The Preparation, Charactization And Cell Experiments In Vitro Of A Somatostatin Receptor Targeted Magnetic Resonance Molecular Probe

Purpose To prepare the carboxymethyldextran modified Fe3O4 magnetic nanoparticles (CMD-MNPs); To prepare superparamagnetic iron oxide crosslinked with octreotide (CMD-MNPs-Oc) by immobiliting the octreotide onto the carboxymethyldextran modified magnetic nanoparticles by means of chemical methods; To determine the physicochemical and magnetic characteristics of the magnetic nanoparticles (CMD-MNPs and CMD-MNPs-Oc) prepared.Methods By means of chemical co-deposition method, modified magnetic nanoparticles were prepared in carboxymethyldextran-water dispersed phase by ultrasonic pretreatment. The Octreotide was immobilitied with the amide linkage on the carboxymethyl-dextran modified magnetic nanoparticles. 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 The experimental results showed that disperse of composite magnetic nanoparticles is improved by ultrasonic pretreatment method. The magnetic nanopaticles are spheric, uniformly disperse, and with an average 50 nm in size. Such modification kept the crystal structure of the Fe3O4 nanoparticles and improved the colloidal stability of nanoparticles in aqueous suspension. The CMD-MNPs and the CMD-MNPs-Oc nanoparticles have the property of supermagnetism with measured saturation magnetization of 35 emu·g-1 and T2 relaxativity (r2) of 146.7 s-1mM-1 and 173.6 s-1mM-1, respectively.Conclusions The carboxymethyldextran modified Fe3O4 magnetic nanoparticles (CMD-MNPs) are successfully prepared, and the octreotide is successfully linked to the CMD-MNPs with the amide linkage. Both of the CMD-MNPs and the CMD-MNPs-Oc nanoparticles have good physicochemical and magnetic properties, and can be servered as potential T2 contrast agents for magnetic resonance imaging.Purpose To compare the difference between the endocytosis of the prepared CMD-MNPs and CMD-MNPs-Oc particles in the somatostatin receptor positive tumor cells, and to determin if the difference is detectable by magnetic resonance imaging in vitro, and to initially investigate the feasibility that CMD-MNPs-Oc be served as a somatostatin receptor targeted magnetic resonance molecular probe.Methods The expression of somatostatin receptor subtypes (SSTR1-5) mRNA for BxPC-3 human pancreatic tumor cell line was testified by reverse transcription polymerase chain reaction (RT-PCR) technique. The BxPC-3 cells were cultured to exponential phase of growth as routine, then the prepared CMD-MNPs or CMD-MNPs-Oc solutions were added to the culture mediums respectively until the mediums reached a final iron concentration of 35μg/ml. There were 2 groups of cells according to the difference of the iron solutions added: targeted group was BxPC-3 cells cultured with CMD-MNPs-Oc and non-targeted group was BxPC-3 cells cultured with CMD-MNPs. They were underwent a succedent continuous culture with the mediums containing iron for labeling. Then all these cells were harvested, and the cell suspensions were centrifugated and washed for several times and were prepared for the following examinations:①The cell sediments were fixed and were made into ultrathin sections with electron staining. The ultrathin sections were then inspected under a Hitachi JEOL-1230 transmission electron microscope.②The cells were underwent conventional HE staining and Prussian blue iron staining, and the stained slide were inspected under a light microscope with specific attention to detect if there were dark blue stained particles in the cells for slides with Prussian blue iron staining, and the cells with dark blue stained particles were considered as positive iron containing cells. Under highpower fields with 400 folds of magnification, five fields were randomly taken to count the numbers of the positive iron containing cells and the percentages of the positive cells were calculated as the ratio of the numbers of the positive iron containing cells to the numbers of the total cells for each group.③The cells were washed and were resuspended with non-iron containing culture mediums, and the resuspensions were adjusted to 1.0×107 cells per milliliter for each group. The resuspension of each group was then divided and added into 5 Ependoff tubes, and 2% gelatin was also added to prevent the cells from settling. The Ependoff tubes containg the cell suspensions or the non-cell containing culture mediums were examined at a 1.5 Tesla magnetic resonance imaging unit. The T2 weighted spin echo sequence imaging (TR 5000 ms, TE 100 ms) was obtained and the signal intensity of each sample was measured. The mean signal intensity of each group and the non-cell containing culture mediums was obtained and was compared with each other. The rate of the changement in signal intensity (△SI%) of each group was calculated using that of the non-cell containing culture mediums as a reference.Results The results of RT-PCR showed that the BxPC-3 tumor cell line was positive for all somatostatin receptor subtypes (SSTR1-5) mRNA expression. The electron microscope inspections of the cells showed that there were lots of electronic high-density particles in the targeted group, and these particles were mainly located in the cytoplasm, with some of them forming the pinosomes, while such particles were barely seen or absent in the non-targeted group with little of them located in the lysosomes if detected. The conventional HE staining of the cells detected diffused distributed particles in the cytoplasm, and on Prussian blue iron staining sections there were blue particles thoughtout the cells of the targeted group with the percentage of the positive cells of 96.7%, while such particles were barely seen in the non-targeted group with the percentage of the positive cells of only 6.7%. The mean signal intensity on T2 weighted spin echo sequence imaging was 382.4±56.4, 866.6±58.55 and 1256.6±31.7 for targeted group, non-targeted group, and the non-cell containing culture mediums respectively, and the calculated△SI% was 69.6%and 31.0%for targeted group and non-targeted group, respectively. The signal intensity of targeted group was lower than that of non-targeted group, and the difference was of statistical significance (P< 0.001).Conclusions BxPC-3 tumor cells are positive for all somatostatin receptor subtypes (SSTR1-5), and can selectively intake CMD-MNPs-Oc particles in bulk but barely intake CMD-MNPs particles, and the difference of intracellular intaking of iron is capable to be detected by T2 weighted MR imaging. This suggests that the octreotide linked on CMD-MNPs may play a critical role to the endocytosis of iron particles, and CMD-MNPs-Oc may potentially suitable to be used as a targeted magnetic resonance contrast agent for SSTR positive tumor.