Tirapazamine br micelles and MP PPPD micelles
micelles and MP/PPPD micelles). Two weeks later, tumors were col-lected and evaluated.
2.6. Immunohistochemical staining of Ki-67 and CD31
Evaluation of proliferation of colon tumor were performed by im-munohistochemical staining of Ki-67. In brief, tumor samples were collected and embedded in paraﬃn and sectioned to slices measuring about 5 μm per one. Then the sections were incubated with rabbit anti-mouse Ki-67 antibody (1: 50; Abcam, USA). Subsequently, there were washed in PBS and stained with secondary antibody. Lastly, images were obtained and the Ki-67 positive Tirapazamine was counted under a fluor-escence microscope.
The apoptosis of colon tumor was investigated by TUNEL staining. Briefly, tumor samples were collected and embedded in paraﬃn and sectioned as mentioned above. We apply a TUNEL kit (Promega, Madison, WI, USA) to analyze the apoptotic cells within tumor sections following the manufacturer's instruction. TUNEL-positive cells were counted under a fluorescence microscope.
2.8. Drug safety evaluation
Organs were dehydrated and embedded in paraﬃn. They were sectioned to slices and stained with hematoxylin-eosin (HE) staining. Images were obtained for observation.
2.9. Statistical analysis
SPSS15.0 software for Windows (SPSS Inc., Chicago, IL) was applied for statistical analysis. Results were recorded as standard deviation (SD). Analysis of variance (ANOVA) were employed for multiple group comparisons. P < 0.05 was considered statistically significant.
3.1. Preparation and characterization of MP/PPPD micelles
The preparation of MP/PPPD micelles was shown in the schematic diagram Fig. 1. First, the interaction between PEG-PCL-PEG and DOTAP was evaluated by molecular dynamics simulation. As shown in Fig. 2 and Fig. 3, the interaction between DOTAP and PEG-PCL-PEG in water made them gradually approach each other. DOTAP tried to find a sui-table place for interaction on the surface of PEG-PCL-PEG by moving its position and adjusting its conformation. Meanwhile, PEG-PCL-PEG continuously adjusted its conformation to provide a good site for DOTAP, and the interaction between them is stable. Hydrophilic MPEG parts were on the surface of MP/PPPD micelles, with cationic DOTAPs inside the micelles binding MP plasmid through electrostatic attraction. They were positive charged and have a zeta potential of 5.4 mv (Fig. 4). The size distribution of MP/PPPD micelles was determined by dynamic light scattering (DLS, Malvern Nano-ZS 90) and the result is shown in Fig. 4. These MP/PPPD micelles were monodispersed in GS and have an average diameter of 102 nm (PDI = 0.21). The morphology of MP/ PPPD micelles was further observed under transmission electron mi-croscopy (TEM) and images were obtained shown in Fig. 4.
The amount of MP plasmid encapsulated by DPPP micelles was determined by gel retardation assays. As shown in Fig. 4D, free DNA not encapsulated was manifested as white band (lane 1 to 3) and no white band was seen from lane 10 to 12. The encapsulation of MP plasmid reaches 98% at weight ratios of MP with DMP micelles (50:1).
Fig. 4. Physicochemical properties of MP/PPPD. A) The structure model of MP/PPPD. B) Morphological characteristics of MP/PPPD by TEM observation. C) Particle sizes of MP/PPPD. D) Zeta-potential of MP/PPPD particleplexes. E) Gel retardation assay of DNA and particleplexes. Lane 0, DNA marker; lane 1 to 3, naked MP; lanes 4 to 12, diﬀerent weight ratios of MP with PPPD. MP was completely incorporated into PPPD at a weight ratio of 1:50, and particleplexes were prepared without free DNA.
3.2. Transfection eﬃciency of GFP/DMP micelles
Ct26 cells were incubated with GS, DPPP micelles and prepared GFP/PPPD micelles for 4 h, respectively. And after 24 h, the qualitative analysis of transfection eﬃciency was determined by fluorescence mi-croscopy. Images of diﬀerent wells were obtained and shown in Fig. 5A. Cells emitting green fluorescence were successfully transfected by GFP/ PPPD micelles. It was further quantified by flow cytometry and the transfection eﬃciency was 42% with GFP/PPPD transfection after 48 h (Fig. 5B). And RT-PCR showed that the MP was expressed after MP/ PPPD transfection (Fig. 5C).
3.3. Antitumor eﬀect of MP/PPPD micelles in vitro
The MTT assay was applied to evaluate the cell viability of Ct26 cells in vitro and the result is shown in Fig. 6A. It is obvious that the viability of cells treated with MP/PPPD micelles was significant
lower than cells in the control group (P < 0.05), which indicated a prominent anti-tumor activity of MP/PPPD micelles in vitro.