Study On Preparation Of SPIO Crosslinked With Octreotide And Differences Of Its Uptake By SSTR Positive And Negative Tumor

Research background:Somatostatin receptor positive tumor cells are commonly seen in neuroendocrine tumours (NETs), they are originated from neuroendocrine cells and are often discovered in digestive system, however, they can also be sporadic in other body organs. This kind of tumour majorly originated in the stomach and intestine and pancreas, all of which account for 75% of the whole and are also known as gastroenteropancreatic neuroendocrine tumors (GEP NETs). The preferred therapy to GEP NETs mainly relies on aggressive surgical operation. However, since the lesion is usually hidden deeply within the gastrointestinal wall or retroperitoneum, it is hard for early diagnosis or accurate localization of the tumor, the effect of clinical treatment is therefore largely affected. Hence, proposing a method for early diagnosis and accurate localization of the neuroendocrine tumor has become a focus of international research.The recent researches in oncology have revealed the high density of somatostatin receptor (SSTR) on the cell membranes of NETS. As a G protein-coupled transmembrane receptor, SSTR has 5 subtypes:SSTR1 to SSTR5; and the subtypes of GEP NETs expression are primarily SSTR2 and SSTR3. The ligand of SSTR is somatostatin (SST),which has analogues called somatostatin analogues (SSTA). SSTA can be ligated to SSTR specifically and transited into cells. Therefore, We can take advantage of the endocytosis mechanism of SSTR, and use SSTA as a carrier protein to transport radioactive isotopes into cells. The enriched radioactive isotopes in tumor cells can be detected by SPECT, which is called somatostatin receptor scintigraphy(SRS). SRS had been studied and applied for clinical practice since 1990s to diagnose primary and metastatic lesions of NETs. As a new examine method, SRS has been widely used overseas but is limited in SPECT. In clinical surgery, it is essential to locate the exact position of the lesion and its relationship with the surrounding organs and tissues, and MRI has its advantage in revealing this. However, MRI doesn't show any specificity and sensitivity to NETs. Thus it has become our goal to develop a method that combines both the advantages of SRS and MRI to reach a high specificity and sensitivity for NETs diagnosis and at the meantime presents high spatial resolutions to provide surgeons with a clearer 3-dementional imaging of the lesion and its relationship with the surrounding organs or tissues.The objective of our study is to develop a new early-dignosis and accurate positioning method for somatostatin receptor positive tumor with somatostatin receptor magnetic resonance molecular imaging (SRMRMI), which uses the coupling of chemically modified contrast medium superparamagnetic iron oxide(SPIO) usually applied in MRI and Octreotide, to form a SPIO-Octreotide complex, and selectively transport Fe3O4 particles into tumor cells by the specific combination of Octreotide with SSTR to form a enrichment of Fe3O4 particles in cytoplasm, which can be detected by MRI because of the change of relaxation time. We hope that SRMRMI has the potentials of both high specificity and sensitivity presented in SRS and high spatial resolutions presented in MRI, which provide us with a new way to early diagnose and locate the somatostatin receptor positive tumors. Our study can be divided into two parts.Objective:To prepare the carboxymethyldextran modified Fe3O4 magnetic nanoparticles (CMD-MNPs), which are of high dispersity and stability, and crosslink octreotide with CMD-MNPs. Methods:By means of chemical co-deposition method, modified magnetic nanoparticles were prepared in carboxymethyldextran-water dispersed phase by ultrasonic pretreatment. Crosslinked with octreotide, the product was characterized by IR spectrum, transmission electron microscopy, x-ray diffractometer, and vibrating sample magnetometer, and the T2 relaxativity in solution is measured.Results:(1) The carboxymethyldextran magnetic nanoparticles was prepared by chemical co-deposition method, and the octreotide was successfully linked to the CMD-MNPs with the amide linkage. The reaction solution which contained ferric chloride and ferrous chloride as the source of iron, and glucan as the modifying agent, was pretreated by ultrasonic equipment. (2) The scanning electron microscope shows that nanoparticles is globular and the radius is about 50nm, accompanied with concentrated distribution of the radius. The ultrasonic pretreatment could stop the gathering of the Fe3O4 nanoparticles. The glucan modifying agent has obviously reinforced the water-solubility of the nanoparticles with no change of the crystal construction of Fe3O4. (3) The composite nanoparticles show the property of superparamagetism. The saturated magnetizing strength is 35emu g-1 in the room temperature.Conclusions:1. The ultrasonic pretreatment can significantly improve the dispersibility of the composite nanoparticles. And it maintains the crystal construction which is similar to that of Fe3O4.2. The stability of the composite nanoparticles in the water is significantly improved. The composite nanoparticles also show the property of superparamagetism. Objective:To test the somatostatin receptor subtypes (SSTR1-5) mRNA expression in BxPC-3 cells and HCT-116cells to determine the somatostatin receptor positive and negative cell lines. To test the specific endocytosis of SSTR positive tumor cells to SPIO--Octreotide complex. To detect the difference of MRI signals in somatostatin receptor positive and negative tumor cells after CMD-MNPs-Oc co-cultivation.Methods:Both the BxPC-3 cells and the HCT-116 cells were cultured respectively, and the intracellular expression of somatostatin receptor subtypes (SSTR 1-5) mRNA was tested by reverse transcription polymerase chain reaction (RT-PCR) technique to determine the somatostatin receptor positive and negative cell lines. Both cells were cultured to logarithmic phase when CMD-MNPs-Oc solutions were added to the culture mediums respectively, which therefore comprises 2 groups of cells:Group A was BxPC-3 cells cultured with CMD-MNPs-Oc; Group B was HCT-116 cells cultured with CMD-MNPs-Oc. With the same incubation time, cell suspensions were collected from each group and after that centrifugated respectively to undergo electron microscopy in order to observe whether the Fe3O4 particles were uptaken into the tumor cells and compare the distribution and quantity of the Fe3O4 particles within the tumor cells. The cells underwent conventional HE staining and Prussian blue iron staining, and the ratio of the numbers of the positive iron containing cells to the numbers of the total cells were calculated for each group. The electron microscope inspection was applied to observe the selective uptake of high-density particles in the cytoplasm of cells in each group. The T2 relaxativity of resuspension in each group was detected by MR imaging. (Group A and B were calculated using Group C as control.)Results:RT-PCR showed positive expressions of SSTR1,SSTR2. SSTR3,SSTR4,SSTR5 in BxPC-3 cell line, and negative expressions of the above receptors in HCT-116 cell line. Electron microscope showed high intake of Fe3O4 particles in group A cells, with high concentration in cytoplasm rather than mitochondria and the formation of pinosome; While group B cells had lower Fe3O4 particles intake and the particles were mainly located in lysosome. HE staining after incubation of CMD-MNPs-Oc showed BxPC-3 cells in group A had many particles in cytoplasm while there is no particle in HCT-116 cell line. Prussian blue staining confirmed that cyan or dark blue particles was Fe3O4 particles, which had a high concentration in cytoplasm(positive rate 97%) in group A and a low concentration in cytoplasm(positive rate under 6%) in group B. MRI showed that T2 weighted spin echo sequence imaging of group A was lower than group C (P<0.001), and the calculatedâ–³SI% was 69.6%, lower than group B, the differences were of statistical significance (P<0.001)Conclusions:1. SSTR had positive expression in BxPC-3 cell line and negative expression in HCT-116 cell line.2. CMD-MNPs-Oc can specifically transport Fe3O4 particles into the cytoplasm of SSTR positive cells by receptor binding mechanism3. SSTR positive and negative cells showed different signals under the detection of MRI due to the specific uptake of Fe3O4 particles into the cytoplasm of SSTR positive cells by means of specific and targeted binding of CMD-MNPs-Oc to somatostatin receptor.