br The nanomedicine has got rapid development
The nanomedicine has got rapid development and wide concern in recent years. Various nanomaterials with diﬀerent sizes, physico-chemical properties and surface morphologies were utilized to design proper delivery systems for compounds, antibodies, siRNAs and other agents in the treatment of cancer [17–20]. In this research, we plan to formulate a gene delivery system PPPD micelles with 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) and poly (ethylene glycol)–poly (3-caprolactone)-poly (ethylene glycol) (PEG-PCL-PEG), and to re-search on therapeutic eﬃcacy of MP on colon cancer and explore the mechanisms.
2. Materials and methods
Materials were purchased from standard sources: 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) (Avanti Polar Lipids Inc., Alabaster, AL, USA); 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyl tetrazolium bromide (MTT) (Sigma, USA); Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) (Gibco BRL, USA); methanol and acetic ITF2357 (HPLC grade) (Fisher Scientific, UK); and dimethyl sulfoxide (DMSO) and acetone (KeLong Chemicals, China). Antibodies used include: rabbit anti-mouse Ki67 antibody (Abcam, USA) and rhodamine-conjugated secondary antibody (Abcam, USA).
PECE (E2000-C4000-E2000) triblock copolymers were synthesized by coupling the MPEG2000-PCL2000 diblock copolymers using dicy-clohexylmethane-4,4′-diisocyanate (HMDI) as the coupling agent. Firstly, MPEG2000-PCL2000 diblock copolymers were prepared through ring-opening polycondensation by adding10 g MPEG (Mn = 2000, 0.005 mol), 10 g ε-CL, and 0.1 g Sn(Oct)2 (0.5 wt % of total reactants) into a reaction vessel under dry nitrogen atmosphere, and the reaction system was kept at 140 °C for 12 h. Secondly, 1.44 g of HMDI (Mw = 262.35, 0.0055 mol, eOH: NCO = 1:1.1) was added to the reaction mixture, and then stirred at 80 °C for 8 h. After degassing under vacuum for another 0.5 h, the resultant copolymer was then cooled to room temperature. Finally, the just-obtained PECE block co-polymers were dissolved in dichloromethane, and the mixture was poured into excess diethyl ether, then filtered and vacuum dried to constant weight at room temperature.
2.2. Preparation of MP/DPPP micelles
Fig. 1. Nuclear magnetic Resonance characterization and preparation of
pDNA/DPPP. PEG-PCL-PEG were characterized by 1H NMR, where the peaks were carefully distributed to the protons A); DPPP micelles were prepared using a one step self-assembly method. DOTAP and PEG-PCL-PEG were dissolved in dehydrated acetone under even stirring, followed by evaporation at 55 °C. Next, the above mixture was dissolved in water. The MP plasmid solution was added and incubated for 20 min at room temperature. During this process, plasmids were encapsulated into DPPP micelles by electrostatic attraction B).
Fig. 2. Interaction modes between copolymer and DOTAP revealed by Langevin dynamics simulation in water. A) The initial conformation of PEG-PCL-PEG copolymer complexed with DOTAP. Conformations B), C), D), E), and F) correspond to snapshots of the complex collected at 100 ps, 200 ps, 300 ps, 400 ps and 500 ps, respectively. Copolymer PEG-PCL-PEG is represented by a thin line. DOTAP is depicted by a thick line, and its carbon atoms are colored green. Two terminal heavy atoms in the PEG-PCL-PEG copolymer are high-lighted using “ball” style.
method. 10 mg DOTAP and 90 mg PEG-PCL-PEG were dissolved in 5 ml dehydrated acetone under even stirring, followed by evaporation at 55 °C. Next, the above mixture was dissolved in water. The MP plasmid solution was added and incubated for 20 min at room temperature. During this process, plasmids were encapsulated into DPPP micelles by electrostatic attraction.