| ObjectiveIn this study, TAT/54R/KDEL, a novel recombinant protein, was enveloped by using folate liposomes, in order to effectively enhance the in vivo stability and targeting effect of anti-tumor.Methods1. The recombinant plasmid of TAT/54R/KDEL/pTAT-HA previously constructed was transformed into colorectal strain BL21 (DE3) and its expression was induced by IPTG. Then, the protein was purified through nickel affinity chromatography and HPLC. The purified protein needs deliquation, dislysis and ultrafiltration for renaturation. The phenotypic knockout activity of TAT/54R/KDEL in highly CXCR4-expressed cell (MOLT-4) was investigated by flow cytometry.2. Liposome was prepared by thin-film dispersion method and frozen ultrasound method, and incubated with FA's arm to develop folate liposome. The envelopment and leakage rate of folate liposome to calcein were determined to evaluate the stability of folate liposome.3. The 4T1 mouse breast cancer cells was transplanted into BALB/c mouse vie the second breast fat pad to establish breast cancer model mouse where CXCR4 is highly expressed. Subsequently, the tumor targeting of wrapped calcein folate liposomes was investigated in this tumor model.4. Setting the envelopment rate of folate liposome to TAT/54R/KDEL as an index, the factors including the amount of phospholipid and cholesterol, the quantity of TAT/54R/KDEL, aqueous temperature and ultrasonic time were respectively investigated to know their effects on the envelopment rate of folate liposome to TAT/54R/KDEL. The systhesis process of folate liposomes was optimized through orthogonal design method.5. The activities of wrapped TAT/54R/KDEL folate liposomes on tumor growth as well as metastasis were evaluated through the 4T1 tumor-bearing model.Results1. SDS-PAGE electrophoresis showed that TAT/54R/KDEL was expressed successfully. After purification with nickel affinity chromatography and HPLC, the purity of TAT/54R/KDEL was more than 95%, with the yield rate of-5 mg/L in the medium. The results of flow cytometry demonstrated that, TAT/54R/KDEL has good knockout phenotype activity on the surface of CXCR4 MOLT-4 cells with concentration-depended activity.2. As the fluorescence microscope and atomic force microscopy showed, the prepared liposomes had uniform folate distribution and size, fluorescence intensity of the enveloped calcein was almost the same. the envelopment rate of folate liposomes to Calcein was 41.2%, and folate liposomes exhited relatively stablity within 48h.3. Pathological study confirmed that breast cancer mouse transplantation model was successfully constructed. Fluorescence microscopy and fluorescence spectrophotometer revealed that the accumulation of folate liposomes enriched in primary and metastatic breast cancer cells was significantly higher than that of liposomes, indicating that folate liposomes possessed favorable tumor targeting.4. Orthogonal design demonstrated that the most optimized experimental scheme for wrapped TAT/54R/KDEL folate liposomes was showed as followed:at a ratio of 3:1 (phospholipid: cholesterol), at the ratio of 20:1 (phospholipid:TAT/54R/KDEL), the temperature oscillation hydration at 4℃,5 min of ultrasond. Folate liposome encapsulation of TAT/54R/KDEL was at the radio of 46.1%, and folate liposomes was relatively stable within 48h.5. In vivo study indicated that the TAT/54R/KDEL enveloped by folate liposomes produced potent antitumor effect on breast cancer cell growth and also inhibited the metastasis.Conclusion1. TAT/54R/KDEL displayed potent phenotype-knockout activity on CXCR4 within the surface of MOLT-4 cell membrane in vitro.2. Folate liposomes exhibited favorable tumor targeting effect in the 4T1 tumor-bearing model mouse.3. TAT/54R/KDEL enveloped by Folate liposomes demonstrated enhanced tumor targeting, and produced potent inhibitory effect on tumor cell growth and metastasis in vivo.
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