摘要
目的分别制备两种多功能超声造影剂：载染料苏丹黑B的聚乳酸羟基乙酸超声微泡造影剂（SB-PLGA）和载阿霉素的超顺磁性聚乳酸羟基乙酸超声微泡造影剂（DOX-SPLGA），并检测其一般特性。
方法采用双乳化法和真空冷冻干燥法分别制备出SB-PLGA及不同铁浓度的DOX-SPLGA,并对其进行粒径，形态，包封率等性质检测；其中对载药微泡DOX-SPLGA还进行了体外释药实验观察，采用原子吸收分光光度计检测造影剂的铁含量；用振动样品磁强计及磁共振仪检测造影剂的磁学性质；体外超声和磁共振显像评价造影剂的显像效果。
结果SB-PLGA外观呈蓬松的蓝色粉末状，溶于水后为均匀的深蓝色乳液，分散度好。扫描电镜观察微泡粒径1～2μm，其形态规则，呈球形，表面光滑，大小均匀。马尔文电位检测其Zeta电位为（20.80±1.60）mV，SB的包封率为（85.67±1.44）%，载药量为（3.43±0.06）%，分散度好。DOX-SPLGA外观呈黄褐色粉末，溶于水后为均匀的乳液，分散度较好。扫描电镜观察微泡呈球形，表面有孔，大小较均匀，透射电镜显示微泡为壳核结构，SPIO纳米颗粒分布于微泡的壳膜中。原子吸收光谱法得到7种不同铁浓度制得的DOX-SPLGA中的铁含量分别为（1.47±0.07）μg/ml（,2.83±0.08）μg/ml,（5.76±0.33）μg/ml（,14.77±0.32）μg/ml,（28.06±0.62）μg/ml，（56.71±0.44）μg/ml，（185.45±1.06）μg/ml，其相对应的DOX的包封率分别为（58.10±1.61）%（,57.73±3.45）%,（60.30±2.67）%,（57.53±0.80）%,（61.13±0.95）%,（56.07±1.55）%,（59.60±1.61）%；DOX的载药量分别为（5.81±0.16）%,（5.77±0.35）%,（6.03±0.27）%,（5.75±0.08）%,（6.11±0.10）%,（5.61±0.16）%,（5.96±0.16）%。造影剂的粒径为800~900nm，具有超顺磁性，其磁敏感效应与壳膜中装载的SPIO的含量有关。体外超声显像实验显示，DOX-SPLGA能够较普通PLGA造影剂产生更强的超声回声。体外磁共振显像实验显示，T2*信号随着造影剂内铁浓度的增高而降低。
结论SB-PLGA造影剂为蓝色粉末，粒径均匀，分散好，为其之后既能外科手术前进行肿瘤转移淋巴结内超声显像，又能手术活检时指示定位淋巴结提供了基础。DOX-SPLGA造影剂具有形态规则,大小较均一，表面光滑有孔，分散均匀,超顺磁性等优点，在体外能够增强超声、磁共振显像，同时能载药辅助化疗，是一种有应用前景的多功能超声造影剂。
目的探讨SB-PLGA造影剂的细胞毒性及被巨噬细胞吞噬的能力；探讨SB-PLGA造影剂对兔肿瘤转移淋巴结的超声显像和手术示踪效果，及其在淋巴结组织内的分布情况。
方法体外培养小鼠单核巨噬细胞RAW264.7，加入同一浓度SB-PLGA造影剂孵育3h、24h和48h后，光镜下观察巨噬细胞对造影剂的吞噬情况，用MTT法检测造影剂对巨噬细胞增殖活性的影响。采用瘤块组织悬液种植法建立兔VX2腘窝肿瘤转移淋巴结模型，建模后14天用于肿瘤淋巴结超声显像。选取肿瘤转移淋巴结模型兔8只，共16个肿瘤淋巴结（实验组8个，对照组8个），实验组给予SB-PLGA造影剂，对照组给予等量生理盐水，分别经足垫皮下间隙注射，行超声造影检查评价腘窝肿瘤转移淋巴结及第二站腹股沟淋巴结的增强效果；并分别于30min后行腘窝和腹股沟淋巴结清扫术，切除蓝染及未染色淋巴结，进行冰冻切片HE染色。
结果光镜下观察，巨噬细胞对SB-PLGA造影剂的吞噬量随着造影剂与细胞孵育时间的增加而增加。MTT结果显示，SB-PLGA造影剂被巨噬细胞吞噬后对其增殖活性无明显影响。超声造影可明显增强兔腘窝肿瘤转移淋巴结显像，而同侧腹股沟淋巴结及注射生理盐水组显像不明显。腘窝淋巴结清扫术中可见，注射SB-PLGA造影剂组淋巴结染色效果好；而腹股沟淋巴结清扫术中均未见第二站淋巴结蓝染。淋巴结冰冻切片HE染色可见，SB-PLGA存在于淋巴窦内，并有大量进入巨噬细胞。
结论SB-PLGA在体外能够被巨噬细胞吞噬，且对细胞增殖活性无明显影响。SB-PLGA能够被淋巴结内巨噬细胞吞噬，增强淋巴结超声显像，同时又能通过染色来指示定位淋巴结，是一种良好的多功能超声造影剂。
目的探讨DOX-SPLGA造影剂对兔肿瘤转移淋巴结的超声和磁共振显像效果；探讨DOX-SPLGA造影剂联合低频超声促进药物释放对兔肿瘤转移淋巴结的治疗效果。
方法新西兰大白兔42只，采用瘤块种植法于大腿外侧植入VX2肿瘤组织块悬液，建立兔腘窝肿瘤转移淋巴结模型。建模后第15天，取12只模型兔（24个肿瘤淋巴结），随机分为三组（每组8个肿瘤淋巴结），分别为DOX-SPLGA组（经足垫皮下注射DOX-SPLGA微泡）、PLGA组（经足垫皮下注射普通PLGA微泡）及对照组（经足垫皮下注射等量生理盐水），行超声和磁共振扫描，并应用图像分析软件评价造影效果。扫描结束后，取淋巴结组织进行HE染色和普鲁士蓝染色，以及透射电镜检查。建模后第15天，取30只模型兔（60个肿瘤淋巴结），随机分为6组（每组10个肿瘤淋巴结），包括空白对照组（C）、载药微泡（DOX-SPLGA）、单纯药物组（DOX）、单纯微泡+超声组（PLGA+US）、药物+超声组（DOX+US）、载药微泡+超声组（DOX-SPLGA+US）。治疗后取肿瘤淋巴结组织，用免疫组化法检测淋巴结组织PCNA、CD34及LYVE-1的表达；TUNEL法检测淋巴结内肿瘤细胞凋亡。
结果在超声显像的造影模式和传统灰阶模式下，与对照组相比，经DOX-SPLGA与普通PLGA微泡造影后，腘窝淋巴结均可见回声增强；且DOX-SPLGA组比普通PLGA组回声更强。在磁共振显像中，DOX-SPLGA组表现出明显负性增强效果，普通PLGA组负性增强效果较弱，而对照组未见明显增强。普鲁士蓝染色和透射电镜结果均证实了DOX-SPLGA存在于淋巴结组织内。免疫组化结果显示，DOX-SPLGA+US组的凋亡指数（AI）显著高于其它各组（P<0.05）；DOX-SPLGA+US组的肿瘤增殖受到明显抑制，其增殖指数明显低于其它各组（P<0.05）。
结论DOX-SPLGA造影剂能显著增强显像超声和磁共振两种模式下的兔肿瘤转移淋巴结，同时联合低频超声促进造影剂内的药物释放，达到治疗转移淋巴结的效果，为寻求一种有效、安全、集显像和治疗
Objective To prepare two kinds of multifunctional ultrasound contrastagents: SB-PLGA and DOX-SPLGA, and to investigate their physicalproperties.
Methods SB-PLGA were prepared by double emulsion andfreeze-drying methods by introducing SB in the oil phase, DOX-SPLGAwere prepared similarly with the addition of DOX in the water phase anddifferent iron concentrations of oleic acid-treated Fe3O4nanoparticles in theoil phase. Several analytical tools were employed to characterize thesecontrast agents. The morphology was observed under the light microscope(LM) and transmission electron microscope (TEM). The mean size and sizedistribution were measured by a laser instrument. Drug encapsulationefficiency of SB and DOX were determined using ultraviolet-visible(UV-Vis) spectrophotometry. Except that, DOX release behavior of DOX-SPLGA in the sound field was performed in vitro sonicationexperiment. The concentration of Fe3O4loading in DOX-SPLGA wasdetected by an atomic absorption spectrophotometer (AAS). Themagnetization properties of DOX-SPLGA were detected by using avibrating smaple magnetometer (VSM). The efficacy of DOX-SPLGA forUS and MR imaging was evaluated by in vitro experiments.
Results SB-PLGA microbubbles were fluffy blue powder, whichbecame to uniform, dark blue and well dispersed solution after dissolved inwater. The SEM images showed the smooth and spherical nature of theSB-PLGA microbubbles with the diameter of1～2μm. The zeta potentialwas (20.80±1.60) mV. The encapsulation efficiency of SB in the PLGAmicrobubbles was85.67%±1.44%. The SB loading efficiencies ofSB-PLGA were3.43%±0.06%. DOX-SPLGA microbubbles were brownpowder, which became to uniform and well dispersed solution afterdissolved in water. The SEM images showed that DOX-SPLGAmicrobubbles exhibited a smooth and uniform spherical morphology withholes on the surface. TEM images demonstrated a typical core-shellstructure and Fe3O4nanoparticles were successfully encapsulated into theshell of the contrast agent. The amount of Fe3O4nanoparticles encapsulatedin the microbubbles measured by atomic absorption spectrometry methodwas0±0,1.47±0.07,2.83±0.08,5.76±0.33,14.77±0.32,28.06±0.62,56.71±0.44,185.45±1.06μg/ml, respectively. The encapsulation efficiencies of DOX in the DOX-SPLGA with different concentration ofFe3O4(1.47±0.07,2.83±0.08,5.76±0.33,14.77±0.32,28.06±0.62,56.71±0.44,185.45±1.06μg/ml) were58.10%±1.61%,57.73%±3.45%,60.30%±2.67%,57.53%±0.80%,61.13%±0.95%,56.07%±1.55%,59.60%±1.61%, respectively. The DOX loading efficiencies ofthese DOX-SPLGA were5.81%±0.16%,5.77%±0.35%,6.03%±0.27%,5.75%±0.08%,6.11%±0.10%,5.61%±0.16%,5.96%±0.16%,respectively. The size of DOX-SPLGA was found to be about200~250nm,DOX-SPLGA contrast agent could preserve the superparamagneticproperties of the encapsulated Fe3O4material, and its magneticsusceptibility effect was associated with the concentration of Fe3O4in theshell. The in vitro US imaging showed that DOX-SPLGA could producehigher echo intensity than non-Fe3O4-loaded PLGA. The in vitro MRimaging showed that T2*-weighted signals was dependent on ironconcentration and decreased as iron concentration increased.
Conclusion SB-PLGA contrast agents were prepared successfully,which provided the basis for preoperative and intraoperative localization oflymph nodes as well as for ultrasonographically guided core needle biopsyof lymph nodes. DOX-SPLGA contrast agents have the properties of smallsize, well dispersion and superparamagnetism, which could enhance theUS/MR imaging and perform drug-loaded adjuvant chemotherapy in vitro.DOX-SPLGA could be used as a novel multifunctional ultrasound contrast agent.
Objective To explore the cytotoxicity of SB-PLGA and the ability ofmacrophages phagocytosis in vitro. To explore the efficacy of SB-SPLGAenhanced US imaging and operation tracer in the detection of VX2tumormetastatic lymph nodes in rabbits, and the distribution of SB-SPLGA inlymph nodes tissue.
Methods Mouse mononuclear macrophages RAW264.7were incubatedfor3h,24h and48h with the same concentrations of SB-PLGA. Themacrophage phagocytosis of contrast agent was observed by lightmicroscope, MTT assay was used to detect the viability of the cells. Tumorpopliteal metastatic lymph nodes models were established by intramuscularinjection of VX2tumor tissue suspension into hind legs. Two weeks aftertumor inoculation,8rabbits (16tumor lymph nodes) were screened by USto evaluate the efficacy of SB-PLGA for enhanced US imaging in thepopliteal lymph node and the inguinal lymph node.16tumor lymph nodes were randomly divided into two groups: experimental group (8tumorlymph nodes) and control group (8tumor lymph nodes). Experimentalgroup was subcutaneous injected by the foot pad with SB-PLGA, controlgroup was injected with sterile saline. After30mins, the blue stained andnon-stained lymph nodes were harvested by popliteal and inguinal lymphnodes dissection for the frozen-section examination and HE staining.
Results A time-dependent SB-PLGA uptake by macrophages can beobserved under the light microscope. The amount of SB-PLGA uptakeincreased with the incubation time increased. MTT results showed the cellsproliferation activity was not affected after SB-PLGA engulfed bymacrophages. The enhancement of popliteal lymph nodes could be detectedafter injected with SB-PLGA by contrast lymphosonography. While theinguinal lymph nodes and control tumor lymph nodes injected with salinedid not show any increase in echogenicity over the duration of the imagingperiod. By gross examination with the naked eye, it is apparent that thepopliteal lymph nodes with SB-PLGA injection were stained blue (thenatural color of SB under visible light), whereas the lymph nodes withsaline injection and the inguinal lymph nodes were not stained. The resultsof HE-stained sections with light microscopy indicated that a significantnumber of SB-PLGA microbubbles were trapped in the popliteal lymphnodes tissue and a large number of SB-PLGA microbubbles were localizedwith macrophages.
Conclusion SB-PLGA could be phagocytized by macrophages in vitro,and had no obvious effect on cell proliferation. The ultrasonographicsignals of the lymph nodes were significantly enhanced by SB-PLGAcontrast agent. More important, the same lymph nodes that were enhancedby SB-PLGA microbubbles were easily located during surgery by thenaked eye because of the accumulation of the SB dye (appears blue)encapsulated in the PLGA microbubbles. SB-PLGA may have the potentialto serve as a multifunctional ultrasound contrast agent for enhanced US andbiopsy indicator.
Objective To explore the efficacy of DOX-SPLGA enhanced US/MRimaging in the detection of VX2tumor metastatic lymph nodes in rabbits.To explore the antitumor effect of DOX-SPLGA combined with lowfrequency US triggered in situ drug release for VX2tumor metastaticlymph nodes in rabbits.
Methods Tumor popliteal metastatic lymph nodes models wereestablished in42rabbits by intramuscular injection of VX2tumor tissuesuspension into hind legs. Two weeks after tumor inoculation,12rabbits(24tumor lymph nodes) were screened by US and MR imaging.24tumorlymph nodes were selected and randomized into three groups (8tumorlymph nodes every group). One group was injected with DOX-SPLGA.The second group was injected with the pure PLGA. The third controlanimal group was used to rule out the possibility of enhancement caused bythe sterile saline. After each in vivo US/MR imaging experiment, all of thepopliteal lymph nodes were harvested for HE and Prussian blue stain andTEM detection. Two weeks after tumor inoculation,30rabbits (60tumorlymph nodes) were randomly divided into6groups: blank control group(C)，DOX-SPLGA microbubbles group (DOX-SPLGA), doxorubicin group(DOX)，pure PLGA microbubbles combined with US group (PLGA+US),doxorubicin combined with US group (DOX+US) and DOX-SPLGAmicrobubbles combined with US group (DOX-SPLGA+US). Tumor lymphnodes for histological analysis were harvested after the initial treatment,protein expression of PCNA, CD34and LYVE-1was detected byimmunohistochemistry, apoptosis was detected by TUNEL.
Results The enhancement of popliteal lymph nodes could be detectedafter injected with DOX-SPLGA by both contrast lymphosonography andconventional gray-scale sonography. While control tumor lymph nodes injected with saline did not show any increase in echogenicity over theduration of the imaging period. Importantly, the control tumor lymph nodesinjected with pure PLGA showed similar increase in echogenicity, but theincreased reflectivity was less than that of DOX-SPLGA. In MR imaging,the overall signal in the tumor lymph nodes was negatively enhanced afterinjection of the DOX-SPLGA, while control tumor lymph nodes injectedwith saline did not show any change in the T2*-weighted signal. Likewise,the control tumor lymph nodes injected with pure PLGA also showed anegative enhancement when T2*-weighted images were acquired, however,to a much less extent. Both Prussian blue stain and TEM showed thatDOX-SPLGA were mainly present in lymph nodes. Theimmunohistochemical staining results showed that the tumor cell apoptoticindex (AI) of DOX-SPLGA+US group was highest among all groups(P<0.05), while the tumor proliferation index (PI), micro blood vesseldensity (MVD) and micro lymphatic vessel density (LMVD) were lowestamong all groups (P<0.05).
Conclusion DOX-SPLGA could markedly enhanced dual-modeUS/MR imaging of lymph node, and combined with low frequency US thatcould triggered drug release for lymph nodes metastases therapy, whichmay give a novel strategy for exploring an effective and safemultifunctional ultrasound contrast agent for imaging and therapy.
引文
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