br Fig Anticolon cancer e ect of MP PPPD
Fig. 6. Anticolon cancer eﬀect of MP/PPPD in vitro. A) MTT test; B) cell apoptosis test with FCM.
Fig. 7. Anticolon cancer eﬀect of MP/PPPD in subcutaneous tumor model. A) Body weight of diﬀerent groups. B) Tumor growth curves. C) Tumor weight. D) Tumor photos of GS, PPPD, pVax/PPPD and MP/PPPD treatment groups. The results represent three independent experiments.
Fig. 8. Anticolon cancer eﬀect of MP/PPPD in peritoneal colon cancer model. A) Body weight. B) Images of mouse and the corresponding tumor. C) Tumor weight of diﬀerent group (GS, PPPD, pVax/ PPPD and MP/PPPD-treated mice). The number of tumor nodes in diﬀerent groups: D) Tumor nude (> 3 mm) of diﬀerent groups; E) Tumor nude
(< 3 mm) of diﬀerent groups. The results represent three independent experiments.
Fig. 9. Antitumor mechanisms of MP/PPPD.
Representative tumor tissue sections following the Ki67 staining A) and TUNEL assay B). Tumor cell proliferation and tumor cell apoptosis were assessed by counting the number of the Ki67-positive index rate and TUNEL-positive cells index rate (three high power fields per slide). MP/PPPD was superior to the controls in increasing tumor apoptosis and inhibiting tumor cell proliferation.
Fig. 10. Toxicity assessment in vivo with pathological section. Histological examinations of HE-stained vital organ sections including A) heart, B) liver, C) lung, D) kidney and E) spleen. No significant pathological changes were de-tected. Scale bars is 50 μm.
DMP and demonstrated a high transfection eﬃciency and the en-hancement of anti-cancer activity of IL-12 . The transfection eﬃ-ciency in this research was even higher, about 60%, than that in our previous research and literature, which provided a solid Ko 143 for further anti-cancer treatment [31,32]. During the first step of assembling, PEG went to the surface of this complex, encapsulating DOTAP inside, and formed PPPD. Then the MP plasmids were attracted into the system by electrostatic adsorption of cationic DOTAP. We got a satisfactory drug loading rate of 98% at weight ratios of MP with PPPD micelles 50:1. MP/PPPD micelles, with a zeta potential of 5.4 mV, was found stable in fluid observed by TEM in our research. Latest literature demonstrated the structure of PEG-coated nano-delivery system was stabled in phy-siological fluid and the PEG shell was detached after the uptake by cells .
It is widely known that the topical administration could elevate the concentration of drugs in target region and decrease the systemic toxicity simultaneously. Considering the possible toxicity to normal tissues caused by MP/PPPD micelles, and the intraperitoneal injection is feasible in clinical treatment for colon cancer, we set up two animal models, subcutaneous tumor model and peritoneal tumor model, chose intraperitoneal injection as the method of administration and compare the eﬃcacy of them. As shown in Figs. 5 and 6, the inhibition of tumor growth was satisfactory in both models, and significant diﬀerences of tumor volume and weight were found among groups after treatment.
The degradable PEG-PCL-PEG micelles are known to be with low toxicity and the content of DOTAP was low, about 10% in this delivery system. However, whether MP without VSV platform is hazardous to normal cells is still unknown. So the drug safety evaluation was carried out to confirm its reliability and security. It is gratifying that no obvious diﬀerence was found in histological examination, which was consistent with the result of PPPD delivery system in our previous research .
The mechanisms of anti-cancer activity of MP were explored in our experiment. The cytotoxicity of MP/PPPD micelles on tumoe cell was demonstrated by MTT assay in vitro, and the proliferation of tumor cells was markedly inhibited by MP/PPPD micelles in vivo. It has been re-ported the apoptosis is one of the principal cytopathic eﬀects of VSV infection [34,35]. In our research, the apoptotic cells are abundant in the MP/PPPD micelles treatment group, whereas few apoptotic cells were observed in the control group both in vitro and in vivo, which were determined by Annexin-V-FITC and PI assay and Tunnel assay, indicated that MP/PPPD micelles were intimately involved in the pro-cess of apoptosis and MP could eﬀectually activate apoptosis without the VSV platform. The MP/PPPD micelles may eﬀectively prevent dis-semination of cancer and the formation of ascites in its clinical treat-ment for colon cancer .
We formulated MP/PPPD micelles to deliver the mainly active gene of VSV for colon cancer treatment without virulence of the virus. This nano-delivery system was proven to have a high transfection eﬃciency of MP plasmid. MP synthesized in host cells could prominently inhibit tumor growth through mechanisms of direct cycotoxicity and apoptosis. The formation of MP/PPPD micelles is promising for future clinical application.