br PECE E C E triblock copolymers were synthesized by
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 ABT-263 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.
Fig. 3. Interaction modes between PEG-PCL-PEG copolymer and DOTAP revealed by Langevin dynamics simulation near water. A) The initial con-formation of PEG-PCL-PEG copolymer complexed with DOTAP. Conformations B), C), D), E), and F) are corresponding to snapshots of the complex collected at 100 ps, 200 ps, 300 ps, 400 ps and 500 ps, respectively. Copolymer PEG and PCL are depicted with a solid surface and colored by elements and green, re-spectively.
2.3. Transfection eﬃciency
Green fluorescent protein (GFP)/PPPD micelles were prepared by pEGFP plasmid and DPPP by the method describe above. Ct26 cells were seeded in six-well plate and transfected with GFP/PPPD for 4 h. After 24 h, images were obtained by a fluorescent microscope under high power field ( × 200) and FACS Calibur flow cytometry (FCM, BD Biosciences, San Jose, CA, U.S.) was applied for quantification of GFP and calculation of transfection eﬃciency.
2.4. Cell toxicity and apoptosis
We apply MTT assay to determine the cell viability. Briefly, Ct26 cells were planted in a 96-well plate and incubated for 20 h. Then, they were treated with normal saline (NS), PPPD micelles, pVax blank plasmid (pVax)/PPPD, matrix protein plasmid (MP)/PPPD micelles. After 48 h, MTT solution was added and incubated for another 4 h and added with DMSO. Subsequently, formazan was quantified by mea-suring the absorbance at 570 nm using a plate reader (OPTImax, Molecular Dynamics).
The apoptosis induced by MP/PPPD micelles was further in-vestigated using Annexin-V-FITC and PI assay (Annexin V-FITC/PI Apoptosis Detection Kit). In brief, Ct26 cells were planted in 6-well plate and treated with NS, PPPD and MP/PPPD micelles. After 48 h, cells were harvested and stained with 5 μl Annexin-V-FITC and PI. It was further quantified by flow cytometry.
2.5. Anti-tumor activity in vivo
Balb/c mice were applied to evaluate of anti-colon activity of MP/ PPPD micelles. Ct26 cells suspended at 1 × 107 cells/ml DMEM were injected subcutaneously in the right flank to build subcutaneous tumor model. Tumors were speculated and measured by a caliper every two days and tumor volumes were calculated according to the formula: V (Volume) = 0.52*(ab2), in which a represented the length and b re-presented the width. When the tumors reached about 0.1 cm3, mice were randomly divided into groups and treated with GS, PPPD micelles, pVax/PPPD and MP/PPPD micelles through intratumor injection, re-spectively. After two weeks, mice were sacrificed and tumors were photographed and weighed.