Multifunctional Ultrasound Contrast Agents For Multi-mode Imaging And Therapy Of Metastasis In Lymph Nodes

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.