Nanoparticles Containing High Loads of Paclitaxel-Silicate Prodrugs: Formulation, Drug Release, and Anticancer Efficacy.

We have investigated particle size, interior structure, drug release kinetics, and anticancer efficacy of PEG-b-PLGA-based nanoparticles loaded with a series of paclitaxel (PTX)-silicate prodrugs [PTX-Si(OR)3]. Silicate derivatization enabled us to adjust the hydrophobicity and hydrolytic lability of the prodrugs by the choice of the alkyl group (R) in the silicate derivatives. The greater hydrophobicity of these prodrugs allows for the preparation of nanoparticles that are stable in aqueous dispersion even when loaded with up to ca. 75 wt % of the prodrug. The hydrolytic lability of silicates allows for facile conversion of prodrugs back to the parent drug, PTX. A suite of eight PTX-silicate prodrugs was investigated; nanoparticles were made by flash nanoprecipitation (FNP) using a confined impingement jet mixer with a dilution step (CIJ-D). The resulting nanoparticles were 80-150 nm in size with a loading level of 47-74 wt % (wt %) of a PTX-silicate, which corresponds to 36-59 effective wt % of free PTX. Cryogenic transmission electron microscopy images show that particles are typically spherical with a core-shell structure. Prodrug/drug release profiles were measured. Release tended to be slower for prodrugs having greater hydrophobicity and slower hydrolysis rate. Nanoparticles loaded with PTX-silicate prodrugs that hydrolyze most rapidly showed in vitro cytotoxicity similar to that of the parent PTX. Nanoparticles loaded with more labile silicates also tended to show greater in vivo efficacy.

Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins.

Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z'-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach.

Silicate esters of paclitaxel and docetaxel: synthesis, hydrophobicity, hydrolytic stability, cytotoxicity, and prodrug potential.

We report here the synthesis and selected properties of various silicate ester derivatives (tetraalkoxysilanes) of the taxanes paclitaxel (PTX) and docetaxel (DTX) [i.e., PTX-OSi(OR)3 and DTX-OSi(OR)3]. Both the hydrophobicity and hydrolytic lability of these silicates can be (independently) controlled by choice of the alkyl group (R). The synthesis, structural characterization, hydrolytic reactivity, and in vitro cytotoxicity against the MDA-MB-231 breast cancer cell line of most of these derivatives are described. We envision that the greater hydrophobicity of these silicates (vis-à-vis PTX or DTX itself) should be advantageous from the perspective of preparation of stable aqueous dispersions of amphiphilic block-copolymer-based nanoparticle formulations.

Flash nanoprecipitation: particle structure and stability.

Flash nanoprecipitation (FNP) is a process that, through rapid mixing, stabilizes an insoluble low molecular weight compound in a nanosized, polymer-stabilized delivery vehicle. The polymeric components are typically amphiphilic diblock copolymers (BCPs). In order to fully exploit the potential of FNP, factors affecting particle structure, size, and stability must be understood. Here we show that polymer type, hydrophobicity and crystallinity of the small molecule, and small molecule loading levels all affect particle size and stability. Of the four block copolymers (BCP) that we have studied here, poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PEG-b-PLGA) was most suitable for potential drug delivery applications due to its ability to give rise to stable nanoparticles, its biocompatibility, and its degradability. We found little difference in particle size when using PLGA block sizes over the range of 5 to 15 kDa. The choice of hydrophobic small molecule was important, as molecules with a calculated water-octanol partition coefficient (clogP) below 6 gave rise to particles that were unstable and underwent rapid Ostwald ripening. Studies probing the internal structure of nanoparticles were also performed. Analysis of differential scanning calorimetry (DSC), cryogenic transmission electron microscopy (cryo-TEM), and (1)H NMR experiments support a three-layer core-shell-corona nanoparticle structure.