A Specific "Switch-on" Type Magnetic Resonance Nanoprobe For High Resolution Imaging Of Tumors

Malignant tumor seriously endangers human life and health.Imaging-mediated diagnosis and localization are essential for the detection,diagnosis and treatment of malignant tumors.Among many imaging techniques,magnetic resonance imaging?MRI?is widely used due to its high intensity,high soft tissue resolution,no significant radiation damage and technical flexibility of arbitrary azimuth tomography.The sensitivity and resolution of MRI are mainly dependent on imaging contrast agents?CAs?.However,MRI is still greatly limited due to the shortcomings of traditional CAs,such as low distribution specificity,poor contrast and short development time.In order to further improve the imaging resolution and sensitivity of tumor,a tumor microenvironment specific"switch on"type nanoprobe is proposed in this paper,which has the ability of tumor targeting and long circulation time in vivo,so as to significantly enhance the sensitivity and specificity of tumor MRI.In order to achieve tumor specific"switch on"MRI,this subject is based on the theory of magnetic resonance tuning?MRET?to control the"on"and"off"state of MRI signal.That is,the"switch on"state conversion of MRI signal can be realized through intelligently controlling the distance between superparamagnetic material?quencher?and paramagnetic CAs?enhancer?.Therefore,the tumor microenvironment specific"switch on"type nanoprobe is mainly divided into two units:?1?MRET effect unit:hollow mesoporous iron oxide nanoparticles?HMINs?with superparamagnetic and mesoporous properties efficiently load with paramagnetic CAs gadolinium?III?chloride?GdCl₃?.?2?Tumor intelligent response gating unit:hyaluronic acid?HA?with hyaluronidase?HAase?-specific responsiveness encapsulates the above nanoparticles,and the tumor microenvironment specific"switch on"type nanoprobe is obtained finally.In normal tissue with low-level HAase,Gd is restricted inside of HMINs,and T₁-field signal is"turned off"because the strong magnetic field generated by HMINs quench Gd spin magnetic field;while in tumor tissue with high-level HAase,the HA blocked on the surface of HMINs is specifically degraded,leading to the opening of channels and the rapid release of Gd,and the T₁-field signal is"turned on"because the distance between Gd and its quencher HMINs is significantly increased.Firstly,HMINs were synthesized by hydrothermal method,and the results of scanning electron microscope?SEM?,transmission electron microscope?TEM?,X-ray diffraction?XRD?proved the successful preparation of HMINs.The average size was around 215 nm and the particle size distribution was uniform.The specific surface area,average pore size and total pore volume of HMINs were calculated to be 139 m2/g,6.05 nm and 0.369 cm³/g respectively by measuring the specific surface area and pore size distribution,proving the existence of hollow mesoporous structure.The result of the magnetic hysteresis loop showed that HMINs had good superparamagnetism.Subsequently,HA was modificated on HMINs surface by amidation reaction.The results of ultraviolet-visible spectroscopy?UV-Vis?,fourier transform infrared spectroscopy?FT-IR?,thermogravimetric analysis?TGA?confirmed the successful modification of HA on the surface of nanoparticles.Then GdCl₃ was loaded in HMINs by crystallization method.The results of energy dispersive spectrometer?EDS-mapping?showed that Gd was successfully loaded and evenly distributed.The loading efficiency of Gd was 12.0%,which determined by inductively coupled plasma mass spectrometry?ICP-MS?.In this subject,the tumor specific"switch on"effect of the nanoprobe was investigated by detecting the MRI signal of the supernatant after release of HA-HMIN@Gd in the presence or absence of HAase in vitro.T₁-weighted image?T₁WI?showed that HA-HMIN quenched the MRI signal of Gd in the absence of HAase,and the longitudinal relaxation rate?r₁?was as low as 0.327 mM^-1s^-1.When co-incubated with HAase,the T₁-field signal strength increased,and r₁ was 11.4mM^-1s^-1,which was about 30 times greater than that without HAase.The thermogravimetric curves showed that the weight loss rate of the HAase treated group was only 12.7%,which was significantly lower than that of HA-HMIN group?21.2%?,indicating that the encapsulated HA in HA-HMIN@Gd was degraded after treated with HAase.Then we investigated the dependence of HA-HMIN@Gd MRI ability on HAase concentration and incubating time.The results showed that as the HAase concentration and incubating time gradually increased the release rate of Gd was significantly increased.At 1.8 nM and 180 nM of HAase treated groups,after release of 8 h,the release rate of Gd was 26.6%and 79.4%,respectively.Furthermore,the T₁-field signal increased as well,which was proved that under the action of HAase,Gd loaded in the channels and the hollow structure of HMINs could achieve specific release and the T₁-field signal was"switch on".The results of MRI experiments in different physiological environments showed that HA-HMIN@Gd had high sensitivity to specific response to HAase in different media.In addition,the results of cytotoxicity showed that HA-HMIN@Gd had no obvious cytotoxicity at the concentration of use and had good cellular compatibility,which laid a foundation for further study in vivo.We used tumor-bearing mice as animal model to investigat the biodistribution of HA-HMIN loaded near-infrared fluorescent dye IR783 by in vivo imaging system.The results showed that HA-HMIN@IR783 had significantly enhanced imaging agent accumulation in tumor tissue compared with free IR783 no matter at which timepoint,and HA-HMIN@IR783 displayed a longer tumor retention time over time.Then we examined the expression of HAase in liver and tumor tissue.The expression of HAase in the tumor tissue was higher than that in normal liver tissue,which laid a physiological foundation for the HA-HMIN@Gd to realize the"switch on"of high-specific MRI signals of tumors.The results of MRI in vivo showed that after injection of HA-HMIN@Gd for 4 h and 8 h,the tumor site produced a high T₁-field signal,while no high-intensity signal was observed in normal tissue,indicating that the nanoprobe had a tumor specific response.The MRI switch specificity of tumor and represent normal tissue?liver tissue,tumor/liver,T/L?was obtained by semi-quantitatively analysis.After injection of HA-HMIN@Gd for 8 h,T/L increased to the highest value?2.17?,which was significantly higher than that of Gd-DTPA group?1.14?.Also,compared with free Gd-DTPA,the imaging contrast of HA-HMIN@Gd in tumor and paracancerous tissue?T/P?was significantly improved,and the maximum imaging contrast was 1.9 times greater than Gd-DTPA.ICP-MS was used to quantify the content of Gd in the liver and tumor tissue.The results showed that the nanoprobes accumulated in liver tissue were more abundant than those in tumor tissue.Combined with the low T₁-field signal in liver tissue,the tumor specific"switch on"effect of HA-HMIN@Gd was further confirmed.And no obvious hemolysis and blood cell aggregation were observed with increasing HA-HMIN@Gd concentration in hemolysis test and blood routine examination,which showed that HA-HMIN@Gd had good blood compatibility.We further examined the histocompatibility of HA-HMIN@Gd by organ coefficient and tissue staining analysis,and there was no abnormality in the main tissues of the mice,indicating well tissue compatibility,suggesting its enormous potential for clinical application.