| Gastric cancer is a common disease in China and a major problem of people health issue. 5 years survival of patients with gastric cancer depends on the stage of cancer. However, most people were diagnosed in advanced stage due to the insignificant and non-specific syndrome of gastric cancer. Currently, early diagnosis of gastric cancer depends on biopsy by endoscopy and immunohistochemistry, and it is important to develop specific and sensitive methods to detect gastric cancer in early stage. It is the best way to use endoscopy to detect cancer lesions in early stage, especially combined with optical molecular imaging technology, target cancer sensitively and specifically in cellular and molecular levels, and molecular probe is one of key factors in these process. Though numerous molecular probes have been developed and applied in targeted molecular imaging in tumor exnografts animal models, its application in clinical practice is restricted due to toxicity, low specificity, limited sensitivity and cost. For these reasons, it is important to develop safe, high biocompatible, high gastric cancer target specific, and lower cost molecular probes and then translate into clinical practice. Near-infrared fluorescence imaging(NIRF, wavelength range from 650 to 900nm), has advantages of deep tissue permeation, lower scattering, and can avoid the interference of autofluorescence of tissue, furthermore, NIRF imaging instrument can integrate with endoscopy to enhance the imaging contrast between tumor and normal tissue. GX1, a cyclic peptide, had been screened by phage-library in our laboratory previously, can target to human gastric cancer endothelial cell in vivo, and can be modify by isotope or fluorescence dyes for in vivo imaging. ICG(indocyanine green) is a NIRF dye approved by FDA for clinical practice in vascular imaging. Human serum albumin(HSA) can load ICG as a dye carrier and functionalize with peptide for targeting imaging.Aims Functionalized HSA-ICG by GX1 peptide, synthesized and characterized gastric cancer vascular target specific NIRF imaging probe GX1-HSA-ICG and evaluated its target imaging ability against Co-HUVEC in vitro and exnograft gastric cancer bearing mouse in vivo.Methods 1. To synthesize GX1-HSA-ICG, ICG was conjugated to HSA by electrostatic and hydrophobic interactions, then GX1 was covalently bond to the HSA-ICG after activation by EDC and sulfo-NHS.2. To characterize GX1-HSA-ICG by SDS-PAGE and Coomassie brilliant blue staining, UV/vis was obtained to confirm the binding of HSA and ICG. The size and zeta potential of GX1-HSA-ICG were measured by dynamic light scattering(DLS) using a Malvern Zeta Sizer.3. To measure the cytotoxity of GX1-HSA-ICG, Typan blue staining was conducted to detected GES cells validity after culture with probe or control.4. To evaluate the cancer vascular target specificity and fluorescence imaging capability, GX1-HSA-ICG was incubated with HUVEC and Co-HUVEC cells in vitro. GX1 without fluorescence dye labelling was mixed with GX1-HSA-ICG to compete the target site in Co-HUVEC cells.5. To estimate the target specificity of GX1-HSA-ICG in vivo, GX1-HSA-ICG was injected via tail vein of gastric cancer xenograft mice in vivo, and fluorescence intensity was detected by IVIS system.6. To evaluate the biodistribution of GX1-HSA-ICG, fluorescence intensity was measured by IVIS system 24 th hour after injected via tail vein of gastric cancer xenograft mice ex vivo.Results 1. GX1-HSA-ICG was synthesized successfully. SDS-PAGE and Coomassie brilliant blue staining revealed that GX1-HSA-ICG molecular weight was larger than that of HSA-ICG. UV/vis spectra of GX1-HSA-ICG showed the ICG and protein characteristic peaks in 280 nm and 800 nm respectively. The size of HSA-ICG could be turned by modified with GX1, the average hydrodynamic particle diameter of GX1-HSA-ICG was increased to 81±4.3nm, with an zeta potential of-35±0.4m V.2. After co-culture with solutions of probe or controls for 4 hours, no significant difference was found in the viability of GES cells among the groups of PBS(96±1.52%), ICG(95±1.15%), HSA-ICG(95±1.37%) and GX1-HSA-ICG(96±1.78%).3. The fluorescence signal was compared by cellular binding and compete assay in HUVEC or Co-HUVEC cells among the group of ICG, HSA-ICG, GX1-HSA-ICG and Block, and the average radiant efficiency [p/sec/cm2/sr]/[μW/cm2] were 3.09±1.09×108 vs 3.76±1.21×108, 1.54±0.21×108 vs 1.78±0.09×108, 8.83 ± 0.83×108 vs 1.51±0.01×109, 1.93±0.27×108 vs 2.31±0.36×108, respectively. The signal intensity of GX1-HSA-ICG in Co-HUVEC was significantly higher than that in HUVEC cells(P<0.01).4. GX1-HSA-ICG accumulation in tumor tissue specifically was confirmed by in vivo imaging, and tumor-to-background ratio(TBR) of GX1-HSA-ICG(7.81±0.32) is significantly higher than that of ICG(3.62±0.16), HSA-ICG(3.51±0.23) and Block(2.89±0.71).5. Biodistribution assay confirmed that TBR in the groups of ICG, HSA-ICG, GX1-HSA-ICG and Block were 7.53±1.11, 11.43±1.30, 22.20±1.09 and 7.11±0.20, respectively. And the TBR of liver is higher than that of other organs.Conclusions 1. Near-infrared fluorescence imaging probe GX1-HSA-ICG which could specifically target gastric cancer vessels is successfully synthesized. 2. GX1 could be conjugated to HSA-ICG. GX1-HSA-ICG had low cytotoxity and could target to Co-HUVEC specifically in vitro.3. GX1-HSA-ICG showed the gastric tumor-targeting ability in vivo, and could play a potential role in the diagnosis of gastric cancer and evaluation of therapy response. |
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