Preliminary Assessment Of Biologic Security Of A Membrane Penetrating Peptide MClock's DNA-BIND

ObjectiveTo investigate the biological security of a novel membrane penetrating peptide (MPP)—mCLOCK's DNA-BIND (CDB) derived from circadian protein mCLOCK's DNA binding domain.MethodExperiment 1 The internalization and cytotoxicity of mCLOCK's DNA-BIND:mCLOCK's DNA-BIND chemically synthesized and labeled with FITC at N-terminal was incubated with NIH3T3 cells in the culture medium. After incubation, the distribution of CDB was observed on a fluorescence microscope at different time, the fluorescence intensity of every cell was measured using the Image-Pro Plus (Version 4.5 for Windows^TM) system, and the cytotoxicity of CDB was detected by MTT.Experiment 2 The influence of mCLOCK's DNA-BIND on circadian system: The circadian genes expression in NIH3T3 fibroblasts was induced by PMA (Phorbol 12-Myristate 13-acetate). The influence of CDB on the expression of mPeriodl was sequently detected by RT-PCR.Experiment 3 The influence of mCLOCK's DNA-BIND on cell cycle, cell proliferation and apoptosis: NIH3T3 fibroblasts cells were cultured and incubated with CDB. 12h or 24h after incubation, the cell proliferation was detected by MTT, and the cell cycle and apoptosis were detected by flow cytometer. On the other hand, the circadian genes expression in NIH3T3 cells was induced by PMA. At the indicated times, the cell proliferation activity was detected by MTT, the cell cycle, the rate of proliferation and apoptosis were detected by flow cytometer.ResultExperiment 1 CDB has the internalization ability into the NIH3T3 cells. The concentration of the CDB in the cells kept increasing as the incubation time increased during the observed time period. And the amount of the internalized peptide increased as the applied peptide concentration increased. Moreover, CDB did not induce any toxicity in all conditions, even at 100μM for 24h, or 10μM for 48h.Experiment 2 The circadian oscillation of mPeriod1 gene induced by PMA in cultured NIH3T3 cells had not been influenced by CDB.Experiment 3 The influence of CDB on the cell cycle, cell proliferation and apoptosis was not observed, whether the circadian rhythm were induced by PMA in NIH3T3 cells or not.ConclusionThere was no toxic and adverse reaction on circadian rhythm, cell cycle, cell proliferation and apoptosis in cultured cells treated with CDB. As a result, CDB can be used as a safe and efficient drug-carrier for the intracellular treatment, and will have extensive perspective in clinical application.