磁性纳米颗粒作为基因转染载体的研究

通过扫描电子显微镜和Zeta电位仪对磁性纳米颗粒的形貌、粒径、表面电位等进行了表征。利用凝胶电泳阻滞试验分析磁性纳米颗粒与DNA的结合情况,研究磁性纳米颗粒对DNA的保护效果,运用MTT和流式细胞术分析磁性纳米颗粒对细胞的毒性。以绿色荧光蛋白基因为报告基因进行293T细胞的转染,研究磁性纳米颗粒与质粒DNA不同比例条件下对293T细胞的转染效率,并与脂质体(Lipofectamine2000)介导的转染进行比较分析。结果表明,磁性纳米颗粒与DNA可以稳定结合,可以保护DNA免受酶的消化作用,当磁性纳米颗粒与DNA比为1 1时,转染效率最高,优于脂质体(Lipotamine2000)介导的转染,且对细胞的毒害作用小于Lipotamine2000。     

Compare Analysis for the Nanotoxicity Effects of Different Amounts of Endocytic Iron Oxide Nanoparticles at Single Cell Level

Developing methods that evaluate the cellular uptake of magnetic nanoparticles (MNPs) and nanotoxicity effects at single-cellular level are needed. In this study, magnetophoresis combining fluorescence based cytotoxicity assay was proposed to assess the viability and the single-cellular MNPs uptake simultaneously. Malignant cells (SKHep-1, HepG2, HeLa) were incubated with 10 nm anionic iron oxide nanoparticles. Prussian blue stain was performed to visualize the distribution of magnetic nanoparticles. MTT and fluorescence based assay analyzed the cytotoxicity effects of the bulk cell population and single cell, respectively. DAPI/PI stained was applied to evaluate death mechanism. The number of intracellular MNPs was found to be strongly correlated with the cell death. Significant differences between cellular MNP uptake in living and dead cells were observed. The method could be useful for future study of the nanotoxicity induced by MNPs.     

Gum Arabic coated magnetic nanoparticles with affinity ligands specific for antibodies

A novel magnetic support based on gum Arabic (GA) coated iron oxide magnetic nanoparticles (MNP) has been endowed with affinity properties towards immunoglobulin G (IgG) molecules. The success of the in situ triazine ligand synthesis was confirmed by fluorescence assays. Two synthetic ligands previously developed for binding to IgG, named as ligand 22/8 (artificial Protein A) and ligand 8/7 (artificial Protein L) were immobilized on to MNPs coated with GA (MNP_GA). The dimension of the particles core was not affected by the surface functionalization with GA and triazine ligands. The hydrodynamic diameters of the magnetic supports indicate that the coupling of GA leads to the formation of larger agglomerates of particles with about 1 µm, but the introduction of the triazine ligands leads to a decrease on MNPs size. The non‐functionalized MNP_GA bound 28 mg IgG/g, two times less than bare MNP (60 mg IgG/g). MNP_GA modified with ligand 22/8 bound 133 mg IgG/g support, twice higher than the value obtained for ligand 8/7 magnetic adsorbents (65 mg/g). Supports modified with ligand 22/8 were selected to study the adsorption and the elution of IgG. The adsorption of human IgG on this support followed a Langmuir behavior with a Q_máx of 344 mg IgG/g support and K_a of 1.5 × 10⁵ M. The studies on different elution conditions indicated that although the 0.05 M citrate buffer (pH 3) presented good recovery yields (elution 64% of bound protein), there was occurrence of iron leaching at this acidic pH. Therefore, a potential alternative would be to elute bound protein with a 0.05 M glycine‐NaOH (pH 11) buffer. Copyright © 2010 John Wiley & Sons, Ltd.     

Effect of the concentration of protein and nanoparticles on the structure of biohybrid nanocomposites

We present colloidal nanocomposites formed by incorporating magnetite Fe₃O₄ nanoparticles (MNPs) with lysozyme amyloid fibrils (LAFs). Preparation of two types of solutions, with and without addition of salt, was carried out to elucidate the structure of MNPs-incorporated fibrillary nanocomposites and to study the effect of the presence of salt on the stability of the nanocomposites. The structural morphology of the LAFs and their interaction with MNPs were analyzed by atomic force microscopy and small-angle x-ray scattering measurements. The results indicate that conformational properties of the fibrils are dependent on the concentration of protein, and the precise ratio of the concentration of the protein and MNPs is crucially important for the stability of the fibrillary nanocomposites. Our results confirm that despite the change in fibrillary morphology induced by the varying concentration of the protein, the adsorption of MNPs on the surface of LAF is morphologically independent. Moreover, most importantly, the samples containing salt have excellent stability for up to 1 year of shelf-life.     

Silver nanocrystals sensitize magnetic-nanoparticle-mediated thermo-induced killing of cancer cells

Magnetic nanoparticles (MNPs) can heat up tumor tissues and induce killing of cancer cells under external AC magnetic field. However, magnetic nanoparticles hyperthermia (MNPH) requires high concentration of MNPs that are injected into the tumor in order to obtain clinically needed thermal dose because of the complicated heat transfer in vivo and the limited heat quality of MNPs. To cut down the dose of MNPs and enhance the effect of this Nanotherapy, we prepared silver nanoparticles (AgNPs) with different sizes and investigated the effects of these AgNPs on cancer cells in MNPH treatment. It was found that AgNPs could enhance thermo-sensitivity of glioma cells and this effect was size dependent. AgNPs could induce cell cycles arrested in G(2)/M phase and enhanced the apoptosis rate of cancer cells after hyperthermia. In glioma bearing rats model, MNPH combined with AgNPs could enhance Bax expression in cancer cells. Our results suggested that AgNPs could be a potential thermo-sensitizer and could be further developed for the design of Ag nanostructure-based thermal seeds for MNPH therapy.     

Immobilization of histidine-tagged proteins by magnetic nanoparticles encapsulated with nitrilotriacetic acid (NTA)-phospholipids micelle

We described the development of functionalized magnetic nanoparticles (MNPs) with PEG-modification, a phospholipids micelle coating, and their use in manipulating histidine-tagged proteins. Highly monodisperse MNPs were synthesized in an organic solvent and could be phase-transferred into an aqueous solution by encapsulating the nanoparticles with a phospholipids micelle. The phospholipids micelle coating rendered the nanoparticles highly water-soluble, and the functional groups of the phospholipids coating allowed for the bioconjugation of various moieties, such as fluorescent molecules and engineered proteins. Functionalized phospholipids, such as nitrilotriacetic acid (NTA)-phospholipids, caused the MNPs to bind and allowed for manipulation of histidine-tagged proteins. Due to their high surface/volume ratio, the MNPs showed better performance (about 100 times higher) in immobilizing engineered proteins than conventional micrometer-sized beads. This demonstrates that MNPs coated with phospholipids micelle can be a versatile platform for the effective manipulation of various kinds of engineered proteins, which is very important in the field of proteomics. It is expected that a combination of MNPs with optical fluorescent molecules can find applications in bimodal (magnetic and optical) molecular imaging nanoprobes.     

Controlling the thickness of polymeric shell on magnetic nanoparticles loaded with doxorubicin for targeted delivery and MRI contrast agent

The polymeric functionalization of superparamagnetic iron oxides nanoparticles is developed for cancer targeting capability and magnetic resonance imaging. Here the nanoparticles (NP) are decorated through the adsorption of a polymeric layer around the particle surface for the formation of core-shell. The synthesized magnetic nanoparticles (MNPs) are conjugated with fluorescent dye, targeting ligand, and drug molecules for improvement of target specific diagnostic and possible therapeutics applications. In this investigation doxorubicin was loaded into the shell of the MNPs and release study was carried out at different pH. The core-shell structure of magnetic NP coated chitosan matrix was visualized by TEM observation. The cytotoxicity of these magnetic NPs is investigated using MTT assay and receptor mediated internalization by HeLa and NIH3T3 cells are studied by fluorescence microscopy. Moreover, compared with T₂-weighted magnetic resonance imaging (MRI) in the above cells, the synthesized nanoparticles are showed stronger contrast enhancements towards cancer cells.     

Interaction between immunoglobulin G and peroxidase-like iron oxide nanoparticles: Physicochemical and structural features of the protein

The study is devoted to the oxidative modification of immunoglobulin G (IgG) on the surface of peroxidase-like iron oxide magnetic nanoparticles (MNPs) under conditions of induced reactive oxygen species (ROS) generation and without them. A pronounced change of thermodynamic parameters of denaturation has been detected for IgG in solutions containing MNPs under hydrogen peroxide action during 24 h of incubation. Dynamic light scattering measurements and UV–Visible spectrophotometry have been used to show aggregation in these solutions. Ferromagnetic resonance (FMR) was used to compare IgG coating thickness on individual MNPs under conditions of induced ROS generation and without them. The similarity between IgG adsorption on MNPs under these conditions after 24 h of incubation has been confirmed by the fluorescence measurements. The sites of IgG oxidative modifications that take place on MNPs surface and some evidences of the influence of oxidative modification and adsorption on the chemical structure of IgG were revealed by HPLC MS/MS analysis.     

Promising insights into the kosmotropic effect of magnetic nanoparticles on proteins: The pivotal role of protein corona formation

Increasing concerns about biosafety of nanoparticles (NPs) has raised the need for detailed knowledge of NP interactions with biological molecules especially proteins. Herein, the concentration-dependent effect of magnetic NPs (MNPs) on bovine serum albumin and hen egg white lysozyme was explored. The X-ray diffraction patterns, zeta potential, and dynamic light scattering measurements together with scanning electron microscopy images were employed to characterize MNPs synthesized through coprecipitation method. Then, we studied the behavior of two model proteins with different surface charges and structural properties on interaction with Fe₃O₄. A thorough investigation of protein–MNP interaction by the help of intrinsic fluorescence at different experimental conditions revealed that affinity of proteins for MNPs is strongly affected by the similarity of protein and MNP surface charges. MNPs exerted structure-making kosmotropic effect on both proteins under a concentration threshold; however, binding strength was found to determine the extent of stabilizing effect as well as magnitude of the concentration threshold. Circular dichroism spectra showed that proteins with less resistance to conformational deformations are more prone to secondary structure changes upon adsorption on MNPs. By screening thermal aggregation of proteins in the presence of Fe₃O₄, it was also found that like chemical stability, thermal stability is influenced to a higher extent in more strongly bound proteins. Overall, this report not only provides an integrated picture of protein–MNP interaction but also sheds light on the molecular mechanism underling this process.     

Dendrimer functionalized magnetic nanoparticles as a promising platform for localized hyperthermia and magnetic resonance imaging diagnosis

Magnetic iron oxide nanoparticles are a well-explored class of nanomaterials known for their high magnetization and biocompatibility. They have been used in various biomedical applications such as drug delivery, biosensors, hyperthermia, and magnetic resonance imaging (MRI) contrast agent. It is necessary to surface modify the nanoparticles with a biocompatible moiety to prevent their agglomeration and enable them to target to the defined area. Dendrimers have attracted considerable attention due to their small size, monodispersed, well-defined globular shape, and a relative ease incorporation of targeting ligands. In this study, superparamagnetic iron oxide nanoparticles were synthesized via a coprecipitation method. The magnetic nanoparticles (MNPs) had been modified with (3-aminopropyl) triethoxysilane, and then polyamidoamine functionalized MNPs had been synthesized cycling. Various characterization techniques had been used to reveal the morphology, size, and structure of the nanoparticles such as scanning electron microscopy, transmission electron microscope, X-ray diffraction analysis, and vibrating sample magnetometer, Fourier-transform infrared spectroscopy and zeta potential measurements. In addition, the cytotoxicity property of G3–dendrimer functionalized MNPs were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide assay which confirmed the biocompatibility of the nanocomposites. Dendrimer functionalized MNPs are able to act as contrast agents for MRI and magnetic fluid hyperthermia mediators. A superior heat generation was achieved for the given concentration according to the hyperthermia results. MRI results show that the synthesized nanocomposites are a favorable option for MRI contrast agent. We believe that these dendrimer functionalized MNPs have the potential of integrating therapeutic and diagnostic functions in a single carrier.     

Development of a multilayered polymeric DNA biosensor using radio frequency technology with gold and magnetic nanoparticles

This study utilized the radio frequency (RF) technology to develop a multilayered polymeric DNA sensor with the help of gold and magnetic nanoparticles. The flexible polymeric materials, poly (p-xylylene) (Parylene) and polyethylene naphtholate (PEN), were used as substrates to replace the conventional rigid substrates such as glass and silicon wafers. The multilayered polymeric RF biosensor, including the two polymer layers and two copper transmission structure layers, was developed to reduce the total sensor size and further enhance the sensitivity of the biochip in the RF DNA detection. Thioglycolic acid (TGA) was used on the surface of the proposed biochip to form a thiolate-modified sensing surface for DNA hybridization. Gold nanoparticles (AuNPs) and magnetic nanoparticles (MNPs) were used to immobilize on the surface of the biosensor to enhance overall detection sensitivity. In addition to gold nanoparticles, the magnetic nanoparticles has been demonstrated the applicability for RF DNA detection. The performance of the proposed biosensor was evaluated by the shift of the center frequency of the RF biosensor because the electromagnetic characteristic of the biosensors can be altered by the immobilized multilayer nanoparticles on the biosensor. The experimental results show that the detection limit of the DNA concentration can reach as low as 10 pM, and the largest shift of the center frequency with triple-layer AuNPs and MNPs can approach 0.9 and 0.7 GHz, respectively. Such the achievement implies that the developed biosensor can offer an alternative inexpensive, disposable, and highly sensitive option for application in biomedicine diagnostic systems because the price and size of each biochip can be effectively reduced by using fully polymeric materials and multilayer-detecting structures.     

Multilayered Magnetic Nanoparticles as a Support in Solid-Phase Peptide Synthesis

The synthesis of multilayered magnetic nanoparticles (MNPs) for use as a support in solid-phase peptide synthesis (SPPS) is described. Silanization of magnetite (Fe₃O₄) nanoparticles with 3-(trimethoxysilyl)propyl methacrylate introduced polymerizable groups on the surface. Polymerization with allylamine, trimethylolpropane trimethacrylate, and trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate provided a polymeric coating and amino groups to serve as starting points for the synthesis. After coupling of an internal reference amino acid and a cleavable linker, the coated MNPs were applied as the solid phase during synthesis of Leu-enkephalinamide and acyl carrier protein (65-74) by Fmoc chemistry. A “high-load” version of the MNP support (0.32 mmol/g) was prepared by four consecutive cycles of Fmoc-Lys(Fmoc)-OH coupling and Fmoc deprotection. Successful synthesis of Leu-enkephalin was demonstrated on the “high-load” MNPs. Chemical stability studies proved the particles to be stable under SPPS conditions and magnetization measurements showed that the magnetic properties of the particles were maintained throughout derivatizations and SPPS. The MNPs were further characterized by high-resolution transmission electron microscopy, inductively coupled plasma atomic emission spectrometry, elemental analysis, and nitrogen gas adsorption measurements.     

Carboxyl-coated magnetic nanoparticles for mRNA isolation and extraction of supercoiled plasmid DNA

Carboxyl-coated magnetic nanoparticles (MNPs) were used to demonstrate dual functionality: isolation of messenger RNA (mRNA) from mammalian cells and extraction of the supercoiled (sc) form of plasmid DNA (pDNA) from agarose gel. These MNPs were attached with 5′-NH₂-tagged oligo-(dT)₂₅ primer and were used to isolate mRNA from breast cancer cells. The isolated mRNA was used for amplification of β-actin to confirm the compatibility. These MNPs were also used to extract the sc form of pDNA from agarose gel. The compatibility of the pDNA was demonstrated by restriction digestion. Both of these methodologies are simple, inexpensive (compared with existing kits), and efficient.     

Optimization of antibody-conjugated magnetic nanoparticles for target preconcentration and immunoassays

Biosensors based on antibody recognition have a wide range of monitoring applications that apply to clinical, environmental, homeland security, and food problems. In an effort to improve the limit of detection of the Naval Research Laboratory (NRL) Array Biosensor, magnetic nanoparticles (MNPs) were designed and tested using a fluorescence-based array biosensor. The MNPs were coated with the fluorescently labeled protein, AlexaFluor647–chicken IgG (Alexa647–chick IgG). Antibody-labeled MNPs (Alexa647–chick–MNPs) were used to preconcentrate the target via magnetic separation and as the tracer to demonstrate binding to slides modified with anti-chicken IgG as a capture agent. A full optimization study of the antibody-modified MNPs and their use in the biosensor was performed. This investigation looked at the Alexa647–chick–MNP composition, MNP surface modifications, target preconcentration conditions, and the effect that magnetic extraction has on the Alexa647–chick–MNP binding with the array surface. The results demonstrate the impact of magnetic extraction using the MNPs labeled with fluorescent proteins both for target preconcentration and for subsequent integration into immunoassays performed under flow conditions for enhanced signal generation.     

Polymerase chain reaction of nanoparticle-bound primers

Using one or two primers respectively bound to the surface of Au nanoparticles (AuNPs) or magnetic nanoparticles (MNPs), polymerase chain reaction (PCR) based on nanoparticles was systemically studied, agarose gel electrophoresis and atomic force microscopy (AFM) were respectively used to detect and observe the PCR product. The results obtained indicated that with either one or two primers respectively bound to the nanoparticle surface, PCR can proceed successfully under optimized condition and is subject to certain rules, consequently a symmetric PCR technique and an asymmetric PCR technique based on nanoparticles have been developed. A kind of nanostructured aggregates can be constructed by a symmetric PCR using two nanoparticle-bound primers.     

Intrinsically superparamagnetic Fe-hydroxyapatite nanoparticles positively influence osteoblast-like cell behaviour

ABSTRACT: BACKGROUND: Superparamagnetic nanoparticles (MNPs) have been progressively explored for their potential in biomedical applications and in particular as a contrast agent for diagnostic imaging, for magnetic drug delivery and more recently for tissue engineering applications. Considering the importance of having safe MNPs for such applications, and the essential role of iron in bone remodelling, this study developed and analysed novel biocompatible and bioreabsorbable superparamagnetic nanoparticles, that avoid the use of poorly tolerated magnetite based nanoparticles, for bone tissue engineering applications. RESULTS: MNPs were obtained by doping hydroxyapatite (HA) with Fe ions, by directly substituting Fe2+ and Fe3+ into the HA structure yielding superparamagnetic bioactive phase. In the current study, we have investigated the effects of increasing concentrations (2000 mug/ml; 1000 mug/ml; 500 mug/ml; 200 mug/ml) of FeHA MNPs in vitro using Saos-2 human osteoblast-like cells cultured for 1, 3 and 7 days with and without the exposure to a static magnetic field of 320 mT. Results demonstrated not only a comparable osteoblast viability and morphology, but increased in cell proliferation, when compared to a commercially available Ha nanoparticles, even with the highest dose used. Furthermore, FeHA MNPs exposure to the static magnetic field resulted in a significant increase in cell proliferation throughout the experimental period, and higher osteoblast activity. In vivo preliminary results demonstrated good biocompatibility of FeHA superparamagnetic material four weeks after implantation into a critical size lesion of the rabbit condyle. CONCLUSIONS: The results of the current study suggest that these novel FeHA MNPs may be particularly relevant for strategies of bone tissue regeneration and open new perspectives for the application of a static magnetic field in a clinical setting of bone replacement, either for diagnostic imaging or magnetic drug delivery.     

Functionalization of whole‐cell bacterial reporters with magnetic nanoparticles

We developed a biocompatible and highly efficient approach for functionalization of bacterial cell wall with magnetic nanoparticles (MNPs). Three Acinetobacter baylyi ADP1 chromosomally based bioreporters, which were genetically engineered to express bioluminescence in response to salicylate, toluene/xylene and alkanes, were functionalized with 18 ± 3 nm iron oxide MNPs to acquire magnetic function. The efficiency of MNPs functionalization of Acinetobacter bioreporters was 99.96 ± 0.01%. The MNPs‐functionalized bioreporters (MFBs) can be remotely controlled and collected by an external magnetic field. The MFBs were all viable and functional as good as the native cells in terms of sensitivity, specificity and quantitative response. More importantly, we demonstrated that salicylate sensing MFBs can be applied to sediments and garden soils, and semi‐quantitatively detect salicylate in those samples by discriminably recovering MFBs with a permanent magnet. The magnetically functionalized cells are especially useful to complex environments in which the indigenous cells, particles and impurities may interfere with direct measurement of bioreporter cells and conventional filtration is not applicable to distinguish and harvest bioreporters. The approach described here provides a powerful tool to remotely control and selectively manipulate MNPs‐functionalized cells in water and soils. It would have a potential in the application of environmental microbiology, such as bioremediation enhancement and environment monitoring and assessment.     

Microstructure study of liposomes decorated by hydrophobic magnetic nanoparticles

Magnetoliposomes, consisting of liposomes and magnetic nanoparticles (MNPs), have been tailored as very promising delivery vehicles in biotechnology and biomedicine applications. In this paper, liposomes with hydrophobic MNPs were prepared. The hydrophobic MNPs were successfully embedded in the lipid bilayer, which was proved by the results obtained from transmission electron microscope, atomic force microscope, differential scanning calorimetry and steady state fluorescence measurements. Moreover, systematic researches were carried out to investigate the effects of hydrophobic MNPs concentration on the morphology and microstructure of liposomes. The results show that the lipid bilayer was saturated with the hydrophobic MNPs when the mass ratio of MNPs to lipid reached 0.002.     

抗人精子蛋白17磁性纳米探针靶向的卵巢癌体内外磁共振成像

用MRI(magnetic resonance imaging)技术探索连接抗人精子蛋白17单克隆抗体(anti-Sp17 mAb)的磁性纳米探针对体外培养及动物体内Sp17+卵巢癌的靶向性。将anti-Sp17mAb连接到表面包覆壳聚糖的超顺磁性氧化铁纳米颗粒上,制成磁性纳米探针anti-Sp17-MNP,用作MRI阴性对比剂。将磁性纳米探针与Sp17+和Sp17-培养的肿瘤细胞共育,进行一系列体外磁共振成像实验。荷瘤小鼠尾静脉注射磁性纳米颗粒,用7T磁共振仪在体成像,观察肿瘤部位的信号变化,并用普鲁士蓝染色肿瘤组织切片,观察有无铁粒子聚集。体外MRI数据显示,anti-Sp17-MNP与细胞靶向结合,并与细胞共育2 h后,Sp17+HO-8910的T2*信号强度比Sp17-HepG2低2倍;anti-Sp17-MNP对肿瘤细胞的靶向作用可被重组人Sp17阻断。7T磁共振仪对动物在体肿瘤成像结果显示,感兴趣区因磁性纳米探针靶向聚集而导致信号降低,并经组织切片普鲁士蓝染色证实。本研究结果表明,用anti-Sp17抗体和新的合成路线制备的纳米探针具有用作MR对比剂进行分子成像的潜能。     

Histochemical changes in neonatal liver caused by vaginal instillation of magnetic nanoparticles in pregnant mice

Drug delivery through the vagina is a novel and effective approach for treating embryonic diseases. Magnetic nanoparticles (MNPs) currently are used as drug delivery systems. The safety of MNPs for use with embryonic tissues remains unclear. We used pregnant mice to investigate the possible toxicity of MNPs toward neonatal liver at three embryonic ages using histochemical and immunohistochemical techniques. MNPs were instilled through the vaginas of pregnant mice at days 12 (E12), 15 (E15) and 17 (E17) after fertilization. We found MNPs in the neonatal liver parenchyma after delivery of the pups on day 20. We observed that MNPs caused mild apoptosis of hepatocytes, cytoplasmic vacuolation and lymphocytic infiltration in the neonatal liver after treatment at E15 compared to instillation at E12 and E17. We observed also that MNPs increased the production of caspase proteins and tumor necrosis factor receptor 2 proteins, which are indicators of apoptosis, in the neonatal liver after instillation of MNPs at E15 compared to instillation at E12 and E17. MNPs also increased the number of collagen fibers and the amounts of connective tissue growth factors in the neonatal liver parenchyma after instillation at E15 compared to instillation at E12 and E17. The general carbohydrates in the neonatal liver were decreased in a time-dependent manner after instillation at E17, E15 and E12 owing to the presence of MNPs in the parenchyma. Overall, we determined that MNPs were mildly toxic to neonatal liver.