Objective:To explore the feasibility of pretargeting technique for immunoPET with epidermal growth factor receptor (EGFR) monoclonal antibody in EGFR positive/negative tumor bearing mice.Methods:Cetuximab- Trans-cyclooctene (TCO)was obtained by modifying Cetuximab with TCO- N-hydroxysuccinimide (NHS). 2, 2′-((6-amino-1-(4, 7-bis-(carboxymethyl)-1, 4, 7-triazonan-1-yl)hexan-2-yl)azanediyl)-diacetic acid (L-NETA)was used as a chelating agent to prepare the radioligand 68Ga-L-NETA-tetrazine (Tz), then the labeling rate and in vitro stability of the product were determined. Human basal breast cancer cells MDA-MB-468 (EGFR+ ) and MDA-MB-231 (EGFR-) were cultured in vitro. In vitro experiments were performed to explore the specificity of the probe and the feasibility of pretargeting technique. Nude mice (Balb/c-nu) bearing xenografts of the above two cell lines were established. Cetuximab-TCO (50 μg) was injected into the tumor-bearing mice in advance, then 68Ga-L-NETA-Tz was injected at different time points (48, 36, 24 and 12 h), and pretargeting was realized through " click chemistry" . Small-animal PET imaging and biodistribution were performed to evaluate pharmacokinetic properties and specificity of the probe. The one-way analysis of variance was used to compare the data. Results:The 68Ga-L-NETA-Tz molecular probe was successfully prepared with the labeling yield >95%, and the radiochemical purity was >95% after 2 h. Cetuximab-TCO and 68Ga-L-NETA-Tz were added to MDA-MB-468 cells successively, and the cell uptake rate reached (0.69±0.04)% at 1 h, which demonstrated the feasibility of the pretargeting technique. PET imaging and biodistribution results showed that the best imaging results were obtained in 36 h pre-injection group, in which the tumor uptake was the highest ((0.77±0.05) percentage activity of injection dose per gram of tissue (%ID/g), 1 h) and the tumor/muscle ratio was optimal (4.67±0.46); the tumor uptake in the blocking group, the group without injecting Cetuximab-TCO, and the MDA-MB-231 group were significantly lower ((0.35±0.01), (0.39±0.05), (0.45±0.10) %ID/g; F=15.50, P=0.002). Conclusions:EGFR targeted immunoPET imaging is successfully performed in mouse models of breast cancer by injecting Cetuximab-TCO and 68Ga-L-NETA-Tz successively. It provides an effective method for immunoPET imaging of monoclonal antibodies.

Objective:To explore the feasibility and conditions of in vitro and in vivo imaging of triple-negative breast cancer using visible light emitted quantum dots(QDs) as the carrier to target epidermal growth factor receptor (EGFR). Methods:The water-soluble QDs reacted with Cetuximab to synthesize the probe QD-Cetuximab. The morphology, particle size, stability and luminescence properties of the probe were examined. Human breast cancer cells MDA-MB-468 (EGFR+ ) and MDA-MB-453 (EGFR-) were cultured. Cytotoxicity assays, in vitro imaging and fluorescence intensity quantification were performed after cells incubation with QD-Cetuximab and QDs. Eight MDA-MB-468 tumor-bearing mice models were constructed, 100 μl QD-Cetuximab and QDs were injected through the tail vein. In vivo imaging and probe distribution were obtained at different time points. Independent-sample t test was used to analyze the data. Results:QD-Cetuximab had a particle size of (40.34±2.44) nm detected by transmission electron microscope (TEM), a hydrated particle size of (57.85±4.69) nm detected by dynamic light scattering (DLS), and a stable structure. When the concentration of QD-Cetuximab was ≤50 nmol/L, the relative survival rate of cells was more than 90%, and when the concentration exceeded 100 nmol/L, the relative survival rate of cells was reduced to (72.52±4.91)% ( P<0.05). The red fluorescence of MDA-MB-468 incubated with QD-Cetuximab was stronger than that of MDA-MB-468 incubated with QDs and MDA-MB-453 incubated with QD-Cetuximab or QDs. The confocal fluorescent intensity quantitative determination showed that the ratio of QD-Cetuximab group/QDs group was 5.1 (863.36/169.97). Flow cytometry showed that the uptake of QD-Cetuximab and QDs by MDA-MB-468 increased with incremental incubating concentration, and the former was more significantly( t values: 12.25-38.11, all P<0.05). When the incubating concentration was 25, 50, 100, and 200 nmol/L, the quantitative average fluorescent intensity ratio of QD-Cetuximab group/QDs group was 5.4, 6.9, 7.4 and 6.2, respectively. The QD-Cetuximab and QDs probes mainly accumulated in the liver in vivo. The fluorescence emitted by tumor was not obvious under the high fluorescence of liver as a background. However, the fluorescence was visible in the isolated tumor tissue, and the quantitative fluorescence intensity of experimental group and control group were (2.46±0.60)×10 4 and (1.29±0.05)×10 4, respectively ( t=3.392, P=0.015). Conclusions:Cetuximab can increase the targeting ability of QDs and promote cell uptake. Although the isolated tumor imaging results are acceptable, further modification of QDs should be considered to reduce the liver uptake and improving in vivo fluorescence imaging efficiency.

ObjectiveTo investigate the mechanism of Yiqi Huoxue Tongluo prescription （YHTP） in the treatment of diabetic neuropathic pain （DNP）. MethodNinety SPF-grade SD male rats were randomized into blank， model， low- （2.25 g·kg^-1）， medium- （4.5 g·kg^-1）， and high-dose （9 g·kg^-1） YHTP， and mecobalamin （0.175 mg·kg^-1） groups. Except those in the blank group， the rats in the remaining 5 groups were fed with a high-fat and high-glucose diet and subjected to intraperitoneal injection of low-dose （35 mg·kg^-1） streptozotocin （STZ） to establish the model of DNP. The sciatic nerve conduction velocity in DNP rats was measured by the neurophysiological method， and the levels of interleukin-6 （IL-6）， IL-1β， and tumor necrosis factor-α （TNF-α） were measured by enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was employed to measure the mRNA levels of glial fibrillary acidic protein （GFAP） and extracellular signal-regulated kinase （ERK） in the spinal cord. Western blot was employed to measure the protein levels of GFAP and phosphorylated ERK （p-ERK）， and immunofluorescence staining to measure the fluorescence intensity of GFAP and p-ERK in the spinal cord. In the cell experiments， 100 mmol·L^-1 high glucose was used to induce the activation of astrocytes （CTX-TNA2） for the modeling of nerve cell injury. The cells were randomized into the normal， model， drug-containing serum （10% YQHT）， inhibitor ［10 mol·L^-1 corynoxeine （COR）］， drug-containing serum + inhibitor （10% YHTP + 10 mol·L^-1 COR） groups. The levels of pro-inflammatory factors （TNF-α and IL-1β） and the anti-inflammatory factor IL-10 in CTX-TNA2 cells were determined by ELISA， and the protein levels of GFAP and p-ERK in CTX-TNA2 cells by Western blot. ResultThe animal experiments showed that compared with the blank group， the model group presented reduced mechanical withdrawal threshold （MWT）， thermal work limit （TWL）， and nerve conduction velocity， elevated levels of fasting blood glucose， IL-1β， TNF-α， and IL-6， and up-regulated protein levels of GFAP and p-ERK， and mRNA levels of ERK1， ERK2， GFAP （P<0.01）. Compared with model group， YHTP increased the MWT， TWL， and sciatic nerve conduction velocity （P<0.01）， lowered the levels of IL-1β， TNF-α， and IL-6 （P<0.01）， and down-regulated the protein levels of GFAP and p-ERK， and mRNA levels of ERK1， ERK2， GFAP in the spinal cord （P<0.05， P<0.01）. The cell experiments showed that compared with the blank group， the model group had decreased survival rate， elevated levels of pro-inflammatory factors， and up-regulated protein levels of ERK and GFAP （P<0.01）. Compared with the model group， the YHTP-containing serum lowered the levels of IL-1β and TNF-α （P<0.05， P<0.01）， elevated the level of IL-10 （P<0.01）， and down-regulated the protein levels of ERK and GFAP （P<0.01）. ConclusionYHTP may inhibit the activation of astrocytes by inhibiting the ERK signaling pathway to reduce inflammation and thus relieve DNP.