Design and synthesis of phospholipase C and A2-activatable near-infrared fluorescent smart probes.

The primary focus of this work was to develop activatable probes suitable for in vivo detection of phospholipase activity. Phospholipases (PLs) are ubiquitous enzymes that perform a number of critical regulatory functions. They catalyze phospholipid breakdown and are categorized as A(1), A(2) (PLA(2)), C (PLC), and D (PLD) based on their site of action. Here, we report the design, synthesis, and characterization of self-quenching reporter probes that release fluorescent moieties upon cleavage with PLA(2) or PLC. A series of phospholipids were synthesized bearing the NIR fluorophore pyropheophorbide a (Pyro) at the sn-2 position. Fluorescence quenching was achieved by attachment of either a positively charged black hole quencher-3 (BHQ-3) to the phospholipid headgroup or another neutral Pyro moiety at the sn-1 position. The specificity to different phospholipases was modulated by insertion of spacers (C(6), C(12)) between Pyro and the lipid backbone. The specificity of the quenched fluorescent phospholipids was assayed on a plate reader against a number of phospholipases and compared with two commercial probes bearing the visible fluorophore BODIPY. While PyroC(6)-PyroC(6)-PtdCho revealed significant background fluorescence, and a 10% fluorescence increase under the action of PLA(2), Pyro-PtdEtn-BHQ demonstrated high selective sensitivity to PLC, particularly to the PC-PLC isoform, and its sensitivity to PLA(2) was negligible due to steric hindrance at the sn-2 position. In contrast, the C(12)-spacered PyroC(12)-PtdEtn-BHQ demonstrated a remarkable selectivity for PLA(2) and the best relative PLA(2)/PLC sensitivity, significantly outperforming previously known probes. These results open an avenue for future in vivo experiments and for new probes to detect PL activity.

High payload delivery of optical imaging and photodynamic therapy agents to tumors using phthalocyanine-reconstituted low-density lipoprotein nanoparticles.

To improve the labeling efficiency of a low-density lipoprotein (LDL)-based photosensitizer (PS) for achieving high probe to protein payload, a tetra-t-butyl silicon phthalocyanine bearing two oleate moieties at its axial positions, SiPcBOA, is designed and synthesized. Using this novel strategy, SiPcBOA reconstituted LDL (r-SiPcBOA-LDL) with a very high payload (SiPcBOA to LDL molar ratio >3000 to 35001:1) is obtained. Using electron microscopy, we find reconstituted LDL (rLDL) with such a high payload essentially retains the mean particle size of native LDL. Since acetylated LDL binds to scavenger receptors of endothelial and microglial cells instead of LDLR, SiPcBOA reconstituted acetylated LDL (r-SiPcBOA-AcLDL) is also prepared to serve as a negative control to validate the LDL receptor (LDLR) targeting specificity. Confocal microscopy studies demonstrate that the internalization of r-SiPcBOA-LDL by human hepatoblastoma G2 (HepG2) tumor cells is mediated by LDLR pathway. The in vitro photodynamic therapy (PDT) response of HepG2 cells to r-SiPcBOA-LDL is compared to SiPcBOA (free drug control) using a clonogenic assay. The slopes of the linear regression fit to the logarithmic data for these two plots are significantly different from each other (p=0.0007), indicating greatly enhanced efficacy of LDLR-targeted PDT.