Study On Gap-enhanced Raman Tags For Involved Sentinel Lymph Node Imaging In Cervical Cancer

Cervical cancer is one of the most common gynecological malignancies in the world.Radical hysterectomy with pelvic lymphadenectomy is still routine procedure for cervical cancer.However,lymph node involvement is found in fewer than one fifth of patients with early cervical cancer,meaning that most of these patients underwent unnecessary pelvic lymphadenectomy which is probably associated with many morbidities such as nerve injury and lymphedema.Moreover,it will reduce significantly the patient’s quality of life.Sentinel Lymph Node（SLN） refers to the first station of lymph node that accept the lymphatic drainage from the primary tumor site and is the most likely involved lymph node.Hence,the assessment of regional lymph nodes can be evaluated by Sentinel Lymph Node Biopsy（SLNB） in theory.Sentinel lymph node biopsy requires the use of sentinel lymph node tracer and the corresponding mapping technique to complete the sentinel lymph node localization,however,all the existing tracers and mapping techniques have some drawbacks.At present,the sentinel lymph node mapping technique can only localize the sentinel lymph node,while fail to directly evaluate the lymph node status during the procedure.Pathological examination is still the"gold standard"for assessing lymph node metastasis,but the pathological examination has an obvious time limitation.Therefore,the development of a new SLN tracer and mapping technique to overcome the shortcomings of the existing techniques is of great importance.On this basis,designing an active targeting tracer to assess the involved sentinel lymph node in cervical cancer surgery will hold promise for future clinical practice.Folate receptorα（FRα） is a membrane receptor overexpressed in various malignant tumors.Tumor cells uptake a large amount of folic acid（FA）to meet their increasing requirement for rapid proliferation.The high affinity of FRαfor binding and transporting FA into cells makes it an ideal target of cancer cells.Therefore,the preparation of FA-conjugated SLN tracer is expected to achieve specific identification of involved SLN during surgery of cervical cancer.Surface-Enhanced Raman Spectroscopy（SERS） refers to the great enhancement of Raman scattering by molecules adsorbed on rough metal surface due to the excitation of the surface plasmons and the surface chemical effect,which can achieve single molecule detection with extremely high sensitivity.In addition,its unique“fingerprint”spectrum makes its signal highly specific,the ultra-narrow half-width peak is in favor of multiplexing detection for optical encoding,and its good photostability without fluorescence quenching is another apparent advantage.Based on these outstanding merits,SERS nanoprobe has a great potential in the biomedical application.Therefore,SERS nanoprobe and Raman imaging are very promising as a new SLN tracer and tracing technique for SLNB.This thesis is divided into three parts:（1）Preparation of Gap-Enhanced Raman Tags and Raman imaging in sentinel lymph node mapping;（2）The expression of folate receptorα in cervical cancer and the preparation of folic acid modified targeting gap-enhanced Raman tags;（3）Study of FA-MS-NBT-GERTs used in the targeting imaging on cervical cancer cells and in cervical cancer with involved SLN animal models.Objective 1.To investigate the preparation of Gap-Enhanced Raman Tags（GERTs）and its use in sentinel lymph node mapping as a sentinel lymph node tracer.2.To investigate the expression of folate receptorα（FRα） in cervical cancer tissues and cell lines,to study on preparation of FA modified active-targeting Gap-Enhanced Raman tags.3.To investigate the targeting effect of FA modified targeting Gap-Enhanced Raman tags（FA-MS-NBT-GERTs）on cervical cancer cells and to investigate its targeting imaging in cervical cancer with involved SLN animal models.Methods 1.A new type of mesoporous silica（MS）-coated Gap-Enhanced Raman tags were prepared by"label embedding method".The microstructure and morphology were characterized by transmission electron microscopy（TEM）,the distribution of particle size and Zeta potential of MS-GERTs were examined by dynamic light scattering（DLS）laser diffraction particle size analyzer,the spectrophotometer（UV-Vis）was also used for characterizing the morphology,particle size and other optical properties of MS-GERTs probes;the Raman signal intensity,optical stability and detection sensitivity of the probes were analyzed by Raman spectroscopy.2.The distribution of the probes in the sentinel lymph nodes at different time points（0.5 h,2 h,4 h,8 h,24 h）and the dynamic process of the probes entering the sentinel lymph node were achieved by three dimensional（3D）Raman imaging.3.A quantitative volumetric Raman imaging（qVRI）data-processing method is employed to acquire a high-resolution 3D margin of SLN as well as the content variation of MS-GERTs in the SLN,the Au content in each lymph node at different time points was determined by the atomic adsorption spectrometry.4.Using a commercial portable Raman scanner to pinpoint the SLN in vivo.5.CCK-8assay was used to evaluate the cytotoxicity of MS-GERTs,biocompatibility of MS-GERTs was analyzed in vivo.6.Immunohistochemistry was used to determine the expression of FRαin cervical cancer and normal cervix and their relationship with clinicopathological parameters.7.The Real-time PCR was used to determine the expression of FRαmRNA in cervical cancer cells,the expression of total FRα protein in cervical cancer cells was detected by Western Blot,the expression of FRαon the membrane of cervical cancer cells was examined by using flow cytometry.8.A new generation of GERTs probes were prepared by embedding another Raman reporter molecules（4-Nitrothiophenol,NBT） via "label embedding method",named FA-MS-NBT-GERTs.The surface of the GERTs was modified to carboxyl group through amidation reaction by using DMF and succinic anhydride reacted with the amino in the surface of GERTs.Transmission electron microscopy（TEM） and UV-Vis were used to observe the morphology,particle size and dispersibility of FA-MS-NBT-GERTs.The particle size distribution and Zeta potential of FA-MS-NBT-GERTs were characterized by DLS laser particle size analyzer.The Raman signal intensity of the probe was analyzed by Raman spectroscopy.9.The targeted effects of FA-MS-NBT-GERTs on FRαoverexpressed HeLa cells were determined by Raman imaging.10.The cytotoxicity of FA-MS-NBT-GERTs was detected by CCK-8 assay,the biocompatibility of FA-MS-NBT-GERTs was analyzed in vivo.11.Cervical cancer with involved sentinel lymph node animal model was established through the implantation of GFP-Luc-HeLa cells in the hind limb paw and the primary tumor spontaneously metastasized to SLN.The mixture of active targeting probes（FA--GERTs） and non-targeting probes（Nt-GERTs） with same dose and concentration were injected locally and undergo 3D Raman imaging of SLN in animals.12.The Raman spectroscopy datasets were divided into different Raman spectra by classical least squares（CLS）,one of machine learning methods,the ratio between the two probes was calculated to determine whether the SLN was metastatic.13.The accuracy of this ratiometric imaging method used for the diagnosis of SLN metastasis was testified by pathological assessment.Results 1.Mesoporous silica coated gap-enhanced Raman tags（MS-GERTs） was successfully prepared.MS-GERTs particle shows a core-shell structure with Au core and Au shell,Raman reporter molecules（1,4-benzenedithiol,BDT） were sandwiched in the sub-nanometer sized gap between the Au core and the Au shell.A mesoporous silica layer was coated on the outer surface of GERTs.The probe was spherical or oval of shape,with an average diameter of 82±4 nm and an average Zeta potential of +20.1 mV,the maximum ultraviolet absorption wavelength was at 545 nm.The characteristic Raman bands of BDT-GERTs were at 732,1055 and 1555 cm^-1.With continuous laser irradiation for 30 min,there was hardly detectable change of the Raman signal intensity after 30 min continuous irradiation compared to the initial one,suggesting that the MS-GERTs probe has extraordinary photostability.The Raman detection threshold of aqueous MS-GERTs solution could be as low as 100 fM,suggesting the extreme sensitivity of the probes.2.The results of sentinel lymph node mapping showed that the MS-GERTs can precisely localize in the sentinel lymph nodes from 2h to 24h after subcutaneous injection in the hind foot pad,and did not enter the higher tier lymph node,suggesting that MS-GERTs met the standard of an ideal SLN tracer.3.The qVRI data processing method could quickly identified different components through specific Raman spectrum obtained from raw dataset and presented as different colors for MS-GERTs,adjacent tissues and the background,then reconstruct a high-contract 3D SLN Raman image,it could also calculate the proportion of MS-GERTs in each lymph node,which was similar to the result calculated by atomic absorption spectrometry.4.The portable Raman scanner successfully achieved the real-time localization of sentinel lymph nodes in living animals,suggesting that MS-GERTs and Raman spectroscopy have the potential for clinical translation.5.Cytotoxicity assay showed that the cell viability of human normal liver cells L-O2 and human normal tubular epithelial cells HK-2 was little changed when co-cultured with MS-GERTs of different concentrations,indicating that MS-GERTs had little toxicity on cell lines.Animal biosafety experiments showed that there was no significant difference in body weight,chemical biochemistry between the control group and the MS-GERTs group,no significant morphological changes were observed in histological analysis of the major organs,these results suggested the good biocompatibility of MS-GERTs.6.The expression of FRα in paraffin embedded specimens of cervical cancer excised from clinical surgery showed an apparent correlation with the FIGO classification and grade in cervical cancer,while no significant correlation with the pathological type,the FRαexpression was apparently higher than that of normal cervix（P<0.001）.7.In cervical cancer cells,Real-time PCR results showed that FRαmRNA was overexpressed in HeLa cells,and was also expressed in SiHa,CaSki and C33A cells,at the level of total protein,the results of Western Blot showed that the expression of FRαin HeLa cells was as high as that of the FRα-positive control cells,while the expression of SiHa,CaSki and C33A cells were weak.Flow cytometry results suggested that HeLa cells overexpressed FRαon the membrane,SiHa,CaSki and C33A cells expressed FRα,but with lower expression than HeLa.8.After confirming the overexpression of FRαin cervical cancer cells,a new generation of FA modified NBT embedded GERTs probes were successfully prepared.The core-shell structure of the probe was petal-like,the outer layer was coated with a layer of mesoporous silica,the gap structure could be observed among the petal branches,the particle size is about 83±6 nm with good dispersion under TEM,the particle size is uniform;the DLS particle size analyzer determined the size was 147.6±1.11 nm and the Zeta potential was（-24.6±0.6）mV.UV-Vis showed an obvious absorption of FA molecules at 280 nm wavelength,indicating that FA was successfully conjugated to GERTs.Raman spectroscopy was used to obtain the Raman spectrum of FA-MS-NBT-GERTs,and the characteristic peaks of the NBT-GERTs were at 1078,1339 and 1568cm^-1.9.The results of cell targeting Raman imaging showed that the uptake of FA-MS-NBT-GERTs by HeLa cells was significantly higher than that of NBT-GERTs and BDT-GERTs without active targeting label.The uptake of FA-MS-NBT-GERTs in HeLa cells was significantly higher than that of A549 cells with negative FRα.The uptake of FA-MS-NBT-GERTs in HeLa cells pretreated with free FA was significantly decreased,suggesting that free FA can competitively inhibit HeLa cells from absorb the targeting probes.10.The results of CCK-8 assay showed little cytotoxicity of FA-MS-NBT-GERTs on cells.The results of biosafety evaluation in vivo showed no significant difference between the control group and FA-MS-NBT-GERTs group,no obvious histopathological changes in the major organs were found,suggesting that the FA-MS-NBT-GERTs have good biocompatibility.11.The ratio of FA-GERTs/Nt-GERTs in the SLN of the control group was 1.07±0.26,suggesting that there was an equal nonspecific uptake of the two probes in SLN.The ratio of FA-GERTs/Nt-GERTs in reactive hyperplasia SLN was 1.15±0.15,and there was no significant difference between the control group and the reactive hyperplasia SLN group（P>0.05）,the ratio of FA-GERTs/Nt-GERTs in metastatic SLN was 2.29±1.03 and was much higher than the control group（P<0.01）,indicating that the uptake of FA-GERTs was more than Nt-GERTs of tumor cells in the SLN.12.The pathological examination confirmed the presence of tumor foci in the metastatic SLN.Conclusions 1.The MS-GERTs probe is an ideal SLN tracer with suitable particle size,ultra-high sensitivity and specificity,excellent photostability and good biocompatibility.2.The portable Raman scanner can realize real-time localization of sentinel lymph nodes during surgery,suggesting that Raman spectroscopy is likely to become a new technique for sentinel lymph node mapping and has a great potential for clinical translation.3.FRα overexpressed in cervical cancer tissues and is negative or low expressed in normal cervical tissues.FRα was found an apparent correlation with the FIGO classification and grade in cervical cancer,while no significant correlation with the pathological type.All the histological types of cervical cancer expressed FRα.4.FRα can be regarded as an active targeting receptor for targeted imaging of cervical cancer.The FA molecule is a specific legend of FRαand can be used as a surface-modified molecule of an active probe.5.A new generation of NBT-GERTs can regulate the Raman intensity and shows superior characteristics than the first generation GERTs.Successfully prepared FA-MS-NBT-GERTs.6.FA-MS-NBT-GERTs is a kind of probe with the ability of actively targeting cervical cancer cells,and the targeting effect is obvious.7.Using FA-GERTs/Nt-GERTs ratiometric model,the specific Raman imaging of involved SLN in cervical cancer was preliminary achieved.