| BackgroundThe Global cancer report 2014, published by the world health organization (WHO), has reported that the incidence and mortality rates of the liver cancer were 6% and 9% respectively. At the same time, liver cacner is the second most common cause of death from cancer in the world. In 2012, there were 782500 new cases of liver cancer. The population of China is about a quarter of the world population, but the liver cancer patients in China have accounted for more than half of these patients in the world. Throughout the past 50 years, although many countries has spended much funds in researching the diagnosis and treatment of tumor, the prognosis of cancer was still poor. Such as the United States, the national cancer institute research budgets has been reach $6 billion, However, the prognosis of the tumor has not significant improvement.The treatment of liver tumor include (1) surgery;(2) liver transplantation;(3) chemical drug treatment;(4) ablation therapy;(5) the traditional Chinese medicine treatment; (6) the combined treatment of the above method. And the surgery or the liver transplantation were the most likely way for curing liver cancer. However, due to the donor liver was very few and the cost was high, the liver transplantation can not be applied in clinic widely. At present, the surgical treatment is considered to be the preferred way for treat liver cancer. The patients could,be cured if the entire solid tumor can be completely removed by surgery. However, the 5 years recurrence rate was about 100% and the 5 years survival rate was only 5% after the liver cancer patients treated by surgical resection. The main reason were listed as following:(1) The conventional diagnostic instrument, such as CT, MRI, Ultra sound and so on, were difficult to make a effective diagnosis of the tumor less than 1 cm, because of the limitation of its resolution; (2) During the surgery, the doctors remembered the preoperative imaging data and comprehesive their inspection or palpation of the tumor (color, shape, texture) to locate the position and margin of the tumor for resecting the tumor. Then, it is difficult to objectively judge the tumor location and edge. The surgion would resect the tumor rely on their subjective judgment, it would be hard to judge whether the surgical margin was clear without any cancer cell residue and whether there is a tiny metastatic carcinoma residue. The recurrence of the cancer can not be advoided. Moreover, the patients with postoperative recurrence, the effect of secondary surgery and postoperative chemotherapy is extremely limited. Therefore, the surgeons need a kind of technology which can help them find small tumor and precise tumor boundary intraoperatively. Then the surgeons can locate the position and the margin of tumor in real-time, accurately, and objectively. It would be very significance for the patients to achieve the complete removal of the tumor, gett negative surgical margin, reduce the postoperative recurrence rate, improve the postoperative survival rate, and improve their quality of life.The optical molecular imaging guided surgery has been concerned by the surgeons since the japanese doctor applied the near-infrared fluorescent imaging guided navigation system to resect liver cancer. Compared with the traditional clinical imaging system, intraoperative fluorescence imaigng has many advantance, including low cost, easy for handling, no radiation, real-time, high resolution, and high sensitivity and so on. Currently, The indocyanine green (ICG) has been used in many clinical research. For example, this technology has been applied to detect the sentinel lymph node of breast cancer, gastric cancer metastasis lymph node, liver cancer, metastatic hepatic carcinoma and so on. However, there are some disadvantances should be considered. The ICG has bad water stability and has not active targeting ability. It would be removed from the body quickly because it combines with lipoprotein easily and its plasma half-life time is only 2-4 min. Then there is only little ICG uptaked by the tumor. The false positive reached 40%-50% in liver cancer surgery. The above disadvantances of the ICG limited the usage of the ICG in the reaserch and clinical. In order to overcome these difficulties and make better use of the fluorescent surgery navigaiton methods, scientists are beginning to use different molecular probes for magnetic resonance imaging (MRI), optical imaging, ultrasonic imaging and nuclide imging fro non-invasive imaging for single cell. Meanwhile, active targeting probe as the method of fluorescence surgery navigation medium is also the international research hot spot. In 2010, Roger tsien’s team applied fluorescence molecular imaging active targeting probe (ACPPDs) to detect the breast cancer tumors and navigational resection. The results found that, compared with traditional methods, this method reduced the residual rate and the postoperative survival rate are improved obviously. In 2013, Roger tsien’s team applied fluorescence molecular imaging active targeting probe (ACPPDs) to detec breast cancer lymph node metastasis in nude mice, the results pointed out that this kind of active targeting fluorescent probe could locate the lymph node metastases of breast cancer, metastasis of lymph node excision and real-time guidance. In 2014, Debadyuti Ghosh’s team used active targeting probe SBP-M13-SWNT to diagnose submillimeter ovarian tumor and achieve the real-time fluorescence surgery navigation. In clinical applications, in 2013, Matthew b. Sturm’s team used active early esophageal tumor targeting of fluorescent probes (ASY*-FITC) complete the first human trials and operation effect is good. In many optical imaging, near-infrared fluorescence imaging is a special type of fluorescence, as well as an important development trend in current optical fluorescence imaging, compared with the conventional optical, near-infrared fluorescence (NIR,650 to 900 nm) affected by the background of biological tissue is small, and scattering attenuation is small in the biological tissues, thus, NIR has good penetration ability.Because it has more advantages, near-infrared fluorescence imaging probe development has been at the forefront of molecular imaging research. However, the tumor-specific targeting near-infrared fluorescence molecular probe is still lack. It is very significant to make near-infrared fluorescence of fluorescent molecular probes for the NIR surgery navigation.In this experimental study, we would fabricate a NIR active targeting probe for liver tumor navigational resection. The mesoporous silical nanoparticles (MSNs) with good biocompatibility were served as the carrier of molecular probe. The ICG was served as the NIR fluorescent dye. Then the molecular probe were modified by the RGD peptides, which could active targeting to integrins αvβ3 receptor. The nude mouse bearing subcutaneous liver tumor or orthotropic liver cancer or breast cancer metastatic liver cancer were applied to study the value of locating tumor position and tumor margin, and the effiency in NIR navigaitonal surgery.Objective:The aim of this study is to fabricate an NIR active targeting probe, which is targeting to integrin αvβ3 receptor. With the help of the targeting probe, the surgeons could locate the tumor and the tumor margin in real-time and accurately during operation. Then, the surgeons would finish the navigational surgery precisely with the objective surgery edge.Methods:1. The synthesis and charactize of MSNsThe MSNs were synthesized by hydrothermal synthesis method. The morphology and the size of MSNs were observed by transmission electron microscope (TEM) (JeoL-1011, Japan) and scanning electron microscopy (SEM) (HITACHI S-4800, Japan). The distribution of hydrodynamic particle size was measured with Dynamic Light Scattering (DLS) by the Malvern Zeta sizer (ZEN 3600, UK)..2. The synthesis and charactize of ICG/MSNs-RGDTEM and SEM were used to scan the morphology and measure the size of the MSNs and ICG/MSNs-RGD with a 100 kV acceleration voltage. The distribution of hydrodynamic particle size was measured with Dynamic Light Scattering (DLS) by the Malvern Zeta sizer. The fluorescence excitation and emission spectrum of the ICG and the ICG/MSNs-RGD were measured by a fluorescence spectrofluorometer (F-7000, Hitachi, Japan). For stability analysis,50μg of ICG/MSNs-RGD probes were added to 1 mL of fetal bovine serum (FBS; Gibco, Australia) and its optical absorbance at 780nm was monitored by the ultraviolet-visible spectroscopy (UV-Vis) (UV-2450, Shimadzu, Japan) at multiple time points for 24 h and compared to that of a serum-only control solution.3. Cell cytotoxicity assays and Integrin binding assaysThe cellular cytotoxicity of the MSNs and ICG/MSNs-RGD probes was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Promega, China). To evaluate the probe specificity to integrin αvβ3 receptor, MDA-MB-231-GFP and MCF-7 (Department of Radiology, Peking Union Medical College Hospital, China) cells with high and low integrin αvβ3 expression, respectively, were used to evaluate the probe’s specificity. MDA-MB-231-GFP and MCF-7 cells were incubated with equivalent concentrations of ICG/MSNs-RGD (0.2 mg/mL) for 3 h. They were washed three times with cell culture medium and two times with PBS, and then 2 mL of cell culture medium was added to the culture dish for culturing the cells continually. A split fluorescent microscope and NIR-fluorescence imaging-guided surgery system were used to observe the cell at excitation wavelength of 780 nm and an emission wavelength of 840nm.4. Mice modelsAll animal studies were performed in compliance with the principles and procedures outlined by the Institutional Animal Care and Use Committee of Zhujiang Hospital of Southern Medical University. All animals were purchased from the Department of Experimental Animals at Peking University Health Science Center (China).The subcutaneous hepatic tumors model were established by subcutaneous injection of 1×107 Hep-G2-GFP cells into left upper elbow back in 5-6 week old BALB/c nu/nu male mice.The orthotopic hepatic cancer model was established by injecting 1×107 Hep-G2-GFP cells into the liver of 5-6 week old male BALB/c nu/nu mice in PBS with an injection volume of 50μL. Tumors were allowed to grow for 14 days, and then visualized with an IVIS Spectrum Imaging System (PerkinElmer, Germany).For the metastatic breast cancer model, 1×107 MDA-MB-231-GFP cells were injected into of 5-6 week old female BALB/c nu/nu mice in PBS with an injection volume of 50μL. Tumors were allowed to grow for 14 days prior to monitoring with IVIS as described above.5. In vivo study the target function of ICG/MSNs-RGDEight mice with subcutaneous hepatic tumors> 5 mm in diameter selected for in vivo fluorescence imaging were anesthetized with 3% isoflurane air mixture, and 150 μL of ICG/MSNs or ICG/MSNs-RGD (0.2 mg/mL dispersed in PBS, n=4/group) was administered via tail vein injections. Mice were monitored by IVIS at 10 min,24, h,48 h,72 h,96 h, and 120 h post-injection with excitation and emission wavelengths of 780 nm and 840 run, respectively. Imaging data was analyzed with IVIS Living Image 3.0 software (PerkinElmer). And within 24 h, the distribution of the different probe in tumor-burdened nude mice, the mice were took off the neck for death, Then the cancer, heart, liver, spleen, kidney and other important organs were took off and observed the distribution of the probe in each important organs.6. The application of the targeting probe in delining the tumor margin precisely.The feasibility of ICG/MSNs-RGD-mediated intraoperative NIR image guidance was first investigated in mice bearing subcutaneous hepatocellular carcinoma (Hep-G2-GFP-fLuc) xenografts.3 subcutaneous liver tumor nude mice model were chosed, the ICG/MSNs-RGD 150μL (0.2 mg/mL) were injected via tail vein, after 48 h, the near infrared fluorescent microscope surgery navigation system were applied to locate the position and the margin of the tumor, the green fluorescence image as the reference standard. The tumor specimens were deal with HE staining, and observed by the near infrared fluorescent microscope surgery navigation system. Then the tumor margin were observed with near-infrared fluorescence.7. Surgical residual verificationSurgical studies were performed by two general surgeons (Zhujiang Hospital, China). For this,12 mice (6 mice per surgeon) were treated with 150μL of 0.2 mg/mL ICG/MSNs-RGD 48 h prior surgery. Surgeons were asked to remove tumor tissue with maximum protection of the normal tissue based on their visual inspection and palpation. After this procedure, NIR images and GFP images were acquired to determine whether tumor residuals were present. If tumor tissue remained, the surgeons continued the resection with the imaging guidance until all tumor tissue was removed. Tumor number and size were recorded, and the resected tissue used for histological examination.8. Intraoperative detection of metastatic lesionsThrough continuous monitoring 3 orthotopic implant liver cancer (Hep-G2-GFP-fLuc) mice models using bioluminescence imaging, we found intrahepatic metastases in 3 mice and peritoneal satellite lesions in other 5 mice. Surgical studies were performed by a general surgeon on animals administered 150 μL 0.2 mg/mL ICG/MSNs-RGD 48 h prior to surgery. Both orthotopic liver cancer model mice were anesthetized with 120μL 4% chloral hydrate (98%, Alfa Aesar, China) solution and fixed on the operational bed with the supine position. The abdominal cavities were exposed with a longitudinal incision in the middle of the abdomen. The surgeon then resected the all primary and metastatic tumor tissue under the guidance of the NIR-fluorescence imaging system. The tumor-to-normal liver tissue ratio for each ICG/MSNs-RGD probe was evaluated by drawing ten regions of interest (ROI) (0.2 mm2) within the tumor nodules and adjacent liver tissue to measure the average fluorescence intensity of each ROI. Tumor-to-normal tissue ratio (TBR) value was then calculated as the average fluorescence intensity of tumor nodules divided by that of surrounding areas.9. The application of the targeting probe in locating the metastatic hepatic carcinoma and abdominal metastatic carcinoma.3 breast cancer liver metastasis tumor nude mice model were chosed, the ICG/MSNs-RGD 150μL (0.2 mg/mL) were injected via tail vein, after 48 h, the metastatic liver cancer model mice were anesthetized with 120μL 4% chloral hydrate (98%, Alfa Aesar, China) solution and fixed on the operational bed with the supine position. The abdominal cavities were exposed with a longitudinal incision in the middle of the abdomen. The surgeon then resected the all primary and metastatic tumor tissue under the guidance of the NIR-fluorescence imaging system. The tumor-to-normal liver tissue ratio for each ICG/MSNs-RGD probe was evaluated by drawing ten regions of interest (ROI) (0.2 mm2) within the tumor nodules and adjacent liver tissue to measure the average fluorescence intensity of each ROI. Tumor-to-normal tissue ratio (TBR) value was then calculated as the average fluorescence intensity of tumor nodules divided by that of surrounding areas.10. Histological analysis and NIR imaging of resected specimensExcised tissues were embedded in OCT, snap-frozen in liquid nitrogen, and stored at -80℃. Sections were cut on a cryostat microtome (Leica, Germany) at a thickness of 8μm and stained with H&E. Following pathological examination, the ICG/MSNs-RGD-based fluorescence images were collected using the NIR-fluorescence imaging system to determine the fluorescence signal differences between tumor tissues and normal tissues.11. Statistical analysisStatistical analysis was performed in SPSS v.20 (IBM Software, USA). Data are expressed as the means ±standard deviation (SD). The overall comparison between groups used analysis of variance of repeated measurement data. Single factor comparison in the group were analyzed with completely random design analysis of variance. Two-tailed, independent two-sample T tests were used to assess differences in the optical densities and fluorescence intensities between groups. Data analysis use IBM SPSS 19.0 software for analysis. Statistical significance was defined as p-values <0.05.Results1. The MSNs has uniform spherical morphorlogy. The size of the MSNs is about 100 nm and has good dispersion. The targeted probe ICG/MSNs-RGD keep the specifications of the ball and the size also about 100 nm. Compared with the ICG, the Excitation wavelength (780 nm) and emission wavelength (837 nm) redshift 3-4 nm.2. the survival rate of Hep-G2-GFP cells revealed that both nanoparticles exhibited excellent biocompatibility. The differences in probe uptake between MDA-MB-231-GFP and MCF-7 with high and low integrin αvβ3 expression, respectively, indicated satisfactory tumor specificity for the ICG/MSN-RGD probe, implicating integrin αvβ3 receptor binding as the mechanism of RGD-conjugated probe uptake.3. In vivo validation of RGD-mediated tumor specificityAfter continuous observation over 120 h, fluorescent imaging indicated a marked accumulation of both probes in tumor lesions up to 96 h; however, the ICG/MSN-RGD probe displayed better optical contrast than ICG/MSN in the tumor region from the 24 h time point onward. After the initial distribution period (<12 h), the majority of the optical signal was emitted from the tumor and abdominal areas and gradually faded away after 24 h post-injection. Notably, the tumor to background (TBR) of the ICG/MSN-RGD peak (4.92±0.2) appeared at 48 h post-injection. These data confirmed ICG/MSN-RGD tumor specificity and indicated that surgeries be performed 48 h after probe injection for optimal NIR-FMI guidance.4. Targeted probe in intraoperative can provide clear tumor location and tumor boundary, provide an objective tumor boundary for the surgeons.5. The results turned out to be that 3 and 2 residual microtumors in total were detected for Surgeon 1 and Surgeon 2, respectively. While it was not difficult to remove major tumor tissues 5.01±2.08 mm in size, it was impossible for the surgeons to identify residual microfoci <0.95±0.07 mm. However, Because of the enhanced ICG loading capacity and great tumor specificity of ICG/MSN-RGD, intraoperative NIR-FMI was able to provide sufficient optical contrast and highly sensitive detection of such small lesions, which allowed for the full removal of tumor tissues. Moreover, the pathological examination of resected tissue confirmed that all specimens (n=17) were true positive tumors.6. For this, the intraoperative NIR-FMI-guided surgery was performed on three mice with intrahepatic metastases 48h after the Ⅳ administration of ICG/MSN-RGD (0.2 mg/mL,150μL). Notably, only implanted tumors (5.09±2.31 mm) could be visually recognized after exposing the liver, and the NIR imaging confirmed this with accurate tumor margin definition, meanwhile NIR-FMI provided for the accurate detection of microtumor lesions (0.4±0.31 mm) with excellent optical contrast (TBR: 4.7±0.21). These highly suspicious satellite foci were impossible to notice without the help of ICG/MSN-RGD. Moreover, H&E staining and NIR fluorescence imaging of the specimens confirmed all foci as tumor nodules, and ICG/MSN-RGD was indeed accumulated in the tumor lesions. Unexpectedly, we also found five other mice with peritoneal satellite lesions. The average size of these lesions were about 1 mm. The TBR of all satellite lesions was 5.23±0.82.7. we also simulated liver metastasis from breast cancer, which is the second most common hepatic metastasis in clinical practice, by transplanting human breast cancer cells (MDA-MB-231-GFP) into mouse livers (n=4). After the administration of ICG/MSN-RGD, the intraoperative NIR-FMI successfully detected the transplant tumor lesions in all mice. All the detected tumor lesions were confirmed with histological analyses. This indicated the great potential of using ICG/MSN-RGD for a wide range of clinical scenarios in the liver cancer surgery, intraoperatively.Conclusion:In this stuty, We have successfully constructed a active targeting tumor αvβ3 receptor probes (ICG/MSN-RGD), which is hight loading NIR dyes (ICG) in MSN, for fluorescence imaging-guided surgery. The targeting probes ICG/MSN-RGD are sensitively and precisely for the detection and definition the tumor location and border of human liver tumors and its tiny metastases or residual tumors in tumor-bearing mice during operation. Meanwhile, it performs best biocompatibility, good stability, high water dispersibility, non-cytotoxicity and the active targeting ability in vitro and in vivo. Overall, the studies assuredly demonstratied the significant potential application of ICG/MSN-RGD as non-invasive fluorescence nanoprobe for in vivo tumor-targeted diagnosis and fluorescence imaging-guided surgery. |
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