Magnetic Resonance Molecular Imaging Experiments Based On Endoglin Target Rat Gliomas

Objectives:The objectives of this study was (1) to develop paramagnetic sterical stabilized liposomes conjugated with monoclonal antibodies targeted to Endoglin on neovascular endothelial cells and evaluate their physical characteristics. (2) to quantify and compare the magnitude of signal intensity enhancement on a rat glioma model applying different contrast agent or molecular tracer using a clinical MRI (3.0 Tesla) scanner. (3) to demonstrate the use of targeted paramagnetic nanoparticles to delineate the glioma margins.Materials and Methods:1. Preparation of paramagnetic sterical stabilized liposomes:(1) A mixture of the appropriate amounts of lipids (DSPC/CHOL/mPEG-DSPE/Gd-DTPA-BSA at a molar ratio of 36/36/3/25) was dissolved in chloroform/methanol 2/1 (v/v) and evaporated to dryness by rotary evaporation at 40?. The lipid film was subsequently hydrated in HEPES-buffered saline at 60?. The resulting lipid dispersion was extruded sequentially 10 times through polycarbonate membrane filters with a pore diameter of 400,200, and 100 using a high-pressure homogenizer. The temperature during extrusion was 60?. The paramagnetic sterical stabilized liposomes were obtained. (2) The size, stability, Gd containing, magnetic resonance relaxivity of the liposomes was evaluated in vitro.2. Coupling the antibodies to liposomes and synthesizing of pretargeting reagents. (1) For monoclonal antibodies conjugated liposomes, PDP-PEG-DSPE were added to the lipid during the preparation of liposomes (at a molar ratio of mPEG-DSPE/PDP-PEG-DSPE=4/1). The pyridyldithio groups on the PDP-PEG-DSPE incorporated liposomes were reduced by adding DTT and the thiolated liposomes (HS-SLs) were obtained. (2) CD105 monoclonal antibodies reacted with SMPB to prepare the MPB-MAb (maleimidophenylbutyrate-MAb). (3) Thiolated liposomes were incubated with MPB-MAb. The antibodies conjugated immunoliposomes (MAb-SLs) were obtained. (4) Biotinylated monoclonal antibodies were synthesized by added Sulfo-NHS-LC-biotin to the antibodies. (5) The preparation of Streptavidin coupling liposomes (SAv-SLs) was similar to that of MAb-SLs. HS-SLs were incubated with MPB-SAv. Gel filtration or centrifugation was used to purify the product in these above-mentioned reactions. (6) The antibodies protein to lipid ratio which denoted the density of antibodies conjunct to the liposomes and antibodies coupling efficiency were evaluated. The morphology of liposomes was studied by Transmission electron microscopy (TEM).3. MR imaging in vivo. (1) 27 male Sprague-Dawley rats were inoculated with 1.5×106 C6 glioma cells in the right caudate nucleus to develop animal model. (2)25 tumor-bearing rats were classified into five groups randomly (5 rates pre group). Gd-DTPA, Gd-SLs, IgG-SLs, MAb-SLs were injected through tail veins respectively in corresponding groups. For two steps pretargeting group, Bio-MAb were administrated 24 hours before SAv-SLs contrast enhancement. (3) MR T1-weighted images of each group were compared to evaluate the enhancement features, the signal enhancement time-intensity curves of arteries, contralateral normal brain tissues, muscular tissues and tumor tissues were analyzed. (4)The degree of contrast enhancement and notable enhancement regions were compared between each group. The degree of enhancement of the tumor central and periphery were also compared for each tumor. (5) The enhancement morphology of the tumors was analyzed. (6) CD 105 immunohistochemistry was performed after MR imaging to detect the difference of microvessel density of the tumor central and periphery.Results:1. Features of Gd-SLs. The size of Gd-SLs was 117.4±31.8nm after homogenized. The incorporation efficiency of Gd-DTPA-BSA was 87%-100%. At 26?(room temperature), the T1 relaxivity of Gd-SLs was 1.16 times that of the solution with free Gd-DTPA. It was 1.25 times at 37?. MR T1 imaging of Gd-SLs in vitro presented similar enhancement effect to that of Gd-DTPA. 2. Characterization of immunoliposomes. The mean size of immunoliposomes after antibodies coupling increased from 116.1nm±33.9nm (Gd-SLs) to 129.9nm±40.9nm (MAb-SLs) and the polydispersity index showed little increase. Hypothermia (4?) showed little influence on the size of the immunoliposomes. The mean size of SAv-SLs increased dramatically to 267nm after incubated with Bio-MAb with a polydispersity index of 0.286. The antibodies coupling efficiency ranged from 52% to 67% and corresponding antibodies protein to lipid ratio was 47?60?g/?mol. Transmission electron microscopy revealed spherical structures of liposomes with homogeneous size. SAv-SLs aggregated after mixed with Bio-MAb.MR imaging and analysis. (1) Arterials enhancement:Cervical arterial showed instantly significantly enhancement after Gd-DTPA was given and declined dramatically after that. The signal intensity returned to precontrast level at 2-hr time point. Thus, time-intensity curve represented as quick washin-early washout pattern. The features of arterials enhancement after liposomes tracers (Gd-SLs. IgG-SLs. MAb-SLs, Bio-MAb/SAv-SLs) injection were similar. Notable contrast enhancement was detected soon after injection and continued to improve through 20 min. It decreased slowly and washout absolutely at 48-hr time point. The time-intensity curve was described as quick washin-platform-slow washout pattern. (2) Characteristics of tumor enhancement:The signal intensity of the tumor peaked soon after the Gd-DTPA injection and sharply decreased subsequently. The intensity was about 30% that of the peak at 2-hr time point and return to the precontrast level at 24-hr. After Gd-DTPA or IgG-DTPA was administrated, the tumor showed no enhancement at early phase. It reached to maximum at 60?120 min and appeared mildly enhancement. As for MAb-SLs and Bio-MAb/SAv-SLs groups, the central and periphery of tumor signal intensity increased post-injection and reached to peak at 8-hr time point. It decreased slowly after that. The signal enhancement of the central and periphery of tumor after MAb-SLs injection were not different. In contrast, the periphery enhancement was significantly higher than that of central in Bio-MAb/SAv-SLs group (P=0.032). (3) The morphology of enhancement:T1 weighted imaging revealed an intensive entire tumor heterogeneous enhancement after Gd-DTPA injection. As for Gd-SLs and IgG-SLs groups, dots or spots enhancement scattered in or around the tumor. Ring-like marked enhancement occurred predominately along the tumor periphery after MAb-SLs or Bio-MAb/SAv-SLs were given. Immunohistochemical assessments of the tumors corroborated that angiogenesis was predominately distributed along the tumor periphery.Conclusions:1. It is easy to prepare the paramagnetic sterical stabilized liposomes which present high relaxivity to improve the MR contrast signal. The liposomes are thermo-sensitive but stable at common temperature. It may potential serve as an ideally useful contrast agent for MR molecular imaging.2. The applied covalent coupling of targeting antibodies is efficient. The biotin-avidin mediate antibodies and liposomes crosslink are certified in vitro.3. MR imaging in a glioma rat model confirmed that:no difference was found in degree between the enhancement of tumor central and periphery after Gd-DTPA contrast, and the tumor showed low enhancement while using non-targeting liposomes. The results demonstrate the limitation of these contrast agents to define the tumor morphology. The tumor extent was well delineated after targeting liposomes (MAb-SLs and Bio-MAb/SAv-SLs) contrast enhancement and the specificity was supported by the competition study in vivo. Moreover, the two-step imaging using biotin-avidin interaction was certified to induce more intensive enhancement, which imply that this novel MR molecular tracer is more suitable for detecting the tumor margins.