L-Selectin Ligand Targeted USPIO-PBCA Nanoparticles:Synthesis And MRI Application In Lymph Nodes

Background and ObjectiveBackgroundLymph nodes are important immune organs in mammalian and the lymphatic pathway is an important route of tumor metastasis. To judge the lymph nodes involved or not, it is a major index for tumor stage, treatment alternatives, disease prognosis. Although with great values, it has many difficulties in lymph nodes biopsies in deep tissue or review after clinical treatment. Nowadays, to find a simple, safe, non-invasive lymph node examination method has been received wide attentions.Traditionally, to judge lymph node metastasis or not, it was often to use the method of determination of lymph node size. But, in the qualitative diagnostic value of lymph node, this method still has considerable controversy in routine Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) scan. Ultrasound examing the vascular change of lymph node has some significance in the identification of the benign or malignant lesions in lymph nodes. Because of the overlap signs between in the benign and the malignant lymph nodes, the diagnostic accuracy of ultrasound is ralertively low. X-ray lymphadenography are complex, time-consuming, with some complications and can not satisfactorily display the details of lymph nodes. PET (Positron Emission Tomography)/CT could determine the lymph node metastasis from the perspective of molecular metabolism, but the resolution is low.Contrast-enhanced MR lymphatic imaging is a new noninvasive detection method. Currently the Superparamagnetic Iron Oxides (SPIO)s contrast agents are the most widely used and the preferred ones which are a kind of passively targeting to reticuloendothelial system contrast agents with negative enhancement effect on the T2WI sequences. Ultrasmall Superparamagnetic Iron Oxides (USPIO)(the diameter less than30nm in SPIO) enhancement MRI scans can find localized metastases in the lymph nodes independent of the sizes of lymph nodes. But USPIO particles were engalfed by the phagocytic cells in the lymph node medulla to image. This enhancement MRI is a passive targeting method with many problems, which may cause false positives and false negtives and affect the accuracy of diagnosis.L-selectins exit in the surface of normal lymphocytes, as the homing receptors which lead peripheral lymphocytes to reach lymph nodes. L-selectin ligands highly express in the endothelial cells of high endothelial veins, labyrinths and medullas of lymph nodes and its core antigens are MECA-79monoclonal antibodies, which have a similar role with L-selectin and highly specific to combine with the areas where L-selectin ligand highly expressed in. Literature suggested that ultrasound contrast agent modified by MECA-79monoclonal antibody could target initiatively to normal lymph nodes. Therefore, superparamagnetic substances (for example USPIO) carried by the antibody may target initiatively to lymph nodes in MR enhancement scan. Being of safe and good biocompatibility characteristics, Polybutylcyanoacrylate (PBCA) was the most important drug carrier. So, after PBCA were modified by this antibody, these nanoparticles which carryed superparamagneticsubstances (such as USPIO) could be prepared as MR contrast agent, this enhanced MR scan should be able to target to the lymph nodes actively. As we known that no similar literatures have reported.Objectives of Study1、In the weakly acidic conditions, the USPIO particles were coated in the PBCA nanospheres. After processing under alkaline conditions to generate carboxyls automatically, USPIO-PBCA particles reacted with streptavidins by crosslinking agent. Then, biotinylated MECA-79monoclonal antibodies were modified to USPIO-PBCA nanoparticles shells by biotin-avidin system, which made nanoparticles initiativly targeting to L-selectin ligands of lymph nodes.2、The animal in vitro experiments:the specific targeting abilities of the biotinylated MECA-79monoclonal antibodies were detected by immunohistochemistry in fresh lymph nodes of normal rabbits.3、Normal live New Zealand white rabbits experiments:by injection of anti-MECA-79-of USPIO-PBCA nanoparticles through the dorsal gap injection, the qualitative enhancement on normal rabbit popliteal lymph nodes at different time points by MRI scan, then by confirmed in pathology.MethodsI Preparation and characterization1、Synthesis and detection of USPIO-PBCA nanoparticlesBCA monomer was diluted by CH2-Cl2(VCH2ci2:VBCA=3:1).The non-ionic surfactant of5%Dextran-70was dissolved into the acidic (pH6.4) buffer solution (KH2PO4-NaOH) solution and the concentration of11.2mg/ml USPIO was dropped to the mixed solution of50ml. Afer stirring5minutes with the stirring speed of800rpm to1200rpm incresingly,1.2%concentration of the butylcyanoacrylate (BCA) monomer was slowly added into the mixture solution drop by drop. After emulsion polymerization continuously for5hours, the reaction was terminated. After this solution was filtered by vacuum pumb, the stable suspension of PBCA magnetic nanoparticles was prepared. Following filtred by0.45μm microporous filtration membrane, the filtrate was kept in the refrigerator of4℃for use.Size and distribution of particle nanoparticles:Malvern-3000HS laser particle size analyzer: Take the above of USPIO-PBCA mixture to dialysis for48hours to remove of free USPIO and Dextran-70. The dialysised USPIO-PBCA solution was detected about the size and distribution by Malvern-3000HS laser particle size analyzer.The morphology of nanoparticles by transmission electron microscopy:Take a small amount of the above of dialysised USPIO-PBCA, then the gross shape of it was observed by transmission electron microscopy.The measured T2relaxation time and the calculated T2relaxation rate by Magnetic resonance scan:appropriate amount of USPIO-PBCA solution was diluted by water to the iron concentration of0.04,0.06,0.08,0.10,0.12,0.14,0.16mmol/L respectively, then scanned in the MR machine (3.0T U.S. company GE, Sigma EXCIT HD) with an8-channel head coil, thickness of3.0mm, interval of0.5mm, the scanning sequence T2map:time of repetion (TR) of3000ms, time of echo(TE) of20ms,40ms,60ms,80ms, all with a coronal scan. Determination of T? values: raw data were imported to ADW4.3workstation and USPIO-PBCA T2relaxation time values were measured.Calculate the T2relaxation rate of USPIO-PBCA:T2relaxation rate is defined as the slope of the linear equation about the iron concentration and1/T2.2、Preparation and detection Streptavidin-linked USPIO-PBCA particlesThe prepared USPIO-PBCA particles were dealed with under alkaline conditions (pH=9) for an hour, so carboxyl groups generated on the surfaces of particles. Thses USPIO-PBCA particles were diluted to the iron concentration of USPIO-PBCA0.52μM/ml with sterile Phosphate Buffered Saline (PBS) and another PBS solution containing of1-ehyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC) was added into. Then the solution was adjusted to pH7.0and streptavidin with PBS was added to reacte at4℃overnight, streptavidin labeling USPIO-PBCA nanoparticles were made. The molar ratioof USPIO-PBCA: streptavidin:EDC is1:10:25(EDC concentration of0.15g/L, optimaztion).Using the reaction of biotinylated Fluorescein Isothiocyanate (FITC) and streptavidin-USPIO-PBCA nanoparticles and observed the degree of green fluorescence by fluorescence microscopy to determine and evaluate whether streptavidins were successfully linked to the USPIO-PBCA nanoparticles shell.The biotinylated FITC was droped into the streptavidin-USPIO-PBCA solution by titration to observe the movement of the fluorescence peak by flow cytometry.3、The connection of the streptavidin labeled USPIO-PBCA and biotinylated MECA-79monoclonal antibody5μg corresponding biotinylated MECA-79monoclonal antibody reacted with containing10’streptavidin-labeled USPIO-PBCA suspensions. After incubating for10min, anti-MECA-79-USPIO-PBCA nanoparticles were made.4、Determination the physical and chemical properties of anti-MECA-79monoclonal antibody labelled USPIO-PBCA particleThe apparent morphological characteristics of the prepared anti-MECA-79-USPIO-PBCA were observed by the transmission electron microscope. The size and distribution of nanoparticles was tested by particle size determination.II MECA-79monoclonal antibody targeted experiments to fresh lymph nodes in normal rabbitThe selected three normal New Zealand white rabbits were anesthetized to take the popliteal lymph nodes in both sides. These six lymph nodes were rinsed in pre-cooling of saline for3-5times to clean the blood and drifted by absorbent filter paper. Freshly removed lymph nodes were immediately snap-frozen at-7℃and fixed by acetone to perform8μm-cryosection. Unspecific binding sites were inhibited (incubation for one hour at37℃, immediate driftion by PBS overnight at4℃) with5%goat serum albumin (prepared by0. lmol/L PBS).Next, sections were incubated for1hour with biotinylated MECA-79antibody in0.1%goat serum albumin (1:100) at room temperature and dealed with H2O2(prepared by0.1mol/L PBS) to block of endogenous peroxidase. After having been rinsed for three times which lasted for5minutes, these sections were incubated with an extravidin peroxidase conjugate for one hour. The peroxidase-based detection system was employed with3-amino-9-ethylcarbazole (6ml20mg/ml solved in dimethylformamide) as the chromogenic substrate. Specificity of staining was confirmed by means of employing an IgM isotype as a negative control. The experiment was divided into two groups:MECA-79monoclonal antibody for the experimental group, another IgM monoclonal antibody as a control group. Respectively, these two groups were investigated with regard to the specificity of for the antibodies to L-selectin ligand immunohistochemically.Ⅲ The targeted study Of MECA-79monoclonal antibody labelled USPIO-PBCA nanoparticles to normal rabbits’nodes1、Experimental groups:18normal rabbits were randomly divided into three groups (n=6).1) Targeted experimental group:Normal New Zealand white rabbits, injection of15μmol Fe MECA-79monoclonal antibody labeled USPIO-PBCA contrast agent with15μmol Fe through the toe clearance by one side.2) Control group1:Normal New Zealand white rabbits, injection of USPIO-PBCA contrast agent with15μmol Fe through the toe clearance by one side.3) Control group2:Normal New Zealand white rabbits, injection of USPIO contrast agent with15μmol Fe through the toe clearance by one side.2、Experimental procedures:1) Anesthesia:3%pentobarbital sodium (20mg/Kg) ear vein administration, where appropriate, add a small amount of diazepam to anesthetize animals.2) Fix:The animals were fixed at a suitable board by supine position, as far as possible symmetry.3、Scanning equipment and parameters1) Scanning equipment:GE Signa Excite3.0T Magnetic Resonance Imaging system, with head8-channel quadrature coil.2) Scan sequences and parameters:coronal T2WI,(TR/TE:5000/85ms echo train length(ETL):16, thickness:2mm, Field of View (FOV):12cm, scan matrix:256×256, number of excitations(NEX):3); axial T2WI2-Dimensional gradient echo (TR/TE:400/24ms, flip of angle:20°, thickness:2mm, FOV:12cm, scan matrix:256×256, NEX:2); axial T1WI spin-echo sequence (TR/TE:400/12ms, thickness:2mm, FOV:12cm, scan matrix:256×256, NEX:2), the above sequences of unenhanced and enhanced scans.3) Contrast-enhanced scan mode:the contrast agent was injected locally in the toe clearance by one side of the rabbit, after injection, at the time point of the No.1,2,8,12,24,48h, imagingly. When MR scans were completed, the data were transmitted to the post-processing workstation to analyze MRI images.4、Data processingLymph nodes signal intensity and muscles signal intensity was measured by the injected side. The larger region of intrist in the backgroud was selected to measure the standard deviation of the background noise. And signal to noise ratio (SNR) of the lymph nodes were calculation.5、Statistical analysis SPSS13.0was used as analyzing software.Repetitive measurement ANOVA (statistical method: Bonferroni) analyzed the difference in SNR of the lymph nodes Bonferrom).analyzed the difference in SNR of lymph nodes and museles among different groups and time points.P>0.05was regarded as no statistiea;dofferemee. P<0.05was regarded as statistical significant difference. P<0.05was regarded as statistical significant difference.6、Pathology examinationAfter scanning by MR enhancement, all animals were sacrificed to take lymph nodes by injection of the air through the ear vein.10-15%neutral formalin fixed, paraffin-embedded sections (slice thickness1.5μm) were prepared. Two series slices were taken from each specimen to undergo Prussion Blue Staining to observe the lymph nodes’iron content in the experimental group and comparison groups through light microscope, and imaging findings were compared.ResultsI By the determination of Malvern-3000HS laser particle size analyzer, the average particle size of USPIO-PBCA was approximately159nm, the polydispersity index was0.23.Ⅱ The transmission electron microscope showed that USPIO-PBCA nanoparticles were spherical or spherical-like, with ralertively uniform distribution, smooth surface and no adhesion between the particles. The size of the average particle size was about60-70nm, which suggested that small particle size and high stability of USPIO-PBCA nanoparticles were prepared.ⅢThe magnetic properties measured by vibrating sample magnetometer showed the prepared USPIO-PBCA nanoparticles were superparamagnetic.Ⅳ By magnetic resonance T2relaxation time scan, T2values of USPIO-PBCA particles gradually decreased with the increasing of iron concentration, and T2relaxation rate of the samples was about0.156×106mol-1s-1.Ⅴ The results of Fluorescence microscopy and flow cytometry all suggested that USPIO-PBCA has been successfully linked with streptavidine, with the reactivity of biotinylated monoclonal antibody.Ⅵ The apparent morphological characteristics of the prepared nanoparticles were observed by scanning electron microscopy:the nanoparticles were round or oval, with even distribution and no adhesion to each other and the particle size of80.0nm. Determination of particle size showed that the distribution of particle size was uniform, with the particle size about of220.3nm and the dispersion coefficient about of0.318.Ⅶ The results of horseradish peroxidase immunohistochemistry of normal rabbits’fresh lymph nodes in vitro showed that MECA-79monoclonal antibody, had specific targeting to normal lymph nodes in targeted experimental group, ordinary IgM monoclonal antibody had no specific targeting in control group.Ⅸ MRI scans of normal rabbits’popliteal lymph nodes and the SPSS statistical analysis results showed that:on T2*WI, T2WI sequences, the decreasing degree of the SNR of lymph nodes in the targeted group lower than in the two control groups with statistical difference; In the same group, the SNR values at different time points had statistical difference. The two factors had interaction effect. On T1WI sequences, the SNR values in different groups and different time points all had statistical differences, but the two factors had no interaction effects. The SNR of muscles in different groups and at different time points were no statistical difference.X The results of pathological examination by Prussian blue staining showed that more blue-stained iron particles existed in the rabbits’lymph nodes in the targeted experimental group, and fewer blue-stained iron particles in the two control groups, especially in the medullas of lymph nodes.ConclusionsI Under the weak acidic conditions, the targeted and superparamagnetic anti-MECA-79-USPIO-PBCA nanoparticles were successfully prepared. With ralertively uniform size, smooth surface, these particles based the next step animal experiments.Ⅱ In vitro experiment, MECA-79monoclonal antibodies specifically targeted to the paracortex of lymph nodes in normal rabbits. This suggested that the prepapared anti-MECA-79-USPIO-PBCA nanoparticles could specifically target to rabbits’normal lymph nodes.Ⅲ The animal experiments in vivo showed that the anti-MECA-79-USPIO-PBCA nanoparticles could specifically target to the normal lymph nodes and the SNR values of lymph nodes decreased on three sequences. More, the results of control experiment showed that the negative enhancement in the targeted group superior to the two groups. The Prussian blue stainning results showed that the more and the broader blue-stainning particles and in the targeted grou. This means that the prepared anti-MECA-79-USPIO-PBCA nanoparticles not only engulfed by Phagocytes in Mononuclear-phagocytic system, but aslo had actively specific targeting to the paracotex of the lymph nodes.