技术领域technical field
本发明属于医学影像材料制备技术领域，具体涉及一种双稀土掺杂碳点磁共振/CT/荧光多模态成像探针及其制备方法。The invention belongs to the technical field of preparation of medical imaging materials, and in particular relates to a double rare earth-doped carbon dot magnetic resonance/CT/fluorescence multimodal imaging probe and a preparation method thereof.
背景技术Background technique
磁共振成像技术因对人体无创、任意方向断层扫描三维图像且分辨率较高、提供形态与功能两方面诊断评价等突出优点，成为了临床上用于疾病诊断的重要手段之一。临床上使用磁共振造影剂可以提高成像的分辨率和灵敏度，提高图像质量，增强对比度和可读性。但是，各种成像技术由于实现原理不同，具有各自的优势和缺陷，靠传统单一的诊断模式无法提供疾病的全面信息，因而在对各种复杂疾病进行诊断时会受到一定的限制。因此，将磁共振成像与其他成像技术如CT成像、荧光成像、超声成像等联合起来使用，则可以达到优势互补的效果，能为疾病的临床诊断提供更快捷精确的信息，同时可将磁共振成像与各种治疗方式结合在一起，即开发基于磁共振成像的诊断治疗一体化试剂，以实现对疾病的及时治疗和实时监控。Magnetic resonance imaging technology has become one of the important methods for clinical diagnosis of diseases due to its outstanding advantages such as non-invasive to the human body, tomographic scanning of three-dimensional images in any direction with high resolution, and the ability to provide both morphological and functional diagnostic evaluations. The clinical use of magnetic resonance contrast agents can improve the resolution and sensitivity of imaging, improve image quality, and enhance contrast and readability. However, various imaging technologies have their own advantages and disadvantages due to their different implementation principles, and the traditional single diagnostic mode cannot provide comprehensive information of the disease, so it will be limited in the diagnosis of various complex diseases. Therefore, combining magnetic resonance imaging with other imaging techniques such as CT imaging, fluorescence imaging, and ultrasound imaging can achieve complementary advantages and provide faster and more accurate information for clinical diagnosis of diseases. Imaging is combined with various treatment modalities, that is, the development of integrated reagents for diagnosis and treatment based on magnetic resonance imaging to achieve timely treatment and real-time monitoring of diseases.
发明内容Contents of the invention
本发明的目的是提供一种新型的双稀土掺杂碳点磁共振/CT/荧光多模态成像探针及其制备方法，该探针是一种适用于MRI/CT/FI多模式成像的Gd/Yb@CDs纳米探针，其纵向弛豫效率可以达到6.65mM-1s-1，高于临床使用的Gd-DTPA(3.69mM-1s-1)，是已报道的Gd@CDs(5.88mM-1s-1)的1.13倍。The purpose of the present invention is to provide a novel double rare earth doped carbon dot magnetic resonance/CT/fluorescence multimodal imaging probe and its preparation method, which is a kind of MRI/CT/FI multimodal imaging probe. The longitudinal relaxation efficiency of Gd/Yb@CDs nanoprobes can reach 6.65mM-1 s-1 , which is higher than that of clinically used Gd-DTPA (3.69mM-1 s-1 ). 5.88mM-1 s-1 ) 1.13 times.
为了实现上述目的，本发明的技术方案具体如下：In order to achieve the above object, the technical solution of the present invention is specifically as follows:
一种双稀土掺杂碳点磁共振/CT/荧光多模态成像探针，是由碳、氮、氧、钆和镱元素组成的钆和镱共掺杂碳点，表达式为Gd/Yb@CDs。A double rare earth doped carbon dot magnetic resonance/CT/fluorescence multimodal imaging probe is a gadolinium and ytterbium co-doped carbon dot composed of carbon, nitrogen, oxygen, gadolinium and ytterbium elements, expressed as Gd/Yb @CDs.
在上述技术方案中，所述镱元素还可以替换为镝或钬元素。In the above technical solution, the ytterbium element can also be replaced by dysprosium or holmium element.
在上述技术方案中，所述双稀土掺杂碳点磁共振/CT/荧光多模态成像探针呈单分散球形，平均粒径为5.26±0.93nm。In the above technical solution, the double rare earth doped carbon dot magnetic resonance/CT/fluorescence multimodal imaging probe is monodisperse spherical, with an average particle diameter of 5.26±0.93nm.
在上述技术方案中，所述碳、氮、氧、钆和镱元素的百分含量分别为30.35％、8.67％、32.20％、7.40％和21.38％。In the above technical solution, the percentage contents of the carbon, nitrogen, oxygen, gadolinium and ytterbium elements are respectively 30.35%, 8.67%, 32.20%, 7.40% and 21.38%.
一种双稀土掺杂碳点磁共振/CT/荧光多模态成像探针的制备方法，包括以下步骤：A method for preparing a double rare earth-doped carbon dot magnetic resonance/CT/fluorescence multimodal imaging probe, comprising the following steps:
步骤1、将Na2EDTA、GdCl3、YbCl3和L-精氨酸溶于去离子水中，磁力搅拌，得到无色透明的溶液；Step 1. Dissolve Na2 EDTA, GdCl3 , YbCl3 and L-arginine in deionized water, and stir magnetically to obtain a colorless and transparent solution;
步骤2、将步骤1得到的无色透明的溶液转移到反应釜中，于200℃下反应10h，待其冷却至室温后，通过离心收集上清液；Step 2. Transfer the colorless and transparent solution obtained in step 1 to a reaction kettle, react at 200° C. for 10 h, and collect the supernatant by centrifugation after it is cooled to room temperature;
步骤3、将步骤2得到的上清液转移到透析膜中，并用超纯水透析；Step 3, transfer the supernatant obtained in step 2 to a dialysis membrane, and dialyze with ultrapure water;
步骤4、将透析后的溶液用微滤膜过滤、冷冻干燥后获得Gd/Yb@CDs。Step 4. Filter the dialyzed solution with a microfiltration membrane and freeze-dry to obtain Gd/Yb@CDs.
在上述技术方案中，所述GdCl3还可以替换为DyCl3或HoCl3。In the above technical solution, the GdCl3 may also be replaced by DyCl3 or HoCl3 .
在上述技术方案中，所述Na2EDTA、GdCl3、YbCl3和L-精氨酸的摩尔比为250.00:3.34:30.06:70.00。In the above technical solution, the molar ratio of Na2 EDTA, GdCl3 , YbCl3 and L-arginine is 250.00:3.34:30.06:70.00.
在上述技术方案中，步骤1中磁力搅拌的时间为15min。In the above technical scheme, the time for magnetic stirring in step 1 is 15 minutes.
在上述技术方案中，步骤2中离心收集上清液时的转速为11000rpm，时间为20min。In the above-mentioned technical scheme, the rotating speed when centrifuging to collect the supernatant in step 2 is 11000rpm, and the time is 20min.
在上述技术方案中，步骤4中所述微滤膜的孔径为0.22μm。In the above technical solution, the pore size of the microfiltration membrane in step 4 is 0.22 μm.
本发明的有益效果是：The beneficial effects of the present invention are:
本发明提供的双稀土掺杂碳点磁共振/CT/荧光多模态成像探针是由碳、氮、氧、钆和镱元素组成的钆和镱共掺杂碳点，该探针适用于磁共振成像、CT成像和荧光成像，纵向弛豫效率可以达到6.65mM-1s-1，高于临床使用的Gd-DTPA(3.69mM-1s-1)。The double rare earth doped carbon dot magnetic resonance/CT/fluorescence multimodal imaging probe provided by the present invention is a gadolinium and ytterbium co-doped carbon dot composed of carbon, nitrogen, oxygen, gadolinium and ytterbium elements, and the probe is suitable for For magnetic resonance imaging, CT imaging and fluorescence imaging, the longitudinal relaxation efficiency can reach 6.65mM-1 s-1 , which is higher than that of Gd-DTPA (3.69mM-1 s-1 ) in clinical use.
本发明提供的双稀土掺杂碳点磁共振/CT/荧光多模态成像探针与Gd@CDs相比，不仅增加了CT成像功能，其纵向弛豫效率也是已报道的Gd@CDs(5.88mM-1s-1)的1.13倍。Compared with Gd@CDs, the double rare earth-doped carbon dot magnetic resonance/CT/fluorescence multimodal imaging probe provided by the present invention not only increases the CT imaging function, but also has the longitudinal relaxation efficiency of the reported Gd@CDs (5.88 1.13 times of mM-1 s-1 ).
本发明提供的双稀土掺杂碳点磁共振/CT/荧光多模态成像探针中的Gd、Yb不会泄露。Gd and Yb in the double rare earth doped carbon dot magnetic resonance/CT/fluorescence multimodal imaging probe provided by the present invention will not leak.
本发明提供的双稀土掺杂碳点磁共振/CT/荧光多模态成像探针具有较高的化学稳定性和较低的毒性。The double rare earth doped carbon dot magnetic resonance/CT/fluorescence multimodal imaging probe provided by the invention has high chemical stability and low toxicity.
本发明提供的双稀土掺杂碳点磁共振/CT/荧光多模态成像探针在MRI/CT/FI成像中作为局部对比增强探针具有很大的应用潜力。The dual rare earth-doped carbon dot magnetic resonance/CT/fluorescence multimodal imaging probe provided by the invention has great application potential as a local contrast enhancement probe in MRI/CT/FI imaging.
本发明提供的双稀土掺杂碳点磁共振/CT/荧光多模态成像探针的制备方法，其制备流程简单，制得的纳米探针不需要进一步修饰，其纵向弛豫效率就可以达到6.65mM-1s-1。The preparation method of the double rare earth doped carbon dot magnetic resonance/CT/fluorescence multimodal imaging probe provided by the present invention has a simple preparation process, and the prepared nanoprobe does not need further modification, and its longitudinal relaxation efficiency can reach 6.65mM-1 s-1 .
附图说明Description of drawings
下面结合附图和具体实施方式对本发明作进一步详细说明。The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.
图1为本发明实施例制备的Gd/Yb@CDs纳米粒子的HRTEM照片。Fig. 1 is an HRTEM photo of Gd/Yb@CDs nanoparticles prepared in the embodiment of the present invention.
图2为本发明实施例制备的Gd/Yb@CDs纳米粒子的XPS谱图。Fig. 2 is the XPS spectrum of the Gd/Yb@CDs nanoparticles prepared in the embodiment of the present invention.
图3为本发明实施例制备的Gd/Yb@CDs纳米粒子的FTIR谱图。Fig. 3 is the FTIR spectrum of the Gd/Yb@CDs nanoparticles prepared in the embodiment of the present invention.
图4为本发明实施例制备的Gd/Yb@CDs纳米粒子中Gd3+的泄露情况图表。Fig. 4 is a graph showing the leakage of Gd3+ in the Gd/Yb@CDs nanoparticles prepared in the embodiment of the present invention.
图5为HeLa细胞和4T1细胞与不同浓度的Gd/Yb@CDs共同孵育4h和24h后的细胞存活率。Figure 5 shows the cell viability of HeLa cells and 4T1 cells incubated with different concentrations of Gd/Yb@CDs for 4h and 24h.
图6为本发明实施例制备的Gd/Yb@CDs的UV-Vis的光谱图，其中图6A为吸收光谱、图6B为激发/发射荧光光谱。Fig. 6 is a UV-Vis spectrum diagram of Gd/Yb@CDs prepared in the embodiment of the present invention, wherein Fig. 6A is an absorption spectrum, and Fig. 6B is an excitation/emission fluorescence spectrum.
图7为HeLa细胞和Gd/Yb@CDs(1mg/mL)共同孵育2h后的细胞图像，其中(A)为亮场图像；(B)为荧光图像。Figure 7 is the cell image of HeLa cells incubated with Gd/Yb@CDs (1 mg/mL) for 2 h, where (A) is a bright-field image; (B) is a fluorescence image.
图8为不同浓度的本发明实施例制备的Gd/Yb@CDs纳米粒子和Gd-DTPA的体外MRI成像。Fig. 8 is the in vitro MRI imaging of Gd/Yb@CDs nanoparticles and Gd-DTPA prepared by different concentrations of the embodiments of the present invention.
图9为本发明实施例制备的Gd/Yb@CDs纳米粒子和Gd-DTPA的纵向弛豫效率1/T1随造影剂浓度变化的线性关系图。Fig. 9 is a linear relationship diagram of the longitudinal relaxation efficiency 1/T1 of Gd/Yb@CDs nanoparticles prepared in the embodiment of the present invention and Gd-DTPA as a function of contrast agent concentration.
图10为不同浓度的本发明实施例制备的Gd/Yb@CDs纳米粒子和碘比醇溶液的体外CT成像。Fig. 10 is an in vitro CT imaging of Gd/Yb@CDs nanoparticles prepared in the embodiments of the present invention and iopicol solutions at different concentrations.
图11为本发明实施例制备的Gd/Yb@CDs纳米粒子和碘比醇溶液的CT值随纳米粒子浓度变化的线性关系图。Fig. 11 is a graph showing the linear relationship between the CT value of the Gd/Yb@CDs nanoparticles prepared in the embodiment of the present invention and the iodine alcohol solution as a function of the nanoparticle concentration.
具体实施方式Detailed ways
下面结合附图对本发明做以详细说明。The present invention will be described in detail below in conjunction with the accompanying drawings.
本发明提供的双稀土掺杂碳点磁共振/CT/荧光多模态成像探针，是由碳、氮、氧、钆和镱元素组成的钆和镱共掺杂碳点，表达式为Gd/Yb@CDs，呈单分散球形，平均粒径为5.26±0.93nm。其中的镱元素还可以替换为镝或钬元素。优选所述碳、氮、氧、钆和镱元素的百分含量分别为30.35％、8.67％、32.20％、7.40％和21.38％。The double rare earth doped carbon dot magnetic resonance/CT/fluorescent multimodal imaging probe provided by the present invention is a gadolinium and ytterbium co-doped carbon dot composed of carbon, nitrogen, oxygen, gadolinium and ytterbium elements, and the expression is Gd /Yb@CDs exhibits a monodisperse spherical shape with an average particle size of 5.26±0.93 nm. The ytterbium element can also be replaced by dysprosium or holmium element. Preferably, the percentage contents of carbon, nitrogen, oxygen, gadolinium and ytterbium elements are 30.35%, 8.67%, 32.20%, 7.40% and 21.38%, respectively.
本发明提供的双稀土掺杂碳点磁共振/CT/荧光多模态成像探针的制备方法，包括以下步骤：The preparation method of the double rare earth doped carbon dot magnetic resonance/CT/fluorescence multimodal imaging probe provided by the present invention comprises the following steps:
步骤1、将Na2EDTA、GdCl3、YbCl3和L-精氨酸溶于去离子水中，磁力搅拌15min，得到无色透明的溶液；优选所述Na2EDTA、GdCl3、YbCl3和L-精氨酸的摩尔比为250.00:3.34:30.06:70.00；所述GdCl3还可以替换为DyCl3或HoCl3；Step 1. Dissolve Na2 EDTA, GdCl3 , YbCl3 and L-arginine in deionized water, and stir magnetically for 15 minutes to obtain a colorless and transparent solution; preferably the Na2 EDTA, GdCl3 , YbCl3 and L- - the molar ratio of arginine is 250.00:3.34:30.06:70.00; the GdCl3 can also be replaced by DyCl3 or HoCl3 ;
步骤2、将步骤1得到的无色透明的溶液转移到反应釜中，于200℃下反应10h，待其冷却至室温后，通过离心收集上清液，转速为11000rpm，时间为20min；Step 2. Transfer the colorless and transparent solution obtained in step 1 to a reaction kettle, and react at 200° C. for 10 hours. After cooling to room temperature, collect the supernatant by centrifugation at a speed of 11,000 rpm for 20 minutes;
步骤3、将步骤2得到的上清液转移到透析膜中，并用超纯水透析；Step 3, transfer the supernatant obtained in step 2 to a dialysis membrane, and dialyze with ultrapure water;
步骤4、将透析后的溶液用孔径为0.22μm的微滤膜过滤、冷冻干燥后获得Gd/Yb@CDs。Step 4. The dialyzed solution was filtered through a microfiltration membrane with a pore size of 0.22 μm, and then freeze-dried to obtain Gd/Yb@CDs.
实施例Example
用天平称量Na2EDTA(93.06mg，0.25mmol)，GdCl3(0.88mg，3.34μmol)，YbCl3(8.48mg，30.06μmol)和L-精氨酸(12.54mg，0.07mmol)溶于20mL去离子水中，将烧杯放在磁力搅拌器上搅拌15min，得到无色透明的溶液。将该溶液转移到50毫升聚四氟乙烯反应釜中，并将其放入烘箱中200℃下反应10h。待其冷却至室温后，通过离心收集上清液，转速为11000rpm，离心20min以除去黑色沉淀，反复三次。将得到的棕黄色上清液转移到透析膜(MWCO为1000)中，并用超纯水透析24h。每4h换一次水，除去多余的未反应的物质。最后得到的溶液用0.22μm微滤膜过滤，使用真空冷冻干燥器将过滤的溶液冷冻干燥，得到Gd/Yb@CDs纳米粒子。元素分析结果证明所得到的Gd/Yb@CDs纳米粒子中碳、氮、氧、钆和镱元素的百分含量分别为30.35％、8.67％、32.20％、7.40％和21.38％。Use a balance to weigh Na2 EDTA (93.06mg, 0.25mmol), GdCl3 (0.88mg, 3.34μmol), YbCl3 (8.48mg, 30.06μmol) and L-arginine (12.54mg, 0.07mmol) dissolved in 20mL In deionized water, the beaker was placed on a magnetic stirrer and stirred for 15 min to obtain a colorless and transparent solution. The solution was transferred to a 50 ml polytetrafluoroethylene reactor, and it was placed in an oven at 200° C. for 10 h. After it was cooled to room temperature, the supernatant was collected by centrifugation at 11000 rpm for 20 min to remove the black precipitate, and this was repeated three times. The resulting brownish-yellow supernatant was transferred to a dialysis membrane (MWCO: 1000), and dialyzed with ultrapure water for 24 h. Change the water every 4 hours to remove excess unreacted material. The finally obtained solution was filtered with a 0.22 μm microfiltration membrane, and the filtered solution was freeze-dried using a vacuum freeze dryer to obtain Gd/Yb@CDs nanoparticles. The element analysis results proved that the percentage contents of carbon, nitrogen, oxygen, gadolinium and ytterbium elements in the obtained Gd/Yb@CDs nanoparticles were 30.35%, 8.67%, 32.20%, 7.40% and 21.38%, respectively.
将上述实施例中GdCl3替换为DyCl3或HoCl3，对应制备得到Gd/Dy@CDs纳米粒子或Gd/Ho@CDs纳米粒子。In the above examples, GdCl3 is replaced by DyCl3 or HoCl3 , correspondingly preparing Gd/Dy@CDs nanoparticles or Gd/Ho@CDs nanoparticles.
图1为实施例制备得到的Gd/Yb@CDs纳米粒子的HRTEM照片，由图可知：Gd/Yb@CDs纳米粒子呈单分散类球形，平均粒径约为5.26±0.93nm(图1A)；通过图1B可以看到清晰的晶格条纹，晶格间距为0.213nm，对应于石墨碳的(100)晶面，这与之前文献报道的相一致。Fig. 1 is the HRTEM photo of the Gd/Yb@CDs nanoparticles prepared in the example. It can be seen from the figure that the Gd/Yb@CDs nanoparticles are monodisperse and spherical, with an average particle size of about 5.26±0.93nm (Fig. 1A); Clear lattice fringes can be seen through Figure 1B with a lattice spacing of 0.213 nm, corresponding to the (100) crystal plane of graphitic carbon, which is consistent with previous literature reports.
图2和3分别为本发明实施制备的Gd/Yb@CDs纳米粒子的XPS和FTIR谱图，由图2可知Gd/Yb@CDs纳米粒子由C 1s(284.5eV)，N 1s(399.2eV)和O 1s(531.1eV)，Gd 3d(1186eV)和Yb 4d(184.6eV)元素组成，证明稀土元素Gd和Yb成功掺入碳点中。由图3可知Gd/Yb@CDs与CDs的FT-IR光谱表现出类似的吸收带：在3500～3200cm-1处的吸收峰对应于O-H和N-H伸缩振动，在1100cm-1处为C-N伸缩振动，而COO-基团的峰从1632cm-1变为1615cm-1，可能由于Gd3+/Yb3+和EDTA/L-精氨酸的羧基之间的相互作用引起的。这些特征峰进一步证明了羟基，羧酸和氨基的存在，与XPS结果一致。Gd3+和Yb3+螯合后，整体FT-IR光谱峰形没有明显变化，说明稀土Gd/Yb的掺杂对碳点表面官能团没有明显影响。Figures 2 and 3 are the XPS and FTIR spectra of the Gd/Yb@CDs nanoparticles prepared by the present invention, respectively. From Figure 2, it can be seen that the Gd/Yb@CDs nanoparticles are composed of C 1s (284.5eV), N 1s (399.2eV) and O 1s (531.1eV), Gd 3d (1186eV) and Yb 4d (184.6eV) elements, proving that the rare earth elements Gd and Yb were successfully incorporated into the carbon dots. It can be seen from Figure 3 that the FT-IR spectra of Gd/Yb@CDs and CDs show similar absorption bands: the absorption peaks at 3500-3200 cm-1 correspond to OH and NH stretching vibrations, and the CN stretching vibrations at 1100 cm-1 , and the peak of COO- group changed from 1632cm-1 to 1615cm-1 , which may be caused by the interaction between Gd3+ /Yb3+ and the carboxyl group of EDTA/L-arginine. These characteristic peaks further prove the existence of hydroxyl, carboxylic acid and amino groups, consistent with the XPS results. After the chelation of Gd3+ and Yb3+ , the overall FT-IR spectrum peak shape does not change significantly, indicating that the doping of rare earth Gd/Yb has no obvious effect on the surface functional groups of carbon dots.
考虑到游离的Gd3+能抑制体内Ca2+的通道进而诱导严重的细胞毒性(如心血管和神经细胞毒性)，因而，我们将含有Gd/Yb@CDs的透析袋(MWCO＝1000)放在血清溶液中，进行透析24h，每隔一段时取出0.4mL，用ICP-OES测量Gd/Yb@CDs的Gd3+泄漏，如图4所示，图中&代表初始纳米粒子中Gd3+的含量。由图4可知，血清溶液中几乎没有检测到的Gd3+的泄露，这可能由于Gd3+与碳点之间存在着强烈的相互作用。Considering that free Gd3+ can inhibit Ca2+ channels in the body and induce severe cytotoxicity (such as cardiovascular and neurocytotoxicity), we placed the dialysis bag (MWCO=1000) containing Gd/Yb@CDs In the serum solution, dialyze for 24h, take out 0.4mL at intervals, measure the Gd3+ leakage of Gd/Yb@CDs by ICP-OES, as shown in Figure 4, in the figure & represents the Gd3+ in the initial nanoparticles content. It can be seen from Figure 4 that there is almost no leakage of Gd3+ detected in the serum solution, which may be due to the strong interaction between Gd3+ and carbon dots.
利用MTT法研究Gd/Yb@CDs对HeLa和4T1细胞的毒性。图5为HeLa细胞和4T1细胞与不同浓度的Gd/Yb@CDs共同孵育4h和24h后的细胞存活率，图5中每个Gd/Yb@CDs浓度对应的细胞存活率均从左至右标记为a、b、c、d，图中仅在Gd/Yb@CDs浓度为0时标注出。由图5可知：当培育4h后，两种细胞系的存活率和增殖几乎不受影响。延长到24h，即使Gd/Yb@CDs的浓度高达1mg/mL，细胞存活率仍然超过85％。这些初步的实验结果表明，制备的Gd/Yb@CDs具有较低的毒性。这种低毒性也源于Gd/Yb@CDs较高的化学稳定性。相比具有重金属毒性的半导体量子点，Gd/Yb@CDs的低毒性使其在生物和医学领域具有潜在的应用前景。The toxicity of Gd/Yb@CDs to HeLa and 4T1 cells was studied by MTT assay. Figure 5 shows the cell survival rate of HeLa cells and 4T1 cells incubated with different concentrations of Gd/Yb@CDs for 4h and 24h, and the cell survival rate corresponding to each concentration of Gd/Yb@CDs in Figure 5 is marked from left to right are a, b, c, d, and only marked when the concentration of Gd/Yb@CDs is 0 in the figure. It can be seen from Figure 5 that the survival rate and proliferation of the two cell lines were almost unaffected after incubation for 4 hours. Extended to 24h, even if the concentration of Gd/Yb@CDs was as high as 1mg/mL, the cell survival rate still exceeded 85%. These preliminary experimental results indicated that the as-prepared Gd/Yb@CDs had lower toxicity. This low toxicity is also due to the high chemical stability of Gd/Yb@CDs. Compared with semiconductor quantum dots with heavy metal toxicity, the low toxicity of Gd/Yb@CDs makes them have potential applications in the fields of biology and medicine.
图6为Gd/Yb@CDs的UV-Vis的光谱图，其中图6A为吸收光谱、图6B为激发/发射荧光光谱。由图6可知：CDs和Gd/Yb@CDs在273nm处呈现相同的吸收峰，这对应于C＝O的n→π*跃迁。在365nm紫外灯照射下，Gd/Yb@CDs淡黄色水溶液呈现明亮的蓝色荧光(图6A插图)。Gd/Yb@CDs也表现出依赖的激发发射行为，这是由不同能量的表面态发射陷阱引起的普遍现象。随着激发波长的变化，相应的表面态发射陷阱占主导地位，从而导致激发波长依赖现象。由图6B可知：当激发波长从320nm变为370nm时，发射峰从407nm移动到463nm。当样品激发340nm时，最大荧光发射强度为418nm。用硫酸奎宁作为参比溶液的量子产率为16.84％，略高于Gd@CDs的13.4％。此外，在365nm紫外灯下研究了Gd/Yb@CDs的荧光稳定性，长达2h曝光于紫外灯下，发光强度仍然没有变化，表明Gd/Yb@CDs具有优异的光学稳定性。Figure 6 is the UV-Vis spectrum of Gd/Yb@CDs, where Figure 6A is the absorption spectrum and Figure 6B is the excitation/emission fluorescence spectrum. It can be seen from Figure 6 that CDs and Gd/Yb@CDs exhibit the same absorption peak at 273 nm, which corresponds to the n→π* transition of C=O. Under the irradiation of 365 nm UV lamp, the pale yellow aqueous solution of Gd/Yb@CDs exhibited bright blue fluorescence (Fig. 6A inset). Gd/Yb@CDs also exhibit dependent excitation-emission behavior, which is a common phenomenon caused by surface state emission traps of different energies. As the excitation wavelength varies, the corresponding surface state emission traps dominate, leading to the excitation wavelength-dependent phenomenon. It can be seen from Figure 6B that when the excitation wavelength changes from 320nm to 370nm, the emission peak moves from 407nm to 463nm. When the sample is excited at 340nm, the maximum fluorescence emission intensity is 418nm. The quantum yield was 16.84% using quinine sulfate as a reference solution, slightly higher than 13.4% of Gd@CDs. In addition, the fluorescence stability of Gd/Yb@CDs was studied under a 365 nm UV lamp, and the luminous intensity remained unchanged after exposure to UV lamp for up to 2 h, indicating that Gd/Yb@CDs has excellent optical stability.
为了考察Gd/Yb@CDs在荧光成像方面的性能，我们将HeLa细胞与1mg mL-1Gd/Yb@CDs共同孵育2h。使用荧光显微镜分别在明场和暗场下捕获图像。图7为HeLa细胞和Gd/Yb@CDs(1mg/mL)共同孵育2h后的细胞图像，其中(A)为亮场图像；(B)为荧光图像。由图7可知在明场下，经过处理的细胞仍然保持着完整的形态；在暗场中，HeLa细胞显示出非常强的蓝色荧光。此外，我们可以看到，Gd/Yb@CDs主要位于细胞质中，这与以前报道的CDs行为相同。这表明在很短的时间内，Gd/Yb@CDs可以通过细胞膜屏障进入细胞内区域。In order to investigate the performance of Gd/Yb@CDs in fluorescence imaging, we incubated HeLa cells with 1mg mL-1 Gd/Yb@CDs for 2h. Images were captured using a fluorescence microscope in brightfield and darkfield, respectively. Figure 7 is the cell image of HeLa cells incubated with Gd/Yb@CDs (1 mg/mL) for 2 h, where (A) is a bright-field image; (B) is a fluorescence image. It can be seen from Figure 7 that under bright field, the treated cells still maintain a complete shape; in dark field, HeLa cells show very strong blue fluorescence. Furthermore, we can see that Gd/Yb@CDs are mainly located in the cytoplasm, which is the same behavior as previously reported CDs. This indicates that Gd/Yb@CDs can enter the intracellular region through the membrane barrier in a short time.
为了考察Gd/Yb@CDs纳米粒子在T1-MR成像方面的性能，首先，将去离子水作为对照，测定不同浓度纳米粒子水溶液的水管成像。Magnevist(Gd-DTPA)，FDA批准并广泛用于临床的造影剂，被选为对照组。随着Gd3+浓度增加(0～0.36mM)，两组T1-加权的MR图像信号逐渐增亮(图8，图中a代表Gd-DTPA，b代表Gd/Yb@CDs)，但在同浓度下本实施制备的Gd/Yb@CDs纳米粒子造影效果更好。为了进一步定量评估对比效果，使用9.4T标准反转恢复脉冲序列测量不同浓度的Gd/Yb@CDs溶液和Gd-DTPA的纵向弛豫时间(T1)。T1的倒数值与Gd3+的浓度之间存在良好的线性关系，拟合线的斜率为纵向弛豫效率(r1)，常用于评价MRI造影剂的性能。Gd/Yb@CDs的r1值为6.65mM-1s-1(图9)，高于临床的Gd-DTPA(r1＝3.69mM-1s-1)，是已报道的Gd@CDs(粒径为12nm，r1＝5.88mM-1S-1)的1.13倍。Gd/Yb@CDs的强弛豫性主要归因于粒径较小，可以增大S/V值进而增加金属离子与水分子的结合量，此外，亲水基团的存在也在一定程度上促进了r1的增强。In order to investigate the performance of Gd/Yb@CDs nanoparticles in T1 -MR imaging, first, using deionized water as a control, the water channel imaging of aqueous solutions of nanoparticles with different concentrations was measured. Magnevist (Gd-DTPA), a contrast agent approved by FDA and widely used clinically, was selected as the control group. With the increase of Gd3+ concentration (0-0.36mM), the two groups of T1 -weighted MR image signals gradually brightened (Fig. 8, in which a represents Gd-DTPA, b represents Gd/Yb@CDs), but in The contrast effect of Gd/Yb@CDs nanoparticles prepared in this practice is better at the same concentration. To further quantitatively evaluate the contrast effect, the longitudinal relaxation times (T1 ) of different concentrations of Gd/Yb@CDs solutions and Gd-DTPA were measured using a 9.4 T standard inversion recovery pulse sequence. There is a good linear relationship between the reciprocal value of T1 and the concentration of Gd3+ , and the slope of the fitted line is the longitudinal relaxation efficiency (r1 ), which is often used to evaluate the performance of MRI contrast agents. The r1 value of Gd/Yb@CDs is 6.65mM-1 s-1 (Fig. 9), which is higher than the clinical Gd-DTPA (r1 = 3.69mM-1 s-1 ), which is the highest among the reported Gd@CDs ( The particle diameter is 12nm, 1.13 times of r1 =5.88mM-1 S-1 ). The strong relaxivity of Gd/Yb@CDs is mainly attributed to the small particle size, which can increase the S/V value and increase the binding amount of metal ions and water molecules. In addition, the presence of hydrophilic groups is also to a certain extent Facilitated enhancement ofr1 .
稀土元素Yb和Gd具有固有的X-射线吸收能力，因此，Gd/Yb@CDs纳米粒子也可用于CT成像。碘比醇，临床上常用的CT造影剂，作为对照组。在120kV的X射线能量下测试两组的水管CT图像(图10)，随着Yb3+和I浓度的增加，CT亮度逐渐增强。相对而言，在相同浓度下，Gd/Yb@CDs表现出比碘比醇更高的对比效果。为了进一步定量评估CT造影效果，测量了Gd/Yb@CDs的X射线衰减能力。如图11所示，HU值与Yb3+或I的浓度之间具有良好的线性关系，Gd/Yb@CDs的斜率约为45.42HU Lg-1，明显地高于碘比醇(31.83HU L g-1)。这主要是由于Yb的衰减系数(100keV时为3.88cm2/g)大于I的衰减系数(100keV时为1.94cm2/g)。总之，结果表明，Gd/Yb@CDs在MRI/CT成像中作为局部对比增强探针具有很大的潜力。The rare earth elements Yb and Gd have inherent X-ray absorption ability, therefore, Gd/Yb@CDs nanoparticles can also be used for CT imaging. Iobiol, a CT contrast agent commonly used in clinical practice, was used as a control group. The CT images of the water pipes of the two groups were tested under the X-ray energy of 120kV (Fig. 10). As the concentration of Yb3+ and I increased, the CT brightness gradually increased. Relatively speaking, at the same concentration, Gd/Yb@CDs exhibited a higher contrast effect than iodomethol. In order to further quantitatively evaluate the CT contrast effect, the X-ray attenuation ability of Gd/Yb@CDs was measured. As shown in Figure 11, there is a good linear relationship between the HU value and the concentration of Yb3+ or I, and the slope of Gd/Yb@CDs is about 45.42HU Lg-1 , which is significantly higher than that of iodine alcohol (31.83HU L g-1 ). This is mainly because the attenuation coefficient of Yb (3.88cm2 /g at 100keV) is greater than that of I (1.94cm2 /g at 100keV). Taken together, the results demonstrate that Gd/Yb@CDs have great potential as local contrast-enhancing probes in MRI/CT imaging.
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。Apparently, the above-mentioned embodiments are only examples for clear description, rather than limiting the implementation. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. And the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.
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