Trastuzumab Phthalocyanine Micelle Preparation And Application On Diagnostic Imaging Of Colorectal Cancer Metastatic Lymph Nodes

Background:Colorectal Cancer(CRC)is one of the digestive system malignancies.Lymphatic metastasis is the main spread route of CRC.Adequate surgical resection is considered as the most favorable therapeutic option for CRC.Such difference in lymphadenectomy is still maintain a certain blindness and with residual metastatic lymph node(LN).Therefore,accurate identification of metastasis LN is extremely helpful for CRC patients' prognosis.Objective:In present study,we developed a surfactant stripped-Trastuzumab pthalocyanine micelle(ss-TPM)which targeted CRC neoplastic cells.ss-TPM could migrate through lymphatic vessels of the CRC nude mice model to bond with CRC neoplastic cells in order to realize real-time image metastatic LN.Methods:1.Construction and Characterization of ss-TPM,Synthesis of COOH-terminated Pluronic F127(F127-COOH).Our study utilized temperature-sensitive critical micelle concentration(CMC)of F127 to generate surfactant stripped-phthalocyanine micelle(ss-PM)at low temperature centrifuge.ss-TPM was synthesized in the condensation reaction of carboxyl and amine of Trastuzumab and then labled with iFluor fluorochrome.F127-COOH was characterized by fourier transform infrared spectrometry(FT-IR)and nuclear magnetic resonance(NMR).iFluor fluorocluome labled ss-TPM was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDSPAGE).The surface structure,size and Zeta potential of ss-PM and ss-TPM were characterized by transmission electron microscopy(TEM)and dynamic light scattering(DLS).The ultraviolet absorption and fluorescence spectrum of ss-PM and ss-TPM were characterized by multifunctional enzyme marking instrument.2.Establishment of nude mouse model of CRC.CRC nude mice orthotopic model was established by using a stably luciferase expressing HT-29-Luc human cell line.The cells suspension were orthotopically inoculated into the subserosa layer of the cecal wall.Our study monitored the tumor development and the metastasis in LN and other major organs using the sensitive bioluminescence imaging(BLI).3.Diagnostic imaging colorectal cancer lymph node metastasis.The expression of Her 2 was characterized by Western Blot.The targeting ability was characterized by cell internalization.The imaging of tumor and LNs were detected by fluorescence imaging(FRI)after intravenous injection of ss-TPM and further confirmed by BLI.The distribution of ss-TPM in major organs were counted by FRI.Surfactant stripped-IgG phthalocyanine micelle(ss-IPM)was as a negative control group.Results:1.Construction and Characterization of ss-TPM.The FT-IR showed that a prominent peak at 1737.3 cm-1.NMR showed that a characteristic peak at 2.6 ppm.The results confirmed the successfully preparation of F127-COOH.SDS-PAGEshowed that Trastuzumab and ss-PM react completely without free antibodies.TEM and DLS showed that the size of ss-PM and ss-TPM is about 20 nm and 30 nm and abound in our system.Multifunctional enzyme marking instrument showed that ss-TPM generated absorption at 600?800 nm and iFluor fluorophore labled ss-TPM was detected emitted light.2.Establishment of nude mouse model of CRC.A correlation between cell number of HT-29-Luc and bioluminescence was demonstrated(R2=0.99).Our study used trypan blue to confirm that the injection technique was reliable.The cells finally grew into an orthotopic tumor,which can be detected through the sensitive BLI after 2 weeks.Our study identified the four anatomical positions of LNs,Gastric Lymph Node(GLN),Pancreaticoduodenal Lymph Node(PLN),Mesenteric Lymph Node(MLN)and Lumbar Lymph Node(LuLN)in the abdomen.The H&E staining further proved that they are LNs.The BLI intensity showed the growth of the tumor till the 3rd-8th week after the tumor cell injection.From 3rd-8th week,the metastasis can be found at MLN.From week 6 to 8,metastasis occurred to more mice and spread from primary LN to secondary LN.At 8th week the tested mice were found skip metastasis.The organ metastasis began to appear from week 6 to 8 and was more prone to liver and lung metastasis,followed by spleen metastasis and heart or kidney metastasis was fewer happened.3.Diagnostic imaging colorectal cancer lymph node metastasis.The cell internalization experiment showed the targeting ability of ss-TPM.In vivo FRI images at different time points(2 h,4 h,8 h,24 h)after intravenous injection of the targeted ssTPM was achieved the peak intensity of tumor at 4 h and FRI intensity was higher than non-targeted ss-IPM.The distribution of ss-TPM was mainly in liver and kidney which achieved the peak intensity of at 4 h.ss-TPM could target metastatic LN accurately and achieved the peak intensity of at 8 h.Non-targeted ss-IPM and ICG is presence of residual metastatic LN.Conclusions:1.We developed a surfactant stripped-Trastuzumab pthalocyanine micelle(ssTPM).2.The cell number of HT-29-Luc have a good correlation with the intensity of BLI.3.An orthotopic nude mice CRC model was established and the tumors could grow up with the formation rate of 100%.4.The four anatomical LN positions in the abdomen and metastasis rules were characterized.5.We confirmed that 4th-5th week is the ideal imaging timepoint with LN metastasis and without organ metastasis.6.ss-TPM could target the HT-29-Luc cell and image tumor and metastatic LN.