| Cancer, one of the major diseases presenting serious harm to human life and health, is the second killer after cardiovascular disease, which resulted in a mortality rate of about13percent worldwide. The incidence and mortality rate of cancer are still on the rise in recent years. Early diagnosis and timely treatment is essential for raising the cure rate and improving the survival rate of tumor patients. Among various imaging diagnostic techniques used in clinical currently, magnetic resonance imaging (MRI) has become a powerful tool for tumor diagnosis, due to the advantages of non-invasiveness, no ionizing radiation, infinite penetration depth, higher spatial resolution, and precise three-dimensional positioning ability. The sensitivity and specificity of MRI diagnosis depends on the using of imaging contrast agents. Contrast agents can enhance the signal intensity and improve the image contrast in local tissues that are magnetically similar but histologically distinct by shortening the relaxation time of water protons around. The small molecular contrast agents used in clinical presently have some defects such as relatively low relaxation rate, non-specific distribution in vivo, rapid renal clearance and short imaging time.An ideal MRI contrast agent shall meet the following conditions:(1) good safety and low toxicity;(2) high relaxation rate;(3) appropriate retention time in the body for imaging diagnosis;(4) selective distribution in target tissue and organs for specific detection of lesion site. In this paper, chitosan and hyaluronic acid, biocompatible and biodegradable natural polysaccharides, were chosen as the nanocarrier material for the preparation of gadolinium-loaded chitosan nanoparticles (Gd-CSNPs) and hyaluronic acid modified gadolinium-loaded chitosan nanoparticles (CS-DTPA-Gd/TPP/HA NPs) by post-modification and pre-modification methods, respectively. The Gd-loaded nanoparticles were expected to use as tumor targeted MRI contrast agents, in order to improve imaging contrast and its non-specific distribution in vivo, and overcome the drawback of rapid renal clearance of small molecular contrast agents. The physical and chemical properties, MRI imaging ability in vitro and in vivo, and the safety in cells and animals of the Gd-loaded nanoparticles were evaluated in this research systematically. The main research methods and results are as follows:1. Gd-loaded chitosan nanoparticles as molecular imaging contrast agentsChitosan nanoparticles (CSNPs) were prepared by ionic-cross linking method using sodium tripolyphosphate (TPP) as the linking agent. The formulation was optimized by single-factor experiments. Diethylene triamine penlaacetic acid (DTPA) was conjugated onto the surface of CSNPs using EDC-NHS reaction, then the DTPA linked CSNPs were incubated with GdCl3solution to obtain Gd-CSNPs. The morphology was observed using transmission electron microscope (TEM). The particle sizes and the zeta potentials were measured by laser particle size and potential analyzer, respectively. The Gd concentration of Gd-CSNPs solution was determined by inductively coupled plasma emission spectrometry. Cytotoxicity was tested by MTT method in B16cells. The MRI imaging abilities in vitro and in vivo were investigated by magnetic resonance instrument.The morphology of CSNPs and Gd-CSNPs were both approximately spherical; the average particle sizes and zeta potentials of Gd-CSNPs were153.0±7.5nm and13.44±1.52mV, respectively. The results of Cytotoxicity test showed that the safety of Gd-CSNPs was good. The results of the MRI imaging test in vitro showed that the longitudinal relaxation rate of Gd-CSNPs was2.46times than that of Magnevist. The imaging signal intensity of16μMMagnevist was same as that of4μM Gd-CSNPs. The results of MRI imaging in B16tumor bearing mice model suggested that the signal intensity were enhanced significantly by tail intravenous injection of Gd-CSNPs at40μM Gd/kg, especially in the tumor and liver. And the imaging time were prolonged to4h. Compared with small molecular contrast agents, Gd-CSNPs improved imaging contrast significantly by increasing the molecular weight of contrast agents, efficiently retarding the rotational motion of Gd complex, and increasing the exchange rate between Gd and water protons by modifying gadoliniumin on the surface of nanoparticles. And Gd-CSNPs also increased the distribution of contrast agents in tumor site by enhanced permeability and retention (EPR) effect, which was conductive to improve the sensitivity of the diagnosis of tumor. In addition, Gd-CSNPs improved the characteristics of rapid renal clearance in vivo of small molecular contrast agents and prolonged the residence time in the body, which widened the time window of imaging diagnosis.2. Gd-loaded chitosan nanoparticles modified with hyaluronic acid as molecular imaging contrast agentsIn the second part, Gd-loaded chitosan nanoparticles modified with hyaluronic acid (CS-DTPA-Gd/TPP/HA NPs) were prepared as molecular imaging contrast agents for tumor diagnosis. In order to further improve the gadolinium loads of a single nanoparticle, pre-modification method was adopted that DTPA was conjugated with chitosan first through chemical reaction and then incubated with free Gd ions to prepare gadolinium labeled chitosan. Then, CS-DTPA-Gd/TPP/HA NPs were prepared via electrostatic interaction using gadolinium labeled chitosan, HA and TPP. The successful synthesis of gadolinium labeled chitosan was verified by Fourier transform infrared spectrum and nuclear magnetic resonance hydrogen spectrum. The gadolinium concentration was determined by inductively coupled plasma emission spectrometer. The optimal prescription was determined by investigating the main influence factor of the preparation of nanoparticles, and its reproducibility was also studied. The morphology was observed by TEM. The particle sizes and the zeta potentials were measured by laser particle size and potential analyzer, respectively. Cytotoxicity was tested using MTT method in B16, HepG2and A549cells. The MRI imaging abilities in vitro and in vivo were investigated by magnetic resonance instrument. The safety of the nanoparticles in mice was evaluated by tissue section method preliminarily.The experimental results showed that gadolinium labeled chitosan was synthesized successfully; the appearance of CS-DTPA-Gd/TPP/HA NPs was clarify, with pale blue light; the morphology of the nanoparticles were approximately spherical, with good dispersion; the average particle sizes and zeta potentials were213.8Π2.6nm and19.92Π1.69mV, respectively; the polydispersity index was0.219, with a narrow particle size distribution. The results of cytotoxicity test showed that the safety of the nanoparticles was good. The results of the MRI imaging test in vitro showed that the imaging signal intensity of16μM Magnevist was same as that of2μM CS-DTPA-Gd/TPP/HA NPs. Compared with Magnevist of the same Gd concentration, the imaging signal intensity of the nanoparticles was improved significantly. The results of MRI imaging in B16tumor bearing mice model suggested that the signal intensity were enhanced significantly after tail intravenous injection of the nanoparticles, especially in the tumor and liver. And the imaging time were prolonged greatly.In conclusion, Gd-CSNPs and CS-DTPA-Gd/TPP/HA NPs increased the imaging ability of small molecule Gd-based contrast agents significantly, enhanced MRI imaging effect in vivo, improved the imaging in the tumor and liver, and prolonged the residence time in the body. They might be promising MRI contrast agents that has the potential of further development and clinical application prospects. |
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