Hypocrellin-modified Nanoparticle-mediated Photochemical Transfection Of Rev-caspases-3 Gene Induce Apoptosis Of Nasopharyngeal Carcinoma Cells

Background:Nasopharyngeal carcinoma(NPC)is one of the most common malignancy in southern China region, Although NPC is susceptible to radiotherapy and chemotherapy, the 5-year survival rate is about 65%. To explore novel therapeutic strategies can improve the clinical outcomes. Photodynamic therapy (PDT) shows considerable promise as a new treatment for malignant tumors on the basis of the accumulation of a light-sensitive molecule called photosensitizer in malignant tissues and a variety of photochemical reactions that cause cell damage and death when the photosensitizer is activated by light at appropriate wavelength. Recent studies have shown that caspase-3 plays a key role in the process of apoptosis. The caspase-3 is an effector - Caspase, which is in inactivation state due to lacking of a long domain, and its activation needs the protein hydrolysis effect from activated caspase. Once activated, they will destroy cells in the regulation of balance, leading to observed apoptosis. Traditional transfection methods were characteristed by lower efficiency, Greater cytotoxicity. Photochemical gene transfection (PGT) was a new way for transfection in vitro and in vivo. PGT was a new tool for gene transfection, which was able to be induced by light and gene transfection into a fixed target area, leading to increase the level of transfection. In this study, we synthesized the hypocrellin B modified cationic nano-liposomes, which demonstrated a highly efficient photosensitive and successfully constructed spontaneously activated Rev-caspase-3. Then we applied the nano-photosensitizer as a vector which transfected plasmid into nasopharyngeal carcinoma cells, and used photochemical gene transfection(PGT) technology to enhance the transfection efficiency to induce apoptosis. Our aims are to explore a new strategy for treating nasopharyngeal carcinoma.Chapter 1:Preparation of hypocrellin-modified cationic nano-liposomesPurpose:Hypocrellin B-modified cationic nano-liposomes: Preparation and characterization.Methods:Nanoparticle was prepared using the film dispersion-ultrasonic emulsification method. High-performance liquid chromatography (HPLC) was used to detect liposome encapsulation efficiency. MALVERN ZETASIZER 3000HS laser scattering particle size analyzer was used to detect plasmid size and voltage. To observe stability of the liposome, the prepared liposome was placed at 4℃in dark room for 7 days to observe the concentration of hypocrellin B with different storage times.Results: Hypocrellin B-modified cation nano-liposomes were prepared using film dispersion-ultrasonic emulsification method. Encapsulation efficiency of nano-photosensitizer rate was 85.8±4.5%. The nano-photosensitizer possessed a positive charge and voltage was 21.1mv, and its particle size range was 400-700 nm in which the average was 532 nm. The concentration of nano-liposome was no significant change when the liposome stored at 4℃for 7 days.Conclusion: We successfully prepared Hypocrellin B-modified cation nano-liposomes using film dispersion-ultrasonic emulsification method. Encapsulation efficiency was 85.8±4.5%. The nano-photosensititer was stable and suit for experimental use.Chapter 2: Photodynamic Action of LED-activated Nanoscale Photosensitizer in Nasopharyngeal Carcinoma CellsPurpose: The aim is to identify LED-activated nano-photosensitizer to inhibit cell proliferation and induce apoptosis in nasopharyngeal carcinoma cells.Method: The optimal uptake time of nano-photosensitizer in the NPC/CNE-2 cells was detected with the fluorospectrophotometer, and it was a basis for the photosensitizer incubation time in next step experiments. The MTT assay was used to observe cell proliferation of LED-activated nano-photosensitizer in the NPC/CNE-2 cells. PI staining and Hoechst33258 staining was used to measure apoptosis induced by nano-photosensitizer-mediated PDT. The expression of caspase-3 was detected to understand the mechanisms of apoptosis induced by PDT with flow cytometry .Results: Fluorescence spectrophotometer test showed that the optimal uptake time of Nano-photosensitizer in the NPC/CNE-2 cells was 6 hours. The LED-activated nano-photosensitizer could significantly inhibited proliferation of the NPC/CNE-2 cells. In the photosensitizer concentration of 10μM and the LED energy density of 1 J/ cm2, we can reach IC50. After 18 h post photodynamic therapy, obvious Sub-G1 peak was detected and apoptotic index was 34.32±1.92%. When the time prolonged to 30 h, apoptotic index increased to 44.91±2.03%. The expression rate of waspase-3 was 43.90% 24 h post PDT by use of FCM. Conclusion: The optimal uptake of the Nano-photosensitizer in the NPC/CNE-2 cells was 6 hours. The nano-photosensitizer-mediated photodynamic therapy can effectively kill the nasopharyngeal carcinoma cell. The mechanism may be associated with the induction of apoptosis. Nano-Photosensitizer-mediated photodynamic therapy induced apoptosis, the mechanisms of which must be the activation of Caspase-3 in mitochondria pathway, causing irreversible apoptosis procession.Chapter 3: Nano-photosensitizer transfected Rev-caspase-3 and photodynamic therapy effect on nasopharyngeal carcinoma cellsPurpose: The aim is to investigate the effects of Nano-photosensitizer transfected Rev-caspase-3 and nano-photosensitizer-mediated PDT on the NPC/CNE-2 cells and its mechanism.Method: Plasmid was amplified using Competence method. Identification of plasmid was performed using DNA double-digestion, and the binding capacity of nano-photosensitizer with plasmids was detected with gel retardation experiments. The expression of Nano-photosensitizer transfected Rev-caspase-3 in the cell was measured using flow cytometry. Nano-Photosensitizer-mediated photodynamic therapy and transfected Rev-caspase-3 into cells, the inhibition of cell proliferation was measured using MTT colorimetric assay. The apoptosis was detected post PDT and transfection by use of PI staining with flow cytometry.Result: Amplified plasmid was cut to two bands by DNA double-enzyme, which represented empty vector and Rev-caspase-3 sequence respectively. Gel retardation experiments showed that the higher the mass ratio of Nano-photosensitizer to plasmid, the stronger the ability of combination plasmid. Nanophotosensitizer-carried Rev-caspase-3 into the NPC/CNE-2 cells,then expression rate of caspase-3 was detected at 63.10% 24h post transfection. Cell survival rate after the combined treatment of Rev-caspase-3 transfection and nano-photosensitizer-mediated photodynamic therapy was significantly decreased compared with these two alone. Sub-G1 peak and apoptotic index was 24.65% after the treatment of Rev-caspase-3 transfection and nano-photosensitizer-mediated photodynamic therapy together.Conclusion: Hypocrellin B-modified nano-photosensititer can effectively combine with Rev-caspase-3 plasmids, and it can transfect Rev-caspase-3 into the NPC/CNE-2 cells, then expressed active caspase-3. Our experimental results showed that nano-liposomes were a good carrier for plasmid transfection. Transfection of Rev-caspase-3 and nano-photosensitizer-mediated PDT can effectively kill nasopharyngeal carcinoma cells, and induce apoptosis. Photodynamic therapy combined with gene transfer may be a promising strategy for cancer treatment.Chapter 4: Nano- photosensitizer-mediated photochemical gene transfection of Rev-caspase-3 causes apoptosis in nasopharyngeal carcinoma cells.Purpose: Hypocrellin B modified nano-photosensitizer-mediated photochemical gene transfection increases the efficiency of transfection,and transfected Rev-caspase-3 in nasopharyngeal carcinoma cells induce apoptosis.Method: The concentration of transfected nano-photosensitizer reduced to 4μM. FCM detected expression of capase-3 post photochemical transfection of Rev-caspase-3. The cell proliferation was detected with MTT assay. FCM detected cell cycle of in nasopharyngeal carcinoma cells after photochemical transfection of Rev-caspase-3.Result: The expression rate of the control group was 1.48±0.51%. when transfected with 4μM of nano-photosensitizer and 4μg of plasmid, the expression rate was 39.78±3.59%.When photochemical transfection with 4μM of nano-photosensitizer and 4μg of plasmid at the energy of 1J/cm2, transfection rate increased to 59.45±3.63%. When light dose increased to 0.2J/cm2, transfection rate reduced to 54.09±2.26%. Cell viability study showed that the expression of actived Caspase-3 post transfection was higher, leading to a lower cell survival rate. Cell cycle analysis showed that the expression of activated Caspase-3 post transfection was higher, leading to a higher apoptotic rate.Conclusion: Nano-photosensitizer can be used as transfection carrier. The Rev-caspase-3 was transfected into cells, and the transfection efficiency has been strengthened by LED-activated photochemical transfection .The efficacy of photochemical transfection could be enhanced by certain level of light energy, but the transfection efficiency is not increased followed with light energy increases. The higher light energy may damage the structure of DNA, leading to DNA inactivation. Transfected Rev-caspase-3 could spontaneously activate, copy, express activated caspase-3 to start the apoptosis cascade and cause cell apoptosis. The rate of apoptosis and activated caspase-3 expression efficiency showed positive correlation relationship.