Arsonic acid-presenting superparamagnetic iron oxide for pH-responsive aggregation under slightly acidic conditions

We report the synthesis and characterization of the arsonic acid-presenting superparamagnetic iron oxide (SPIO). We used arsonoacetic acid as the ligand for SPIOs in aqueous media. The surface modification of the SPIOs was accomplished via the ligand exchange from undecanoic acid to the carboxyl moiety of arsonoacetic acid. Consequently, the well-dispersed arsonic acid-presenting SPIOs in water were obtained. We found that the dispersion state of the arsonic acid-presenting SPIOs can be sharply regulated by pH changes in the biological significant region. The well dispersion state of the arsonic acid-presenting SPIOs can be maintained at the neutral pH region. In contrast, the arsonic acid-presenting SPIOs can sensitively form the aggregation below pH 6.1. Moreover, these dispersion states can be controlled reversibly by the pH alteration in the narrow region.     

Formulation of novel lipid-coated magnetic nanoparticles as the probe for in vivo imaging

Background  

Application of superparamagnetic iron oxide nanoparticles (SPIOs) as the contrast agent has improved the quality of magnetic resonance (MR) imaging. Low efficiency of loading the commercially available iron oxide nanoparticles into cells and the cytotoxicity of previously formulated complexes limit their usage as the image probe. Here, we formulated new cationic lipid nanoparticles containing SPIOs feasible for in vivo imaging.     

Labeling stem cells with ferumoxytol, an FDA-approved iron oxide nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes. A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo. Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM(-1) sec(-1) strength. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure. Our group has applied this agent in an "off label" use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.     

(⁹⁹m)Tc-bisphosphonate-iron oxide nanoparticle conjugates for dual-modality biomedical imaging

The combination of radionuclide-based imaging modalities such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) with magnetic resonance imaging (MRI) is likely to become the next generation of clinical scanners. Hence, there is a growing interest in the development of SPECT- and PET-MRI agents. To this end, we report a new class of dual-modality imaging agents based on the conjugation of radiolabeled bisphosphonates (BP) directly to the surface of superparamagnetic iron oxide (SPIO) nanoparticles. We demonstrate the high potential of BP-iron oxide conjugation using (??m)Tc-dipicolylamine(DPA)-alendronate, a BP-SPECT agent, and Endorem/Feridex, a liver MRI contrast agent based on SPIO. The labeling of SPIOs with (??m)Tc-DPA-alendronate can be performed in one step at room temperature if the SPIO is not coated with an organic polymer. Heating is needed if the nanoparticles are coated, as long as the coating is weakly bound as in the case of dextran in Endorem. The size of the radiolabeled Endorem (??m)Tc-DPA-ale-Endorem) was characterized by TEM (5 nm, Fe?O? core) and DLS (106 ± 60 nm, Fe?O? core + dextran). EDX, Dittmer-Lester, and radiolabeling studies demonstrate that the BP is bound to the nanoparticles and that it binds to the Fe?O? cores of Endorem, and not its dextran coating. The bimodal imaging capabilities and excellent stability of these nanoparticles were confirmed using MRI and nanoSPECT-CT imaging, showing that (??m)Tc and Endorem co-localize in the liver and spleen In Vivo, as expected for particles of the composition and size of (??m)Tc-DPA-ale-Endorem. To the best of our knowledge, this is the first example of radiolabeling SPIOs with BP conjugates and the first example of radiolabeling SPIO nanoparticles directly onto the surface of the iron oxide core, and not its coating. This work lays down the basis for a new generation of SPECT/PET-MR imaging agents in which the BP group could be used to attach functionality to provide targeting, stealth/stability, and radionuclides to Fe?O? nanoparticles using very simple methodology readily amenable to GMP.     

Magnetic and contrast properties of labeled platelets for magnetomotive optical coherence tomography

This article introduces a new functional imaging paradigm that uses optical coherence tomography (OCT) to detect rehydrated, lyophilized platelets (RL platelets) that are in the preclinical trial stage and contain superparamagnetic iron oxides (SPIOs) approved by the U.S. Food and Drug Administration. Platelets are highly functional blood cells that detect and adhere to sites of vascular endothelial damage by forming primary hemostatic plugs. By applying magnetic gradient forces, induced nanoscale displacements (magnetomotion) of the SPIO-RL platelets are detected as optical phase shifts in OCT. In this article, we characterize the iron content and magnetic properties of SPIO-RL platelets, construct a model to predict their magnetomotion in a tissue medium, and demonstrate OCT imaging in tissue phantoms and ex vivo pig arteries. Tissue phantoms containing SPIO-RL platelets exhibited >3 dB contrast/noise ratio at ≥1.5 × 10⁹ platelets/cm³. OCT imaging was performed on ex vivo porcine arteries after infusion of SPIO-RL platelets, and specific contrast was obtained on an artery that was surface-damaged (P < 10⁻⁶). This may enable new technologies for in vivo monitoring of the adherence of SPIO-RL platelets to sites of bleeding and vascular damage, which is broadly applicable for assessing trauma and cardiovascular diseases.     

Monitoring of in vivo function of superparamagnetic iron oxide labelled murine dendritic cells during anti-tumour vaccination

Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4(+) T cells but, most importantly, they primed cytotoxic CD8(+) T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer.     

Magnetic resonance contrast and biological effects of intracellular superparamagnetic iron oxides on human mesenchymal stem cells with long-term culture and hypoxic exposure

Background aimsHuman mesenchymal stem cells (hMSCs) have gained interest for treatment of stroke injury. Using in vitro culture, the purpose of this study was to investigate the long-term detectability of hMSCs by magnetic resonance imaging (MRI) after transfection with a superparamagnetic iron oxide (SPIO) and evaluate the effects of SPIO on cellular activity, particularly under an ischemic environment.MethodshMSCs were exposed to low doses of SPIOs. After a short incubation period, cells were cultured for additional 1, 7 and 14 d to evaluate proliferation, colony formation and multilinear potential. Labeled cells were imaged and evaluated in agarose to quantify R₂ and R₂1 contrast at each time point. Cells were placed in a low-oxygen, low-serum environment and tested for cytotoxicity. In addition, labeled cells were transplanted into an ischemic stroke model and evaluated with ex vivo MRI and histology.ResultsCellular events such as proliferation and differentiation were not affected at any of the exposures tested when cultured for 14 d. The low iron exposure and short incubation time are sufficient for detectability with MRI. However, the higher iron dosage results in higher calcification and cytotoxicity under in vitro ischemic conditions. Transplantation of the hMSCs labeled with an initial exposure of 22.4 μg of Fe showed excellent retention of contrast in stroke-induced rats.ConclusionsAlthough SPIO labeling is stable for long-term MRI detection and has limited effects on the multilineage potential of hMSCs, high-dose SPIO labeling may affect hMSC survival under serum and oxygen withdrawal.     

Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

Background

Rhodium (II) citrate (Rh₂(H₂cit)₄) has significant antitumor, cytotoxic, and cytostatic activity on Ehrlich ascite tumor. Although toxic to normal cells, its lower toxicity when compared to carboxylate analogues of rhodium (II) indicates Rh₂(H₂cit)₄ as a promising agent for chemotherapy. Nevertheless, few studies have been performed to explore this potential. Superparamagnetic particles of iron oxide (SPIOs) represent an attractive platform as carriers in drug delivery systems (DDS) because they can present greater specificity to tumor cells than normal cells. Thus, the association between Rh₂(H₂cit)₄ and SPIOs can represent a strategy to enhance the former's therapeutic action. In this work, we report the cytotoxicity of free rhodium (II) citrate (Rh₂(H₂cit)₄) and rhodium (II) citrate-loaded maghemite nanoparticles or magnetoliposomes, used as drug delivery systems, on both normal and carcinoma breast cell cultures.

Results

Treatment with free Rh₂(H₂cit)₄ induced cytotoxicity that was dependent on dose, time, and cell line. The IC₅₀ values showed that this effect was more intense on breast normal cells (MCF-10A) than on breast carcinoma cells (MCF-7 and 4T1). However, the treatment with 50 μM Rh₂(H₂cit)₄-loaded maghemite nanoparticles (Magh-Rh₂(H₂cit)₄) and Rh₂(H₂cit)₄-loaded magnetoliposomes (Lip-Magh-Rh₂(H₂cit)₄) induced a higher cytotoxicity on MCF-7 and 4T1 than on MCF-10A (p < 0.05). These treatments enhanced cytotoxicity up to 4.6 times. These cytotoxic effects, induced by free Rh₂(H₂cit)₄, were evidenced by morphological alterations such as nuclear fragmentation, membrane blebbing and phosphatidylserine exposure, reduction of actin filaments, mitochondrial condensation and an increase in number of vacuoles, suggesting that Rh₂(H₂cit)₄ induces cell death by apoptosis.

Conclusions

The treatment with rhodium (II) citrate-loaded maghemite nanoparticles and magnetoliposomes induced more specific cytotoxicity on breast carcinoma cells than on breast normal cells, which is the opposite of the results observed with free Rh₂(H₂cit)₄ treatment. Thus, magnetic nanoparticles represent an attractive platform as carriers in Rh₂(H₂cit)₄ delivery systems, since they can act preferentially in tumor cells. Therefore, these nanopaticulate systems may be explored as a potential tool for chemotherapy drug development.     

Identification of epidermal growth factor receptor-positive glioblastoma using lipid-encapsulated targeted superparamagnetic iron oxide nanoparticles in vitro

Background

Targeted superparamagnetic iron oxide (SPIO) nanoparticles have emerged as a promising biomarker detection tool for molecular magnetic resonance (MR) image diagnosis. To identify patients who could benefit from Epidermal growth factor receptor (EGFR)-targeted therapies, we introduce lipid-encapsulated SPIO nanoparticles and hypothesized that anti-EGFR antibody cetuximab conjugated of such nanoparticles can be used to identify EGFR-positive glioblastomas in non-invasive T₂ MR image assays. The newly introduced lipid-coated SPIOs, which imitate biological cell surface and thus inherited innate nonfouling property, were utilized to reduce nonspecific binding to off-targeted cells and prevent agglomeration that commonly occurs in nanoparticles.

Results

The synthesized targeted EGFR-antibody-conjugated SPIO (EGFR-SPIO) nanoparticles were characterized using dynamic light scattering, zeta potential assays, gel electrophoresis mobility shift assays, transmission electron microscopy (TEM) images, and cell line affinity assays, and the results showed that the conjugation was successful. The targeting efficiency of the synthesized EGFR-SPIO nanoparticles was confirmed through Prussian blue staining and TEM images by using glioblastoma cell lines with high or low EGFR expression levels. The EGFR-SPIO nanoparticles preferentially targeted U-251 cells, which have high EGFR expression, and were internalized by cells in a prolonged incubation condition. Moreover, the T₂ MR relaxation time of EGFR-SPIO nanoparticles could be used for successfully identifying glioblastoma cells with elevated EGFR expression in vitro and distinguishing U-251 cells from U-87MG cells, which have low EFGR expression.

Conclusion

These findings reveal that the lipid-encapsulated EGFR-SPIO nanoparticles can specifically target cells with elevated EGFR expression in the three tested human glioblastoma cell lines. The results of this study can be used for noninvasive molecular MR image diagnosis in the future.

     

阿根廷转基因作物安全管理制度概况及进展

阿根廷是最早采用转基因技术的国家之一,目前已成为全球第三大转基因作物种植国。阿根廷是全球尤其是拉美国家在生物技术产品监管和审批方面的先驱,其在转基因作物监管问题方面的丰富经验以及联合国粮食及农业组织的认可使阿根廷成为全球转基因作物监管的领导者之一。介绍了阿根廷转基因作物研发和应用、转基因作物监管体系、新型育种技术监管体系、转基因作物进出口情况以及追溯体系,讨论了转基因技术的引进对阿根廷的影响,旨在全面了解阿根廷转基因作物及新型育种技术的监管体系,为我国转基因作物安全管理提供参考。     

Chemical modification of spinach ferredoxin with diethylpyrocarbonate: evidence for the essential nature of the histidyl residue

Spinach ferredoxin contains a single ferredoxin which can be chemically modified with diethylpyrocarbonate. By varying the concentration of diethylpyrocarbonate modified ferredoxins could be prepared which had only one or both of the imidazole nitrogens of the histidine modified. A small amount of tyrosine was also modified. Ferredoxin with only one of the imidazole nitrogens modified was fully active in NADP photoreduction by chloroplast membranes. This activity was lost as the second imidazole nitrogen was modified. The results suggest an essential role for the single histidine of ferredoxin.     

巨噬细胞对氧化和丙二醛修饰的低密度脂蛋白结合与降解特性的比较研究

用Cu~(2+)(引发氧化修饰)和脂质过氧化降解产物丙二醛对低密度脂蛋白(LDL)进行修饰,分别测定了巨噬细胞系P~(300)D_1和小鼠腹腔巨噬细胞对两种被修饰LDL的结合量(包括内移量)和降解量。结果显示:LDL经氧化修饰和丙二醛修饰后被两类巨噬细胞的结合量与降解量均高于正常LDL,在修饰程度相近(琼脂糖电泳迁移率相近)时,两类巨噬细胞对氧化修饰LDL的结合量与降解量高于丙二醛修饰的LDL。竞争性抑制结果显示,丙二醛修饰的LDL和乙酰化修饰的LDL均可部分抑制巨噬细胞对氧化修饰LDL的结合与降解。     

巨噬细胞对氧化和丙二醛修饰的低密度脂蛋白结合与降解特性的比较研究

用Cu~(2+)(引发氧化修饰)和脂质过氧化降解产物丙二醛对低密度脂蛋白(LDL)进行修饰,分别测定了巨噬细胞系P~(300)D_1和小鼠腹腔巨噬细胞对两种被修饰LDL的结合量(包括内移量)和降解量。结果显示:LDL经氧化修饰和丙二醛修饰后被两类巨噬细胞的结合量与降解量均高于正常LDL,在修饰程度相近(琼脂糖电泳迁移率相近)时,两类巨噬细胞对氧化修饰LDL的结合量与降解量高于丙二醛修饰的LDL。竞争性抑制结果显示,丙二醛修饰的LDL和乙酰化修饰的LDL均可部分抑制巨噬细胞对氧化修饰LDL的结合与降解。     

修饰血红素提高血红蛋白类过氧化物酶活性

利用苯酚或对羟基联苯对血红蛋白的血红素辅基进行化学修饰,将修饰后的血红素与脱辅基血红蛋白进行重组得到新的血红蛋白。以光吸收扫描分析修饰血红素和重组血红蛋白,证明新的重组血红蛋白构建成功。酶活力测定表明,修饰血红素得到的重组血红蛋白的类过氧化物酶活性都比天然血红蛋白的酶活力高,用对羟基联苯修饰血红素得到的重组血红蛋白的酶活提高明显,约是天然血红蛋白的1.6倍。     

Analysis of the rye chromosome constitution and the amount of telomeric heterochromatin in the widely and narrowly adapted hexaploid triticales

Summary Investigations were made on the rye chromosome constitution and on the presence of telomeric heterochromatin in rye chromosomes of the 26 most widely and 24 most narrowly adapted triticale strains. Among widely adapted lines, 22 (85%) had a complete rye genome and four triticales only had chromosomal R-D genome substitutions. Twenty-three (96%) of the 24 most narrowly adapted triticales had substitutions between the chromosomes of the R and D genomes. The most widely adapted triticales accumulated fewer modified rye chromosomes in comparison to narrowly adapted lines. They had from one to three rye chromosomes with heterochromatic deletions: 46% of widely adapted lines had two modified rye chromosomes; 34% had three modified rye chromosomes, and 19% had a single modified rye chromosome. In widely adapted strains, the 1R, 4R, 5R and 6R modified chromosomes were observed; they were present in 80%, 73%, 50% and 11% of the cases, respectively. The most narrowly adapted triticales had from two to four modified rye chromosomes: 58% of the strains had three modified rye chromosomes; 29% had four modified rye chromosomes and 12% had two modified rye chromosomes. The modified 4R and 5R chromosomes were present in all of these lines. The 1R (modified), 6R (modified) and 7R (modified) were found in 83%, 25% and 16%, respectively, of the narrowly adapted strains.Results support the previous observations (Pilch 1980b) that a wide adaptation of hexaploid triticales is associated with the presence of the full potential of rye genome, and that it is independent of the amount of telomeric heterochromatin possessed by rye chromosomes.     

The effect of cleaving the reactive-site peptide bond Lys-15--Ala-16 on the conformation of bovine trypsin-kallikrein inhibitor (K unitz) as revealed by solvent-perturbation spectra, circular dichroism and fluorescence.

Spectroscopic measurements of virgin bovine trypsin-kallikrein inhibitor and its modified species (in which the reactive-site peptide bond Lys-15--Ala-16 is split) indicate a conformational difference between both proteins. The inhibitor contains four tyrosines but no tryptophan. In the modified inhibitor a tyrosyl blue shift is seen in the difference absorption spectrum of modified against virgin inhibitor. The solvent perturbation spectra show an increase of the fraction of exposed tyrosyls from 0.45 in the virgin inhibitor to 0.59 in the modified form. Comparison of the circular dichroism spectra of the modified and virgin inhibitors reveals a decrease of the mean residue ellipticity in the tyrosine and peptide bond region of the modified inhibitor. In the fluorescence spectra a 50% increase in the quantum yield of the tyrosine fluorescence is observed in the modified inhibitor. All these spectroscopic data support the idea, which is also evidenced by the X-ray crystallographic model, that in the modified inhibitor up to five residues from Ala-16 to Arg-20 gain rotational freedom.     

Carboxylate groups on the manganese-stabilizing protein are required for its efficient binding to photosystem II

The effects of the modification of carboxylate groups on the manganese-stabilizing protein of photosystem II were investigated. Carboxylate groups (including possibly the C-terminus) on the manganese-stabilizing protein were modified with glycine methyl ester in a reaction facilitated by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The manganese-stabilizing protein that was modified while associated with NaCl-washed photosystem II membranes contained 1-2 modified carboxylates, whereas the protein that was modified while free in solution contained 4 modified carboxylates. Both types of modified protein could reconstitute oxygen evolution at high manganese-stabilizing protein to photosystem II reaction center ratios. However, the protein that had been modified in solution exhibited a dramatically altered binding affinity for photosystem II. No such alteration in binding affinity was observed for the protein that had been modified while associated with the photosystem. Mapping of the sites of modification was carried out by trypsin and Staphylococcus V8 protease digestion of the modified proteins and analysis by matrix-assisted laser desorption/ionization mass spectrometry. These studies indicated that the domains (157)D-(168)D and (212)E-(247)Q (C-terminus) are labeled only when the manganese-stabilizing protein is modified in solution. Modified carboxylates in these domains are responsible for the altered binding affinity of this protein for the photosystem.     

Dyeing and diffusion properties of modified novel cellulose with triazine derivatives containing cationic and anionic groups

Cellulose is chemically modified with the compounds containing cationic and anionic groups. Dyeing and diffusion properties of modified cellulose are discussed. The exhaustion and fixation of reactive dyes on modified cellulose are higher than those on unmodified cellulose. Compared with unmodified cellulose, the dyed modified cellulose also gets good washing fastness. The diffusion coefficients of dyes at different temperature are calculated. Compared with unmodified cellulose, the diffusion of dyes in the modified cellulose shows significant change.     

右旋糖酐对Savinase蛋白酶的化学修饰及酶学性质研究

采用经高碘酸钠活化的右旋糖酐修饰Savinase蛋白酶,通过凝胶过滤层析(GPC)和圆二色性光谱(CD)表征了修饰后蛋白酶分子量和结构的变化,测试了修饰酶的反应动力学参数,并考察了温度及pH对修饰酶活力的影响。凝胶过滤层析结果证明修饰后蛋白酶分子量明显提高,圆二色光谱分析表明修饰后蛋白酶的结构有所改变,进一步验证了右旋糖酐和蛋白酶发生了反应。与原酶相比,修饰酶对底物的亲和力增加。原酶和修饰酶的最适温度均为40℃,在30℃~50℃之间修饰酶表现出优于原酶的热稳定性。在pH8.5~9.5之间,修饰酶的稳定性高于原酶。