Asialoglycoprotein receptor-targeted superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide (SPIO) nanoparticles are primarily used as contrast agents in magnetic resonance imaging. SPIO have also been derivatized to add targeting and drug-carrier functionality as drug delivery devices. The preparation and characterization of amino-functionalized SPIO (ASPIO) and lactose-derivatized galactose-terminal-ASPIO are now reported. The target for galactose-terminal-ASPIO is the cell-surface asialoglycoprotein receptor (ASGPR) expressed by hepatocytes. Two batches of ASPIO with average particle sizes of 61 [42]nm and 127 [125]nm [full-width half maximum; FWHM] were prepared. The small ASPIO increased from 61 nm to 278 [309]nm upon lactosylation (Gal-ASPIO-278) and to 302 [280] by N-acetylation (NAcASPIO-302); the larger ASPIO afforded galactosyl-terminal ASPIO of 337 [372]nm and N-acetylated ASPIO of 326 [308]nm. The LD50 of Gal-ASPIO-278 was 1500 microg/mL to HepG2 cells; Gal-ASPIO-278 associated with HepG2 cells in vitro, whereas NAcSPIO-302, prepared from the same ASPIO batch, did not. Gal-ASPIO-278 and NAcASPIO-302 were not bound by ASPGR non-expressing 143B cells. The association of Gal-ASPIO-278 to HepG2 cells was reduced by free galactose, supporting the model of ASGPR-mediated binding. These data underline the potential application of Gal-ASPIO as a targeted ligand for ASPGR-expressing cells in vivo.     

Investigation of superparamagnetic iron oxide nanoparticles for MR-visualization of surgical implants

For the development of a surgical mesh implant that is visible in magnetic resonance imaging (MRI), superparamagnetic iron oxides (SPIOs) are integrated into the material of the mesh. In order to get a high quality mesh regarding both mechanical and imaging properties, a narrow size distribution and homogenous spatial distribution, as well as a strong magnetization of SPIOs within the filament of the mesh are required. In this work, six different samples of SPIOs composed of a magnetite core are synthesized with and without stabilizing dodecanoic acid and analyzed using a superconducting quantum interference device (SQUID), transmission electron microscope (TEM) and a magnetic force microscope (MFM) to determine the properties that are beneficial for the assembly and imaging of the implant. These analyses show the feasibility of visualization of surgical implants with incorporated SPIOs and the influence of the agglomeration of SPIOs on their magnetization and on a homogenous spatial distribution within the polymer of the mesh.     

Preparation and characterization of superparamagnetic iron oxide nanoparticles stabilized by alginate

SPION with appropriate surface chemistry have been widely used experimentally for numerous in vivo applications. In this study, SPION stabilized by alginate (SPION-alginate) were prepared by a modified coprecipitation method. The structure, size, morphology, magnetic property and relaxivity of the SPION-alginate were characterized systematically by means of XRD, TEM, ESEM, AFM, DLS, SQUID magnetometer and MRI, respectively, and the interaction between alginate and iron oxide (Fe(3)O(4)) was characterized by FT-IR and AFM. The results revealed that typical iron oxide nanoparticles were Fe(3)O(4) with a core diameter of 5-10 nm and SPION-alginate had a hydrodynamic diameter of 193.8-483.2 nm. From the magnetization curve, the Ms of a suspension of SPION-alginate was 40 emu/g, corresponding to 73% of that of solid SPION-alginate. This high Ms may be due to the binding of Fe(3)O(4) nanoparticles to alginate macromolecule strands as visually confirmed by AFM. SPION-alginate of several hundred nanometers was stable in size for 12 months at 4 degrees C. Moreover, T1 relaxivity and T2 relaxivity of SPION-alginate in saline (1.5 T, 20 degrees C) were 7.86+/-0.20 s(-1) mM(-1) and 281.2+/-26.4 s(-1) mM(-1), respectively.     

体外评价羧甲基壳聚糖超顺磁氧化铁纳米粒的细胞毒性和巨噬细胞的摄取

目的 体外评价自制羧甲基壳聚糖超顺磁氧化铁纳米粒(OCMCS-SPIO-NPs)的细胞毒性和逃避巨噬细胞的吞噬能力.方法 以菲立磁和未包被的超顺磁氧化铁纳米粒为对照,采用四唑盐(MTT)比色法考察OCMCS-SPIO-NPs对LO2细胞(正常肝细胞株)和A549(人肺腺癌细胞株)的细胞毒性;用菲洛嗪法及普鲁士蓝法考察OCMCS-SPIO-NPs纳米粒并评价其体外的抗吞噬能力.结果 SPIO-NPs经羧甲基壳聚糖共价修饰后,对LO2和A549细胞的细胞毒性明显降低,OCMCS-SPIO-NPs的细胞毒性和dextran-SPIO的细胞毒性无显著性差异(P>0.05),其细胞毒性与培养基中SPIO的浓度呈正相关;与3种SPIO纳米粒孵化24h后,RAW264.7细胞内铁含量随培养基中SPIO的含量增加而增加,细胞内铁含量依次为:未包被SPIO-NPs>dextran-SPIO-NPs>CMCS-SPIO-NPs组(P<0.05).结论 超顺氧化铁纳米粒经羧甲基壳聚糖共价修饰后能显著降低细胞毒性和吞噬细胞摄取,提高了生物相容性,显著降低了巨噬细胞对其的摄取.     

Adsorption kinetics of plasma proteins on ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles

In this study the kinetics of plasma protein adsorption onto ultrasmall superparamagnetic iron oxide (USPIO) particles have been analyzed and compared to previously published kinetic studies on polystyrene particles (PS particles), oil-in-water nanoemulsions and solid lipid nanoparticles (SLNs). SPIO and USPIO nanoparticles are commonly used as magnetic resonance imaging (MRI) enhancers for tumor imaging as well as in drug delivery applications. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to determine the plasma protein adsorption onto the citrate/triethylene glycol-stabilized iron oxide surface. The results indicate that the existence of a Vroman effect, a displacement of previously adsorbed abundant proteins, such as albumin or fibrinogen, respectively, on USPIO particles has to be denied. Previously, identical findings have been reported for oil-in-water nanoemulsions. Furthermore, the protein adsorption kinetics differs dramatically from that of other solid drug delivery systems (PS, SLN). More relevant for the in vivo fate of long circulating particles is the protein corona after several minutes or even hours. Interestingly, the patterns received after an incubation time of 0.5 min to 240 min are found to be qualitatively and quantitatively similar. This leads to the assumption of a long-lived ("hard") protein corona around the iron oxide nanoparticles.     

In vitro cytotoxicity study of superparamagnetic iron oxide and silica nanoparticles on pneumocyte organelles

Inhalation is the main route of nanoparticles (NP) exposure during manufacturing. Although many mechanisms of toxicity have been described, the interaction of NP with relevant pneumocytes organelles is not widely understood. Considering that the physicochemical properties of NP influence their toxicological responses, the objective of this study was to evaluate whether exposure to different NP, crystalline Fe₃O₄ NP and amorphous SiO₂ NP could alter pneumocytes organelles in alveolar epithelial cells. To achieve this goal, cell viability, ultrastructural changes, lysosomal damage, mitochondrial membrane potential (MMP), lipid droplets (LD) formation and cytokines production were evaluated by MTT, electron microscopy, lysotracker red staining, JC-1, Oil Red staining and Milliplex® assay respectively. Both NP were observed within lamellar bodies (LB), lysosomes, and cytoplasm causing morphological changes. Exposure to SiO₂ NP at 6 h induced lysosomal activation, but not Fe₃O₄ NP. MMP decreased and LD increased at the highest concentrations after both NP exposure. Pro-inflammatory cytokines were released only after SiO₂ NP exposure at 48 h. These results indicate that SiO₂ NP have a greater impact than Fe₃O₄ NP on organelles responsible for energy, secretion, degradation and metabolism in pneumocytes leading to the development of respiratory disorders or the exacerbation of preexisting conditions. Therefore, the established biocompatibility for amorphous NP has to be reconsidered.     

In vitro study of CD133 human stem cells labeled with superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) are applied in stem cell labeling because of their high magnetic susceptibility as compared with ordinary paramagnetic species, their low toxicity, and their ease of magnetic manipulation. The present work is the study of CD133+ stem cell labeling by SPIONs coupled to a specific antibody (AC133), resulting in the antigenic labeling of the CD133+ stem cell, and a method was developed for the quantification of the SPION content per cell, necessary for molecular imaging optimization. Flow cytometry analysis established the efficiency of the selection process and helped determine that the CD133 cells selected by chromatographic affinity express the transmembrane glycoprotein CD133. The presence of antibodies coupled to the SPION, expressed in the cell membrane, was observed by transmission electron microscopy. Quantification of the SPION concentration in the marked cells using the ferromagnetic resonance technique resulted in a value of 1.70 x 10(-13) mol iron (9.5 pg) or 7.0 x 10(6) nanoparticles per cell (the measurement was carried out in a volume of 2 muL containing about 6.16 x 10(5) pg iron, equivalent to 4.5 x 10(11) SPIONs).     

In vitro and in vivo study of solid lipid nanoparticles loaded with superparamagnetic iron oxide

Solid Lipid Nanoparticles (SLN) are already under investigation as a pharmaceutical tool able to change the pharmacokinetic and biodistribution of carried molecules. SLN are able to target drugs to lymph after duodenal administration and to overcome the Blood Brain Barrier (BBB). In this study, superparamagnetic SLN have been prepared, have colloidal size, in vitro analysis showed relaxometric properties similar to Endorem. In vivo Magnetic Resonance Imaging (MRI) of the central nervous system (CNS) with both SLN and Endorem showed that superparamagnetic SLN have slower blood clearance than Endorem. MRI data are consistent with CNS uptake of SLN lasting up to the end of the experiment (135 min). These findings confirm the ability of SLN to overcome the BBB; SLN might be used as a CNS MRI contrast agent.     

Endoplasmic reticulum stress mediates inflammatory response triggered by ultra-small superparamagnetic iron oxide nanoparticles in hepatocytes

Abstract

Ultra-small superparamagnetic iron oxide nanoparticles (USPIO-NPs) are widely used as clinical magnetic resonance imaging contrast agents for hepatic diseases diagnosis. USPIO-NPs often damage the hepatocytes and affect the function of liver but its mechanism of action remains unclear. In the present study, USPIO-NPs caused higher cytotoxicity and lactate dehydrogenase (LDH) leakage in hepatic L02 cells than SPIO-NPs. Subsequently, USPIO-NPs affected more genes’ expression than SPIO-NPs analyzed through microarray and bioinformatics analysis. The affected genes were involved in several biological processes, including calcium ion homeostasis, inflammatory response-related leukocyte chemotaxis, and migration. In addition, the level of endoplasmic reticulum (ER) calcium ion was increased by USPIO-NPs. USPIO-NPs also upregulated the genes related to acute-phase inflammation, including IL1B, IL6, IL18, TNFSF12, TNFRSF12, SAA1, SAA2, JAK1, STAT5B, and CXCL14. Furthermore, interleukin-6 (IL-6) secretion was elevated by USPIO-NPs as detected using ELISA. On the other hand, USPIO-NPs changed the morphology of ER and triggered the ER stress and unfolded protein response PERK/ATF4 pathway. Furthermore, blocking ER stress with inhibitor or ATF4 small interfering RNA counteracted IL-6-related acute-phase inflammation and cytotoxicity caused by USPIO-NPs. Taken together, we found that the USPIO-NPs could trigger stronger IL-6-related acute-phase inflammation than SPIO-NPs in hepatocytes. We demonstrated, for the first time, that IL-6-related acute-phase inflammation caused by NPs was regulated by PERK/ATF4 signaling. The PERK/ATF4 pathway explored in this study could be a candidate for diagnostic and therapeutic target against NPs-induced liver injury and cytotoxicity, which would be helpful for USPIO-NPs medical application.     

Very small superparamagnetic iron oxide nanoparticles: Long-term fate and metabolic processing in atherosclerotic mice

We investigated the biotransformation of very small superparamagnetic iron oxide nanoparticles (VSOP) in atherosclerotic LDLR^?/? mice. Transmission electron microscopy revealed an uptake of VSOP not only by macrophages but also by endothelial cells in liver, spleen, and atherosclerotic lesions and their accumulation in the lysosomal compartment. Using magnetic particle spectroscopy (MPS), we show that the majority of VSOP's superparamagnetic iron was degraded within 28?days. MPS spectrum shape indicated changes in the magnetic properties of VSOP during the biodegradation process. Experiments with primary murine bone marrow derived macrophages, primary murine liver sinusoidal endothelial cells, and primary human aortic endothelial cells demonstrated that loading with VSOP induced a differential response of cellular iron homeostasis mechanisms with increased levels of ferritin and iron transport proteins in macrophages and increased levels of ferritin in endothelial cells.     

Pulmonary toxicity and kinetic study of Cy5.5-conjugated superparamagnetic iron oxide nanoparticles by optical imaging

Recent advances in the development of nanotechnology and devices now make it possible to accurately deliver drugs or genes to the lung. Magnetic nanoparticles can be used as contrast agents, thermal therapy for cancer, and be made to concentrate to target sites through an external magnetic field. However, these advantages may also become problematic when taking into account safety and toxicological factors. This study demonstrated the pulmonary toxicity and kinetic profile of anti-biofouling polymer coated, Cy5.5-conjugated thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPION) by optical imaging. Negatively charged, 36 nm-sized, Cy5.5-conjugated TCL-SPION was prepared for optical imaging probe. Cy5.5-conjugated TCL-SPION was intratracheally instilled into the lung by a non-surgical method. Cy5.5-conjugated TCL-SPION slightly induced pulmonary inflammation. The instilled nanoparticles were distributed mainly in the lung and excreted in the urine via glomerular filtration. Urinary excretion was peaked at 3 h after instillation. No toxicity was found under the concentration of 1.8 mg/kg and the half-lives of nanoparticles in the lung and urine were estimated to be about 14.4 ± 0.54 h and 24.7 ± 1.02 h, respectively. Although further studies are required, our results showed that Cy5.5-conjugated TCL-SPION can be a good candidate for use in pulmonary delivery vehicles and diagnostic probes.     

MRI measurement of regional lung deposition in mice exposed nose-only to nebulized superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles show potential in magnetic targeting of inhaled aerosols to localized sites within the lung. These particles are also used as contrast agents in magnetic resonance imaging (MRI). In the present work, we examine the feasibility of measuring regional lung deposition of iron oxide nanoparticles using MRI. Mice were exposed nose-only to nebulized superparamagnetic iron oxide nanoparticles. The droplet size distribution in the inhalation chamber was measured using a time-of-flight device. Regional concentrations of iron in the left and right lung were assessed with MRI by measuring the longitudinal relaxation times (T(1)) of the lung tissue in exposed mice, compared to a baseline group. Regional concentrations of iron in the lungs of the mice ranged from 1.1 +/- 0.8 microg/cm(3) (mean +/- one standard deviation, n = 6) in peripheral lung regions to 2.7 +/- 1.4 microg/cm(3) in the central lung, with no significant difference between the left and right lung. The nebulized droplets in the inhalation chamber had mass median aerodynamic diameter (MMAD) of 5.6 +/- 0.8 microm, with a geometric standard deviation (GSD) of 1.30 +/- 0.03 (both values expressed as mean +/- one standard deviation, n = 6). MRI shows promise for in vivo measurement of regional lung concentrations of superparamagnetic iron oxide nanoparticles, and may be useful in studies of lung deposition and clearance.     

Novel superparamagnetic iron oxide nanoparticles for tumor embolization application: preparation, characterization and double targeting

The goal of this study was to develop novel embolic nanoparticles for targeted tumor therapy with dual targeting: magnetic field-guided and peptide-directed targeting. The embolic nanoparticles SP5.2/tTF-OCMCs-SPIO-NPs were prepared by surface-modifying of superparamagnetic iron oxide nanoparticles (SPIO-NPs) with o-carboxymethylchitosans (OCMCs) and SP5.2/tTF (SP5.2: a peptide binding to VEGFR-1; tTF: truncated tissue factor) to improve their stability and to target over-expressing VEGFR-1 cells. The physicochemical characterization results showed that the OCMCs-SPIO-NPs have a spherical or ellipsoidal morphology with an average diameter of 10-20 nm. And they possess magnetism with a saturation magnetization of 66.1 emu/g, negligible coercivity and remanence at room temperature. In addition, the confocal microscopy, Prussian blue staining and FX activation analysis respectively demonstrated the peptide-directed targeting, magnetic field-guided targeted and blood coagulation activity of the SP5.2/tTF-OCMCs-SPIO-NPs. These properties separately belong to SP5.2, Fe(3)O(4) and tTF moieties of the SP5.2/tTF-OCMCs-SPIO-NPs. Thus these SP5.2/tTF-OCMCs-SPIO-NPs with double-targeting function should have a potential application in embolization therapy of tumor blood vessels.     

超顺磁性葡聚糖氧化铁纳米颗粒中葡聚糖含量测定方法研究

目的新型磁共振造影剂的研究是目前国内外的一个热点,本研究建立了超顺磁氧化铁中葡聚糖含量测定方法。方法用共沉淀法制备氧化铁纳米粒子,通过强酸性阳离子交换树脂纯化,提取,葡聚糖在苯酚-硫酸作用下脱水并显色,于488nm波长处测定吸收度。结果葡聚糖在9.8~98.0μg/mL范围内呈良好的线性关系(r=0.9991)。平均回收率分别为97.3%,104.3%,102.6%。结论本法简便、快速、重现性好,可用于超顺磁氧化铁中葡聚糖的检测。     

The interaction of bovine serum albumin with doxorubicin-loaded superparamagnetic iron oxide nanoparticles: spectroscope and molecular modelling identification

Abstract

To take a comprehensive evaluation of the bio-safety of doxorubicin-loaded superparamagnetic iron oxide nanoparticles (SPION), the interaction of bovine serum albumin (BSA) with the drug delivery was investigated by multi-spectroscopic techniques and molecular modelling calculation. Ultraviolet absorption and synchronous fluorescence results elucidate that DOX-SPION unfold the framework conformation of BSA, leading to changes in the microenvironment of amide moieties. Circular dichroism (CD) data show that the content of α-helix decreases from 68.62% to 62.76%, which shows the changes of protein's secondary structure quantificationally. Through Stern–Volmer analysis, the quenching mode is determined to be static interaction, forming a stable bioconjugate. The molecular model illustrates that DOX prefers a highly polar binding site at the external region of domains □ of BSA, and the hydrogen bonds are marked. This work elucidates that the drug delivery has deleterious effects on the frame conformation of protein, affecting its physiological function.     

磁标记骨髓间充质干细胞的实验研究

目的探讨不同时间及浓度超顺磁性氧化铁(SPIO)标记大鼠骨髓间充质干细胞(MSCs)标记效率、标记后细胞活力等情况,寻找最佳标记浓度和时间。方法配制含不同浓度SPIO培养基,加入MSCs孵育,于标记后不同时间行铁染色、细胞活力、铁含量测定。结果细胞活力检测显示标记1d和3d时,0μg/ml组与25μg/ml组,0μg/ml组与50μg/ml组,25μg/ml组与50μg/ml组的细胞拒染率差异无统计学意义(P>0.05)。铁含量测定显示标记浓度在50μg/ml,标记时间3d时铁含量最高。结论50μg/ml浓度,标记3d不仅标记效率高对细胞活力无影响,而且细胞内铁含量最多,为最佳标记浓度和时间。     

氧化铁纳米粒子的制备

目的用共沉淀法制备氧化铁纳米粒子。方法用JEM— 2 0 0 0EXII场发射电子显微镜观察纳米微粒的形貌和粒子大小 ;为了证实磷脂在纳米氧化物表面吸附 ,又进行了红外光谱研究 ;用X -ray分析确定晶体结构和其结晶性质。 结果在磷脂水溶液中 ,用缓慢 -氧化法成功的制备出氧化铁纳米粒子。通过TEM测量粒径为 7nm。纳米氧化物的红外图谱表明 :纳米氧化物的表面已经被磷脂层所覆盖。XRD相分析表明 ,纳米氧化物微粒已经形成     

Visualization and analysis of superparamagnetic iron oxide nanoparticles in the inner ear by light microscopy and energy filtered TEM

Nanoparticles as potential carriers for local drug transfer are an alternative to systemic drug delivery into the inner ear. We report on the first in vitro tests of a new ferrogel consisting of superparamagnetic iron oxide nanoparticles (SPIONs) and a Pluronic(?) F127 (PF127) copolymer. Pluronic copolymers possess a unique viscosity-adjustable property that makes PF127 gels easy to handle compared to conventional cross-linked hydrogels. This ferrogel was successfully tested in cadaver human temporal bones as well as in organotypic explant cultures of mouse inner ears. SPIONs were identified by light microscopy and localized with different imaging modes in energy-filtered transmission electron microscopy. Our approach shows a promising possibility to use iron oxide nanoparticles, which are suitable for visualization and characterization at both the light- and electron-microscopic levels. FROM THE CLINICAL EDITOR: The authors report the first in vitro tests of a new ferrogel consisting of superparamagnetic iron oxide nanoparticles (SPIONs) and a Pluronic? F127 (PF127) copolymer for drug delivery in the inner ear, demonstrasting a promising possibility to use iron oxide nanoparticles, which are suitable for visualization and characterization at both the light- and electron-microscopic levels.     

超顺磁性四氧化三铁长循环脂质体的制备及表征

目的：构建磁共振对比剂超顺磁性四氧化三铁长循环脂质体（PS）,并对其理化性质与稳定性进行初步评价。方法：采用化学共沉淀法合成柠檬酸修饰的超顺磁性四氧化三铁纳米粒,并用脂质材料将纳米粒外层包被,制备得PS。采用透射电镜、动态光散射、凝胶色谱及磁共振成像等,对PS的形态、粒径、Zeta电位、包封率及弛豫率进行评价,并考察PS在4 ℃条件下的稳定性。结果：制备的PS为圆形或类圆形粒子,粒径为（141±1）nm,PDI为0.242±0.006,Zeta电位为（-17.8±1.0）mV,包封率为（91.73±5.18）%。体外MR成像结果表明,PS的弛豫率为355.20 mM^-1·s^-1。稳定性试验结果表明,PS在4 ℃条件下贮藏10 d,其粒径 [（151±3.2）nm]、PDI（0.273±0.012）、Zeta电位[（-19.9±0.3）mV]和包封率[（93.05±6.52）%],均无显著性变化。结论：本研究成功制备了超顺磁性四氧化三铁长循环脂质体MR对比剂,为进一步构建MR可视化肿瘤靶向药物传递系统提供了有效的物质基础。     

新型超顺磁性氧化铁在干细胞活体示踪中的应用研究

目的：研究超顺磁性氧化铁（superparamagnetic iron oxide,SP10）标记干细胞的效率及其在磁共振活体示踪中的应用价值。方法：以3-氨丙基三乙氧基硅烷（APTS）修饰Fe2O3配制成SP10,用SP10转染标记骨髓间充质干细胞（MSCs）,对标记后的MSCs行普鲁士蓝染色鉴定、MTT测试细胞相对数量。最后将标记后的MSCs移植入大鼠脑内,磁共振（MRI）扫描示踪显示。结果：普鲁士蓝染色可直接显示SP10高效率标记MSCs,MTT测试表明SP10标记对MSCs增殖数量及其活力无明显影响,MRI检查发现脑内移植的SP10标记MSCs在T2上呈明显的低信号。结论：APTS修饰Fe2O3配制而成的新型SP10对细胞无明显毒性,没有多聚赖氨酸（PLL）等转染剂仍可直接高效率标记MSCs,MRI活体显影示踪效果良好。