Anti-VEGFR2 Immunoliposomal Oxaliplatin-Targeted Therapy In Colorectal Carcinoma

PART ONE PREPARATION OF ANTI-VEGFR2 IMMUNOLIPOSOMAL OXALIPLATINObjective We aimed to investigate a novel method for the preparation of anti-VEGFR2 immunoliposomal oxaliplatin to elucidate the bridging role of Streptavidin-biotin.Methods Biotinylated PEG-liposomes were prepared using reverse rotary evaporation. The biotinylated PEG-liposomes were linked to streptavidin molecules that act as a bridge between the biotinylated VEGFR2 antibody and the biotinylated PEG-liposomes. The size and zeta-potential of the liposomes were determined by a Laser particle size analyzer and by scanning electron microscopy; Laser confocal microscopy and flow cytometry detection were used to determine the characteristics and antibody activity of anti-VEGFR2 immunoliposomes; and the encapsulation efficiency and in-vitro drug release rate of anti-VEGFR2 immunoliposomal oxaliplatin were detected by HPLC.Results We obtained immunoliposomes with a particle size of 185.34±8.08 nm and zeta-potential of 19.98±2.96 mv. The evenly shaped liposomes had smooth surfacesand were evenly dispersed without aggregation or adhesion into groups. Confocal microscopy revealed a strong interaction between the rhodamine-labeled secondary antibodies and the anti-VEGFR2 primary antibodies. Flow cytometry analysis revealed binding of the secondary antibody to over 75% of the liposomes. The entrapment efficiency of the liposomes was (58.07±6.94)%, and after 120 h we observed an increase in cumulative percentage release of over 89%, as determined by HPLC.Conclusion The bridging role of Streptavidin-biotin is a feasible method for biotinylated VEGFR2 antibody binding to biotinylated PEG-liposomes. The preparation of anti-VEGFR2 immunoliposomal oxaliplatin is important as it determines the entrapment efficiency and in-vitro release rate of the therapy.PART TWO ANTI-VEGFR2 IMMUNOLIPOSOMAL OXALIPLATIN TARGETING IN HUMAN COLORECTAL CARCINOMA THC8307/L-OHP CELLS Objective We aimed to investigate anti-VEGFR2 immunoliposomal oxaliplatin induction of THC8307/L-OHP cell apoptosis and to elucidate the underlying mechanism.Methods We used immunofluorescence, Western blot, and RT-PCR to analyse the effect of VEGFR2 expression on THC8307/L-OHP cells, and the binding of anti-VEGFR2 immunoliposomes to cells was analyzed by flow cytometry, immunofluorescence, and scanning electron microscopy. MTT analysis was used to determine the administration drug dose and the cytotoxicity of anti-VEGFR2 immunoliposomal oxaliplatin. Apoptosis or proliferation was detected by flow cytometry and TUNEL analyses; RT-PCR analysis was performed to measure the mRNA expression of Bcl-2, Bax, Caspase-3, Ki-67, P38, ERK, and JNK. We performed Western blot analysis to determine protein expression of Bcl-2, Bax and activation of Caspase-3/P17, P38, ERK, and JNK. Whereas Ki-67 positive cells were visualized using immunohisto- chemisty. SP600125 was used as the pretreatment for THC8307/L-OHP cells in order to detect the induction of apoptosis by anti-VEGFR2 immunoliposomal oxalipolatin.Results We found that VEGFR2 was highly expressed in THC8307/L-OHP cells, compared with HUVE cells. The anti-VEGFR2 immunoliposomes were proactively combined with cells than non-immunoliposomes (P<0.05). THC8307/L-OHP cells were treated with free oxaliplatin (50μg/ml), PEG-liposomal oxaliplatin (containing oxaliplatin 50μg/ml), or anti-VEGFR2 immunoliposomal oxaliplatin (containing oxaliplatin 50μg/ml) for 12 h and the apoptosis rate was (10.02±0.69)%, (15.29±1.70)% and (55.89±8.90)%, respectively. There was statistical significance, compared with each group (P <0.01, P <0.05). The results of apoptosis analyzed by TUNEL were similar to the results obtained through flow cytometry. The mean CFSE relative fluorescence intensity ratio was 1.83±0.21, 1.56±0.16, and 1.29±0.10, respectively. It was statistical significance (P <0.05). The mRNA and protein expression of Bcl-2 and Ki-67 were significantly down-regulated, however, Bax and Caspase-3/P17 were significantly up-regulated (P <0.01, P<0.05). In the MAPK signaling pathway, the mRNA and protein expression of P38 were not changed significantly (P >0.05) and expression of ERK was down-regulated, while apoptosis was enhanced and JNK was up-regulated (P <0.05). After SP600125 pre-treatment, THC8307/L-OHP cells, apoptosis induced by anti-VEGFR2 immunoliposmal oxaliplatin was not inhibited by SP600125. Additionally, the mRNA and protein expression of Bcl-2 were down-regulated significantly (P <0.01), whereas Bax and Caspase-3/P17 were up-regulated (P <0.01).Conclusion The anti-VEGFR2 immunoliposomes were fortified combined with THC8307/L-OHP cells, and anti-VEGFR2 immunoliposomal oxaliplatin significantly induced apoptosis. These findings indicated that the ERK and JNK signaling pathways may be involved in apoptotic regulation.PART THREE ANTI-VEGFR2 IMMUNOLIPOSOMAL OXALIPLATIN TARGETING IN A NUDE MOUSE TUMOR-XENOGRAFT MODEL OF COLORECTAL CARCINOMAObjective We investigated the antitumor efficiency of anti-VEGFR2 immunoliposomal oxaliplatin potentialization in a nude mouse tumor-xenograft model of colorectal carcinoma.Methods In a tumor-bearing nude mouse model, we observed intravenous tail vein injections of (Dio)-labeled anti-VEGFR2 immunolipsomes using fluorescence imaging and in-vivo imaging systems. Mice were treated with free oxaliplatin (5μg/g), PEG-liposomal oxaliplatin (containing oxaliplatin 5μg/g), and anti-VEGFR2 immunoliposomal oxaliplatin (containing oxaliplatin 5μg/g) via the tail vein intravenous injections, followed by analysis of the accumulation of oxaliplatin in tumor tissues by HPLC, observation of the tumor volume, and the survival rate. Cell apoptosis and the proliferation of tumors was detected by the TUNEL assay, immunohistochemistry, and Western blot. Results The Dio-labeling anti-VEGFR2 immunoliposomal aggregation was greater than that of the non-immunoliposomal aggregation in the tumor tissues as visualized by the in-vivo imaging system: the majority of anti-VEGFR2 liposomes were found in the tumor cells and the surrounding capillary vessels in the tumor. The tumor volume suppression rate was determined to be (39.55±9.38)%, (50.34±8.86)%, and (61.20±9.77)%, respectively. There was statistical significance, compared with each group (P <0.01, P <0.05). Longer survival was observed in the nude mice that were treated with anti-VEGFR2 immunoliposomal oxaliplatin. After intravenous injection of free oxaliplatin, tumor tissue accumulation of oxaliplatin quickly decreased at 2 h. Upon administration of PEG-liposomal oxaliplatin and anti-VEGFR2 immunoliposomal oxaliplatin, the maximum value was detected at 24 h. Moreover, treatment with anti-VEGFR2 immunoliposomal oxaliplatin led to greater values than that treatment with PEG-liposomal oxaliplatin, with values maintaining a high level 72 h. The apoptotic index in tumor tissue was higher compared to the other groups (P <0.001, P <0.01 vs. P <0.05, respectively), but the proliferation index predominance was reduced (P <0.001, P <0.01vs. P <0.05, respectively). Additionally, protein expression of Bcl-2 was down-regulated, whereas Bax and P17 expression was up-regulated (P <0.01, P <0.05).Conclusion In this study, we demonstrated that anti-VEGFR2 immunoliposomes exhibit a strong aggregation ability. Moreover, treatment with anti-VEGFR2 immunoliposomal oxaliplatin is able to suppress growth of tumor tissue, contribute to tumor cellular apoptosis, and suppress tumor cell proliferation in a nude mouse tumor-xenograft model of colorectal carcinoma.PART FOUR ANTI-VEGFR2 IMMUNOLIPOSOMAL OXALIPLATIN INFLUENCES MULTIDRUG RESISTANCE OF HUMAN COLORECTAL CARCINOMA THC8307/L-OHP CELLSObjective We next analysed the effect of anti-VEGFR2 immunoliposomal oxaliplatin on the multidrug resistance of human colcrectal carcinoma THC8307/L-OHP cells.Methods A significant level of drug resistance reversal was observed in THC8307/L-OHP cells treated with anti-VEGFR2 immunoliposomal oxaliplatin as analyzed by MTT. Immunohistochemistry, RT-PCR, Western blot were performed to determine the expression of P-gp/MDR1, and ABCG2 when THC8307/L-OHP cells were treated with free oxaliplatin, various doses of Anti-VEGFR2 immunoliposomal oxaliplatin at 12 h, or one dose of Anti-VEGFR2 immunoliposomal oxaliplatin at distinct time points. The protein expression of ABCG2, P-gp/MDR1, GST3, Bcl-2, and Caspase-3/P17 were obtained by Western blot after THC8307/L-OHP cells were treated with either free oxaliplatin or various doses of anti-VEGFR2 immunoliposomal oxaliplatin for 48 h. P-gp/MDR1 and ABCG2 positive cells were analyzed by immunohistochemistry.Results A 1.21-fold reversal of drug resistance was observed. After THC8307/L-OHP cells were treated with free oxaliplatin (25μg/ml) and anti-VEGFR2 immunoliposomal oxaliplatin (containing oxaliplatin 25μg/ml vs. 50μg/ml) at 12 h, the observed mRNA expression of P-gp/MDR1 and ABCG2 was 2.22±0.33 and 2.67±0.40 vs. 3.26±0.47; and 2.85±0.43 and 7.74±1.16 vs. 8.68±1.30, respectively. Group comparison, P<0.05 vs. P<0.01. Furthermore, the protein expression of P-gp/MDR1 and ABCG2 was 5.68±0.85 and 10.04±1.51 vs. 17.62±2.64 (P <0.01); and 2.06±0.31 and 3.74±0.56 vs. 4.56±0.68 (P <0.05 vs. P <0.01), respectively. THC8307/L-OHP cells treated with anti-VEGFR2 immunoliposomal oxaliplatin (containing oxaliplatin 25μg/ml) at 12 h, 24 h, 48 h, and 72 h exhibited P-gp/MDR1 protein expression levels of 8.57±1.28, 16.36±2.45, 3.42±0.51, and 0.75±0.11, respectively. There was statistical significance, compared with each group (P <0.01, P <0.05). Protein expression levels of ABCG2 were found to be 1.27±0.19, 2.13±0.32, 1.63±0.24, and 0.55±0.08 (P <0.05), respectively. The P-gp/MDR1 and ABCG2 mRNA expression of resistance genes was significantly increased at 24 h, followed by a significant reduction in expression at 72 h (P <0.01 and P <0.05, compared with the control group). After treatment with free oxaliplatin (25μg/ml) at 48 h, the protein expression levels of P-gp/MDR1, ABCG2, GST3, Bcl-2, and Caspase-3/P17 were not changed significantly (P >0.05, vs. control). After treatment with anti-VEGFR2 immunoliposomal oxaliplatin (containing oxaliplatin 25μg/ml vs. 50μg/ml), the protein expression levels of P-gp/MDR1, ABCG2, GST3, and Bcl-2 were reduced, whereas Caspase-3/P17 was up-regulated. Compared with each group, P <0.01 vs. P <0.05. There were positive cells of P-gp/MDR1 and ABCG2 in tumor tissue. Nevertheless, the protein expression was to weaken following administration agent augmentation.Conclusion We found that the anti-VEGFR2 immunoliposomal oxaliplatin reversed multidrug resistance of human colorectal carcinoma THC8307/L-OHP cells, this may be attributed to the down-regulation of P-gp/MDR1, ABCG2, and GST3. Moreover, anti-apoptotic proteins Bcl-2 and Caspase-3 may be involved in the anti-VEGFR2 immunoliposomal oxaliplatin regulation leading to the multidrug resistance of THC8307/L-OHP cells.