Experimental And Clinical Study Of Carbon Nanoparticles In Axillary Lymph Nodes For Breast Cancer

Breast cancer is the most common malignancy in women, metastasis is the main reason of death. Regional lymphatic channel metastasis is the first occurrence and the most common way of transferring, axillary lymph node status is an important independent and crucial prognostic factor. Therefore, to accurately determine the axillary lymph node status, control or eradicate lymph node metastasis is an important part of diagnosis and treatment for breast cancer. Due to the conditions and technical limitations, sentinel lymph node biopsy has not yet been popularized in China, axillary lymph node dissection is still the most common of treatment with breast cancer.Metastasis lymph nodes will inevitably be left out during the axillary lymph nodes dissection, while, the amount of drug is lower of traditional intravenous chemotherapy can enter the lymphatic system, tumor cell can not be effectively killed, resulting in recurrence and metastasis, effect prognosis and survival. So, finding a lymph tracer that its not only can trace lymph nodes but also carry chemotherapy drugs into lymphatic system and releasing drugs slowly is very important. According to reports, carbon nanoparticles suspension(CNP) is a safe and effective lymphatic tracer and ideal drug carrier. Epirubicin(EPI) is a common and effect of drug for breast cancer treatment.In response to these problems, we explore and research the tracing and targeted chemotherapy effect of carbon nanoparticles-epirubicin suspension injection(CNP-EPI) in axillary lymph nodes for breast cacer in this study. First, the most appropriate ratio of adsorption and desorption will be experimented with carbon nanopaticles and epirubicin, following MDA-MB-231 and MCF-7 cells will be cultured in vitro, study on cytotoxicity or proliferation with CNP-EPI, finally, clinical cases and lymph tissues were collected according to the inclusion criteria, evaluation of the tracing and targeted chemotherapy effect of CNP and CNP-EPI in axillary lymph nodes dissection for breast cancer treatment.1. Adsorption and desorption will be experimented with carbon nanopaticles and epirubicinMethods: Adsorption and desorption effects of different concentrations of CNP and EPI will be detected with high-performance liquid chromatography mass spectrometry(LC-MS/MS) and dialysis method.Results: CNP and EPI has good adsorption effects, 1ml(50mg) CNP and 2ml EPI, concentration is 2.5mg/ml, 5mg/ml, 10mg/ml, 15mg/ml, 20mg/ml, adsorption rate is 94.66%, 85.96%, 80.12%, 71.09%, 69.03%, respectively. With the increasing of drug concentration, adsorption rate decreasing, the adsorption rate stabilizing will reach saturation. 1 ml(50mg) CNP and 2 ml EPI(5mg/ml) can slowly release EPI after adsorption, no burst release phenomenon finding, tend to balance after 48 h, the cumulative release rate can reach 65%.2. Inhibition effect of carbon nanoparticles-epirubicin suspension on the proliferation of human breast cancer cellsMethods: MDA-MB-231 and MCF-7 cells were cultured with different concentrations of CNP-EPI in vitro. CCK-8 assay was used for determinate inhibition effect of CNP-EPI on the proliferation of MDA-MB-231 and MCF-7 cells at different concentration and different time; According to the determination of IC50, 5μg/ml CNP-EPI, 25μg/ml CNP and only add culture medium were selected, cell morphology were observed and cell apoptosis rates were determinated by flow cytometry after 24 h.Results: The inhibition of CNP–EPI on the proliferation of MDA-MB-231 and MCF-7 cells was gradually strengthened in a dose-dependent relation when the concentration was from 1μg/ml to 200μg/ml within same time point, the inhibition effect showed increasing trend after 48 hours, but gradually became stabilization. The inhibition effect of the CNP-EPI was stronger significantly in CNP-EPI group than in normal control group at all time point(P<0.05), and the inhibition effect of different concentration of CNP was not obvious on MDA-MB-231 and MCF-7 cells; Obvious changes of cell morphology were observed under inverted microscope such as: a lot of dead cells, cell adherent growth poor, cell morphology became round and karyopycnosis etc, in 5μg/ml CNP-EPI group after 24 h. However, no obvious abnormity of cell morphology was observed in normal control group and corresponding CNP group; Two cells apoptosis rate was(14.3±6.25)%,(14.57±2.41)%, Methods & Results the mortality rate could reach(80.70±6.20)%,(78.63±0.55)% in 5μg/ml CNP-EPI group after 24 h, respectively.The mortality rate and apoptosis rate of cells was higher significantly in CNP-EPI group than in CNP group and normal control group(P<0.05).3. Clinical Research of the tracing effect of carbon nanoparticles in axillary lymph nodes dissection for breast cancerMethods: A total of 60 breast cancer patients were randomly divided into three equal groups: A, B, and C group from May 2012 to June 2014. A, B, and C group patients were subcutaneously injected with 1ml CNP at four separate sites around the areola 1 day, 3 days and 7 days before surgery, respectively. Modified radical mastectomy, endoscopic subcutaneous mammary resection plus axillary lymph nodes dissection and immediate reconstruction with implants, or breast-conserving surgery were performed. The total number of excised lymph nodes and the number of stained and unstained lymph nodes were counted and recorded for each patient.Results: A number of lymph nodes were dissected in the A(430), B(384) and C(380) groups. The mean number of dissected lymph nodes per patient were not statistically significant in the A(21.5±5.8), B(19.2±4.9) and C(19.0±5.8) groups(P>0.05). Most lymph nodes in the three groups were stained black(78.4%, 64.6% and 43.7%, respectively. The lymph node black dye rate was significant difference among the three groups(P<0.05), A group is the highest and C group is the lowest. Most metastatic lymph nodes were also stained black in the A group(70.0%), B group(61.1%) and C group(56.4%), and with no significant difference among the groups(P>0.05).4. Evaluation of the targeted chemotherapy effect of carbon nanoparticles-epirubicin suspension in axillary lymph nodes dissection for breast cancerMethods: A total of 90 breast cancer patients were randomly divided into three equal groups: control, tracer, and drug-load group from February 2014 to December 2014. Control group patients did not receive any lymphatic tracers, tracer group patients were subcutaneously injected with 1 ml CNP, and drug-load group patients, with 3 ml CNP-EPI at four separate sites around the areola 24 hours before surgery. Modified radical mastectomy, endoscopic subcutaneous mammary resection plus axillary lymph node dissection and immediate reconstruction with implants, or breast-conserving surgery were performed. Two nodes were not more than selected for each patient. Each lymph node was divided into two equal parts, one part was sent for routine pathological examination, and the other part was divided into two small portions, one portion was operated such as: formalin-fixed, paraffin-embedded and immunohistochemistry examination, the other would be detected epirubicin concentration and placed in a cold storage at-70℃. The remaining lymph nodes were sent to pathology examination. Lymph nodes metastasis were screened by HE from three groups, and then were examinated by Ki-67 and TUNEL staining, respectively.Results: The mean EPI concentration of lymph nodes was(2063.27±1236.39)ng/g stained and(108.77±96.39)ng/g unstained in the drug-load group,(143.72±89.36)ng/g stained and(110.68 ± 56.00)ng/g unstained in the trace group, and(125.85±72.29)ng/g in the control group, respectively. The mean EPI concentration of stained lymph nodes was significantly higher in the drug-load group than stained or unstained in the tracer group and the control group(P<0.05). The mean EPI concentration of umstained lymph nodes in the drug group was no significant difference among stained or unstained in the tracer group and the control group(P>0.05). The mean EPI concentration of stained lymph nodes was significantly higher than unstained in the drug-load group(P<0.05).The mean Ki-67 cell proliferation index of lymph nodes metastasis was(2.34±0.54)% stained in the drug-load group,(2.49±1.37)% stained in the trace group and(4.81±3.56)% in the control group, respectively. There was no significant difference among in the drug-load group, trace group and control group(P> 0.05).The mean cells apoptosis index of lymph nodes metastasis was(14.19±6.96)% stained in the drug-load group,(7.59±2.84)% stained in the trace group and(10.24±3.47)% in the control group, respectively. There was no significant difference among in the drug-load group, trace group and control group(P> 0.05).Conclusion:1) CNP and EPI has good adsorption and slow release effects, 1ml(50mg) CNP and 2ml EPI was more appropriate mixed with 5mg/ml~10mg/ml.2) CNP-EPI could obviously inhibit the proliferation or kill human breast cancer MDA-MB-231 and MCF-7 cells, and the inhibition effect of CNP-EPI on proliferation of breast cancer cells might be the result of delayed releasing of EPI.3) The majority of axillary lymph nodes were stained black with CNP subcutaneously injected around the areola 1 days before surgery, which helped identify the lymph nodes from the surrounding tissues and enabled their complete dissection.4) Axillary lymph nodes could be traced and chemotherapy drug concentration of stained lymph nodes metastasis could be increased with CNP-EPI, and could prolong targeted lymphatic chemotherapy effect in the lymph nodes.