| Background and ObjectionToday, there are 3 popular methods for sentinel lymph node biopsy, through the injection of blue dye, radioactive tracer, or both. In foreign countries, the blue dyes used most widely are patent blue, vital dye and Isosulfan, while methylene blue is used most often in our country. This method is simple and cheap without the need of special equipment, but it is partly blind and mostly depends on the experience and skills of the surgeon.99mTc combined with sulfur colloid or dextran is the most used radioactive tracer. In this method, the sentinel lymph node can be detected exactly before the operation. But it needs expensive equipment and exposure to the radiation. There is no significant difference between the two methods in detection rate and accuracy to the experienced surgeons. But simultaneously using the two methods can detect the sentinel lymph node more exactly, increasing the detection rate and accuracy and decreasing the false negative rate.Compared with blue dye, the new method through the injection of fluorescence tracer can detect the sentinel lymph node more exactly without the danger from radiation. When the fluorescence tracer is injected into or around the primary tumor in the breast, it will enter the lymphatic system through the endothelial gaps or by the phagocytosis of endothelial cell and stay in the sentinel lymph node. With the irradiation of exciting light, the tracer will emit fluorescence which will be collected by the imaging system. Then, the image of the sentinel lymph node will be on the screen for the detection. Indocyanine green (ICG) is the representative of fluorescence tracer. It is a three indole carbon dye with near-infrared absorption peak. Its max emission wavelength is 795-845nm. It is hydrophilic and lipophilic. But its hydrodynamic diameter is so small that it can only stay in the lymph node for a short time. So it will quickly pass the sentinel lymph node into the next lymph node, which will increase the false negative rate.Activated carbon nanoparticles (ACNP) can arrive at the targeted lymphatic system accurately and stay in the lymphatic system for a long time. It has been used as a new tracer for sentinel lymph node biopsy. This method is simple and safe, but not do well in sensibility. Besides, due to its honeycombing structure, it can absorb medicine excellently. It has been reported to be medicine vehicle in the chemotherapy targeted at lymphatic system. In this study, ICG will be absorbed by ACNP to be ICG-ACNP, which will be injected into mice to investigate whether ACNP could improve the effect of ICG.Methods1. The preparation of ICG-ACNP suspensionACNP mixed with PVP-K30 in the saline were dispersed in ultrasound to achieve ACNP suspension, which was observed by transmission electron microscope. Its stability was tested under normal conditions. ICG-ACNP suspension was achieved when ICG was absorbed by ACNP. The absorbance of ICG in 780nm was measured by spectrophotometer. With the standard curve, the concentration of ICG was got. Then, the adsorption rate was figured out. The optimal adsorption time and mass ratio of ACNP and ICG were obtained. The hydrodynamic diameter and zeta potential ofICG-ACNP were detected by Zetasizer3000HS.2. The concentration of ICG20 BALB/c mice were randomly divided into 4 groups and injected with ICG solution of different concentration into the footpad of left hind limb. They were observed in the image system 15min after the injection.3. The effect of ICG and ICG-ACNP when injected into the mice10 BALB/c mice were randomly divided into 2 groups and injected with ICG solution or ICG-ACNP suspension into the footpad of left hind limb. They were observed in the image system 5,15,30,60,120min after the injection.Results1. The physical characterization and stability of ACNP suspensionACNP were spherical particles with smooth surface and less impurities while observed by transmission electron microscope. Their diameter were about 40nm and some masses were seen. ACNP suspension was black and well-distributed. No mass or sedimentation were seen by macroscopic observation. The suspension after centrifugation can be restored to well-distributed when dispersed in ultrasound again.2. The preparation of ICG-ACNP suspensionThe max adsorption rate was achieved when the concentration of ICG was 250mg/L.93% of ICG was absorbed by 10mg ACNP. It can be figured out that the optimal mass ratio of ICG:ACNP was 1:4.52% of ICG was absorbed 30min after the ultrasonic dispersion. The speed of absorption slowed down significantly 90min after the ultrasonic dispersion. The adsorption equilibrium was achieve at 120min after the ultrasonic dispersion.3. The concentration of ICGThere was no significant difference in fluorescence intensity when the concentration of ICG was 20,40 or 80ug/ml (P>0.05). But the fluorescence intensity of the concentration above were all significantly stronger than that of lOug/ml (P<0.05).20ug/ml was chosen as the concentration to use in the study.4. The effect of ICG and ICG-ACNP when injected into the mice5min after the injection, the popliteal lymph nodes of the mice in two groups were all detected. But the fluorescence intensity in ICG-ACNP group was weaker than in ICG group (P<0.05).30,60 and 120min after the injection, the fluorescence intensity in ICG-ACNP group was stronger than in ICG group (P<0.05).60min after the injection, the medial iliac lymph node was detected in 1 mouse in ICG group. 120min after the injection, the medial iliac lymph node was detected in 3 mice in ICG group. No medial iliac lymph node was detected in ICG-ACNP group.Conclusions1. The stable ACNP suspension was achieved successfully. By investigating the effect of the concentration of ICG and absorption time to the absorption of ACNP, the optimal preparation conditions of ICG-ACNP was confirmed, which is helpful for the further study.2. By combining ICG to ACNP, the ability of ICG to target at lymphatic system was improved, which made ICG can stay in the sentinel lymph node for a long time and not easy to pass the sentinel lymph node into the next lymph node. Theoretically, it will decrease the false negative rate. As the combination was physical adsorption, the effect to the fluorescence of ICG was not found in the study. So it can be predicted that it won’t decrease the detection rate of ICG. Further study is needed to confirm its clinical feasibility. Meanwhile, our study proved that ACNP can target at lymphatic system exactly and is the ideal vehicle to carry medicine into lymphatic system. |
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