activity of PLP in catabolizing cysteine in the absence of multivalent ions is unknown. In this study, we show that PLP reacts with cysteine to form a thiazolidine product, which is supported by quantum chemical calculations of the absorption spectrum. The reaction of PLP with cysteine is dependent on ionic strength and pH. The thiazolidine product slowly decomposes to produce H2S and the PLP regenerates to its active form with longer reaction times (> 24 h), suggesting that PLP can act as a catalyst. We propose an enzyme-independent plausible reaction mechanism for PLP catalyzed cysteine breakdown to produce H2S, which proceeds through the formation of thiazolidine ring intermediates that later hydrolyzes slowly to regenerate the PLP. This work demonstrates that PLP catalyzes cysteine breakdown in the absence of enzymes, base, and multivalent metal ions to produce H2S.