The Study Of The Biodegradabal Nanoparticles As The Molecular Imaging Contrast Agent For Early Diagnostic Of Liver Cancer

Hepatocellular carcinoma (HCC) is on the list of the higher mortality in China. Early diagnostic and radical cure in the primary stage is the best way to improve the cure rate. The common method of detection of liver cancer is to detect the alpha fetal protein (AFP) level or distinguish the pathological changes of liver using a variety of imaging technologies on clinic. While, the diagnostic of the primary hepatic carcinoma by the contrast agent enhanced imaging technology such as magnetic resonance imaging (MRI) is poor because this technology is good at distinguishing the pathological changes of the mature vessel. Therefore, it is hard to discriminate the earlier pathological changes of the immature vessel of the liver cancer, especially in the early stage. Thus, designing specific diagnosis system might be a promising way to improve the early detection accuracy, thus, to improve the therapeutic effects based on the characteristics of early lesions. One of the feasible methods to get the specific diagnosis program is using ligand modified targeting imaging contrast agent, or so called molecular imaging contrast agent, which could specific bind with the receptor overexpressed on the hepatic carcinoma cell membrane, facilitate the accumulation of the contrast agent in the diseased region, thereby, to enhace the imaging intensity and improve the early detection accuracy.The ideal molecular imaging contrast agent should to hold the properties such as (1) to increase the sensitivity and specificity of the visualization procedure; (2) to control the biodistribution; (3) to decrease the incidence rate of image artifacts; and (4) to collect more information through the imaging processes. Hence, four novel molecular MRI imaging contrast agents were prepared using biocompatible material as the carrier, and then chelated with gadolinium (Gd) in this study. The first system is the cationic solid lipid nanoparticles (SLN)/Gd-diethylenetriamine-pentaacetic acid (DTPA) nanocomplexes; the second one is the poly (lactic acid)-poly (ethylene glycol) (PLA-PEG)/Gd-DTPA nanocomplexes; the third one is the gadolinium labeled PLA-PEG nanoparticles and the fourth is antibody modified gadolinium labeled poly (lactic acid)-poly (ethylene glycol)-polylysine (PLA-PEG-PLL) nanoparticles. The four biodegradable nanocarriers based target contrast agents were evaluated in terms of pharmaceutical characteristics and bio-characteristics, respectively. The main methods and results were as follows:1. Preliminary Studies on Cationic Solid Lipid Nanoparticles/Gd-DTPA Complexes as molecular imaging contrast agentNanoparticles as MRI contrast agent in molecular imaging and targeted therapeutics is one of the most promising clinical application of nano-technology. The aim of this study is to prepare self-assembled nanoparticles for molecular imaging using cationic SLN supported Gd-DTPA (SLN/Gd-DTPA). The pharmaceutical characteristics of SLN/Gd-DTPA were investigated to verify its property as new molecular imaging agent. Cationic SLN was prepared by emulsifying-solvent evaporation method, the SLN/Gd-DTPA was formulated by self-assembly nanotechnology via electrostatic force between the cationic SLN and anionic Gd-DTPA. The morphology of SLN/Gd-DTPA was observed by transmission electron microscopy. The particle sizes and the polydispersity were measured by laser particle size analysator. The zeta potential was measured by ZETASIZER3000. The MRI imagination determination and the stability studies in plasma were carried out to evaluate the potential of SLN-Gd-DTPA as MRI molecular imaging agent. The morphology of SLN and the SLN-Gd-DTPA complex were approximately spherical. The average particle sizes of SLN and the complex were 188.5±2.98nm and 196.1±2.01nm, respectively. The zeta potentials were +19.83mV and +17.12mV, respectively. The results of MRI imaging intensity determination in vitro have shown that the SLN/Gd-DTPA possessed the ability of MRI imagination, and the direct correlation between the MRI imaging intensities and the SLN/Gd-DTPA concentrations was observed (r=0.977). The result of blood plasma stability investigation of SLN-Gd/DTPA has shown that Gd-DTPA was fully combined with cationic SLN, and the complex was stable enough as MRI contrast agent in the further imaging application in vivo. These results indicated that the self-assembled SLN/Gd-DTPA could be prepared easily with perfect pharmaceutical characteristics as new molecular imaging agent which has shown the potential to do well in early hepatoma diagnosis.2. Biocompatible PLA-PEG/Gd-DTPA nanocomplexes as molecular targeted MRI contrast agentAccurate diagnosis in early stage is vital for the treatment of HCC. The aim of this study was to investigate the potential of PLA-PEG/Gd-DTPA nanocomplexes as biocompatible molecular MRI contrast agent. The PLA-PEG/Gd-DTPA nanocomplexes were obtained using self-assembly nanotechnology by incubation of PLA-PEG nanoparticles and the commercial contrast agent, Gd-DTPA. The physicochemical properties of nanocomplexes were measured by atomic force microscopy (AFM) and photon correlation spectroscopy (PCS). The Tl-weighted MR images of the nanocomplexes were obtained in a 3.0 T clinical MR imager. The stability study was carried out in human plasma and the distribution in vivo was investigated in rats. The mean size of the PLA-PEG/Gd-DTPA nanocomplexes was 187.9±2.30 nm, and the polydispersity index was 0.108, the zeta potential was-12.36±3.58 mV. The results of MRI test confirmed that the PLA-PEG/Gd-DTPA nanocomplexes possessed the ability of MRI imaging, and the direct correlation between the MRI imaging intensities and the nano-complex concentrations was observed (r=0.987). The signal intensity was still stable within 2 hours after incubation of the nanocomplexes in human plasma. The nanocomplexes gave much better image contrast effects and longer stagnation time than that of commercial contrast agent in rat liver. A dose of 0.04 mmol of gadolinium per kilogram of body weight was sufficient to increase the MRI imaging intensities in rat livers by 5 fold compared with the commercial Gd-DTPA. PLA-PEG/Gd-DTPA nanocomplexes could be prepared easily with small particle sizes. The nanocomplexes had high plasma stability, better image contrast effect, and liver targeting property. These results indicated that the PLA-PEG/Gd-DTPA nanocomplexes might be potential as molecular targeted imaging contrast agent.3. Gadolinium labeled biocompatible PLA-PEG nanoparticles as liver targeted molecular MRI contrast agentA molecular magnetic resonance imaging (MRI) contrast agent targeted to liver was developed by conjugated biocompatible poly (L-lactide)-block-poly (ethylene glycol) nanoparticles (PLA-PEG) with DTPA-gadolinium. The mean size of the nanoparticles prepared by solvent diffusion method was 265.9±6.7nm. The relaxivities of the gadolinium labeled nanoparticles was measured and the distribution in vivo was evaluated in rats. Compared with conventional contrast agent, the gadolinium labeled PLA-PEG nanoparticles showed significant enhancement both on liver targeting ability and imaging signal intensity. The T1 and T2 relaxivities per [Gd] of the gadolinium labeled nanoparticles was 18.865 mM-1 s-1 and 24.863 mM-1 s-1 at 3 T, respectively. The signal intensity in vivo was intensified comparing with the Gd-DTPA and the T1 weight time was lasting for 4.5 hours. The liver targeting efficiency of the gadolinium labeled PLA-PEG nanoparticles in rats was 14.57. The gadolinium labeled nanoparticles showed the potential as targeting molecular MRI contrast agent for further clinical utilization.4. Antibody-gadolinium labeled PLA-PEG-PLL nanoparticles as the active target contrast agentPLA-PEG-PLL presents more active amido terminal than PLA-PEG-NH2, thus, it is superiority to prepare active target contrast agent using PLA-PEG-PLL. DTPA was conjugated to the PLA-PEG-PLL to obtain a novel polymer for formulating molecular MRI contrast agent because DTPA is the most commonly used chelate for conjugating Gd. The PLA-PEG-PLL-DTPA nanoparticles were fabricated by solvent diffusion method. The Gd labeled nanoparticles was prepared by incubating nanoparticles with GdCl3 and Gd was anchored on the surface of the nanoparticles through the self-link with the exposed DTPA. The anti-VEGF was selected as the active targeting ligand and was immobilized on the surface of the Gd labeled nanoparticles by the conjugation of the carboxy group with the amido terminal of PLL to obtain the anti-VEGF-gadolinum labeled PLA-PEG-PLL nanoparticles. The FITC labeled anti-VEGF modified nanoparticles were synthesized using avindin-biotin conjugate between the avidin-FITC and biotin-PLA-PEG-PLL nanoparticles. The mean size of the Gd labeled PLA-PEG-PLL nanoparticles, anti-VEGF-FITC labeled PLA-PEG-PLL nanoparticles and anti-VEGF-Gd labeled PLA-PEG-PLL nanoparticles was 69.8±5.3nm,82.5±5.8nm and 85.8±7.2nm, respectively. The zata potential of the three nanoparticles was 23.03±4.6mv,22.77±2.5mv, and 21.63±2.4mv, respectively. The T1 and T2 relaxivities of the anti-VEGF-Gd labeled PLA-PEG-PLL nanoparticles was 18.394mM-1 S-1 and 24.35mM-1 S-1 at 3.0 T, respectively. The retention time of the anti-VEGF-gadolinium labeled PLA-PEG-PLL nanoparticles was more than 12 hours in mice of grafting H22 cancer. The target effective of the anti-VEGF-Gd labeled PLA-PEG-PLL nanoparticles to the H22 cancer was 46.9 compared with Gd-DTPA. And compared with the Gd labeled PLA-PEG-PLL nanoparticles, the target effecgtive was 3.2. The anti-VEGF-Gd labeled PLA-PEG-PLL nanoparticles were effetively target to mice of grafting H22 cancer.In conclusion, the SLN/Gd-DTPA nanocomplexes and the PLA-PEG/Gd-DTPA nanocomplexes showed better imaging intensity in rats than the commercial MRI contrast agent, Gd-DTPA. The target effect and T1 relaxivity of the Gd labeled PLA-PEG nanoparticles was further improved. In addition, the anti-VEGF-Gd labeled PLA-PEG-PLL nanoparticles showed specific tumor targeting efficiency. Therefore, the nanoparticles presented in this paper could significantly enhace the imaging intensity and improve the early detection accuracy. Among them, the anti-VEGF-Gd labeled PLA-PEG-PLL nanoparticles might be a promising targeting molecular MRI contrast agent for early detection of the HCC.