Sensing Drug Molecule Interactions With Lipid Membranes By Local Viscosity Modulation

Herein, we utilized a label-free sensing platform based on viscosity modulation to detect the interactions between tetracaine(TTC) and dimyristoylphosphatidylcholine(DMPC) lipid membranes.This work is mainly divided into the following sections:1. Two viscosity sensitive probes, CCVJ-C and CCVJ-E, have been designed and synthesized. The probe’s fluorophore is 9-(2-carboxyl-2-cyanovinyl) julodine(CCVJ). CCVJ is connected to the hydrophilic hydroxyl portion of cholesterol by linker to call as CCVJ–C, while that is connected to the ketone group of estrone to call as CCVJ-E.2. The property characterization of the viscosity fluorescent probes, CCVJ-C and CCVJ–E. The fluorescence spectra of the probes in organic solvents were determinated. As a result, attaching various anchor group to CCVJ does not affect the fluorescence properties of the fluorophore. The fluorescence spectra of the probes in different viscosity solutions were determinated. The results showed that CCVJ-E posesses a higher viscosity sensitivity than that of CCVJ-C. Then the probes CCVJ-C and CCVJ-E were embedded into DMPC liposomes to determine the fluorescence spectra. The data showed that the two types of probes could be well embedded into DMPC liposomes and remained their fluorescence properties.3. Determination of the probe induced phase trasition of DMPC lipid membranes. When the CCVJ-E was embedded into DMPC liposomes, three phase transitions including subtransition(Ts), pre-transition(Tp) and main transition(Tm) were observed along with temperature increased. The phase transition temperatures were observed at 7.3 oC, 13.7 oC and 24.5 oC, respectively, which are in good agreement with the literature. Whereas no inductions to the exact phase transition temperature of the containing CCVJ-C DMPC liposomes. This experiment indicated that the probe CCVJ-E allows the flurophore located at the hydrophobic region of lipids membranes, assuring adequate sensitivity of the dye to the phase transition. Therefore, CCVJ-E probe was choosen to detect the interaction between drugs and lipid membranes.4. The interaction between drug and lipid membranes were detected. CCVJ-E could sensitively detect the strongest perturbation on DMPC membranes after the addition of TTC. This change reflects the phase transition change of lipid membranes. The results showed that TTC has a significant chain ordering effect on liquid-crystalline phase lipids than that on solid-gel phase lipids, especially subgel phase lipids. Moreover, the LβI phase observed between the L′? and the P′? phase suggests the ability of TTC to induce interdigitation of DMPC bilayer. The LβI phase substitutes the P′? and L′? in a TTC-dependent fashion, and the low-temperature end merged to the subtransition at TTC concentrations above 17 mM. Furthermore, our data clearly show that the incorporation of CHOL progressively abolishes the interdigitation and causes the formation of Lo phase in DMPC bilayer.Viscosity sensing probe CCVJ-E is highly sensitive to the local viscosity of the lipid membrane microenvironment, providing a facile and efficient new tool to study drugmembrane interactions. Hence, it may be developed into a rapid high-throughput screening assay for drug development and analysis methods.