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1.  Enhanced cellular uptake of aminosilane-coated superparamagnetic iron oxide nanoparticles in mammalian cell lines 
Purpose
To compare the cellular uptake efficiency and cytotoxicity of aminosilane (SiO2-NH2)-coated superparamagnetic iron oxide (SPIO@SiO2-NH2) nanoparticles with three other types of SPIO nanoparticles coated with SiO2 (SPIO@SiO2), dextran (SPIO@dextran), or bare SPIO in mammalian cell lines.
Materials and methods
Four types of monodispersed SPIO nanoparticles with a SPIO core size of 7 nm and an overall size in a range of 7–15 nm were synthesized. The mammalian cell lines of MCF-7, MDA-MB-231, HT-29, RAW264.7, L929, HepG2, PC-3, U-87 MG, and mouse mesenchymal stem cells (MSCs) were incubated with four types of SPIO nanoparticles for 24 hours in the serum-free culture medium Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 μg/mL iron concentration. The cellular uptake efficiencies of SPIO nanoparticles were compared by Prussian blue staining and intracellular iron quantification. In vitro magnetic resonance imaging of MSC pellets after SPIO labeling was performed at 3 T. The effect of each SPIO nanoparticle on the cell viability of RAW 264.7 (mouse monocyte/macrophage) cells was also evaluated.
Results
Transmission electron microscopy demonstrated surface coating with SiO2-NH2, SiO2, and dextran prevented SPIO nanoparticle aggregation in DMEM culture medium. MCF-7, MDA-MB-231, and HT-29 cells failed to show notable iron uptake. For all the remaining six cell lines, Prussian blue staining and intracellular iron quantification demonstrated that SPIO@ SiO2-NH2 nanoparticles had the highest cellular uptake efficiency. SPIO@SiO2-NH2, bare SPIO, and SPIO@dextran nanoparticles did not affect RAW 264.7 cell viability up to 200 μg Fe/mL, while SPIO@SiO2 reduced RAW 264.7 cell viability from 10 to 200 μg Fe/mL in a dose-dependent manner.
Conclusion
Cellular uptake efficiency of SPIO nanoparticles depends on both the cell type and SPIO surface characteristics. Aminosilane surface coating enhanced the cellular uptake efficiency without inducing cytotoxicity in a number of cell lines.
doi:10.2147/IJN.S28316
PMCID: PMC3289449  PMID: 22393292
magnetic nanoparticles; SPIO; iron oxide; surface coating; cellular uptake
2.  Magnetic poly(lactide-co-glycolide) (PLGA) and cellulose particles for MRI-based cell tracking 
Biodegradable, superparamagnetic micro- and nanoparticles of poly(lactide-co-glycolide) (PLGA) and cellulose were designed, fabricated and characterized for magnetic cell labeling. Monodisperse nanocrystals of magnetite were incorporated into micro- and nanoparticles of PLGA and cellulose with high efficiency using an oil-in-water single emulsion technique. Superparamagnetic cores had high magnetization (72.1 emu/g). The resulting polymeric particles had smooth surface morphology and high magnetite content (43.3 wt% for PLGA and 69.6 wt% for cellulose). While PLGA and cellulose nanoparticles displayed highest r2* values per millimole of iron (399 s-1mM-1 for cellulose and 505 s-1mM-1 for PLGA), micron-sized PLGA particles had a much higher r2* per particle than either. After incubation for a month in citrate buffer (pH 5.5), magnetic PLGA particles lost close to 50% of their initial r2* molar relaxivity, while magnetic cellulose particles remained intact, preserving over 85% of their initial r2* molar relaxivity. Lastly, mesenchymal stem cells and human breast adenocarcinoma cells were magnetically labeled using these particles with no detectable cytotoxicity. These particles are ideally suited for non-invasive cell tracking in vivo via MRI and due to their vastly different degradation properties, offer unique potential for dedicated use for either short (PLGA-based particles) or long term (cellulose-based particles) experiments.
doi:10.1002/mrm.22765
PMCID: PMC3097259  PMID: 21404328
3.  Magnetically driven plasmid DNA delivery with biodegradable polymeric nanoparticles 
The FASEB Journal  2007;21(10):2510-2519.
Targeting gene therapy remains a challenge. The use of magnetic force to achieve this was investigated in the present study. It was hypothesized that nanoparticles with both controllable particle size and magnetic properties would enable magnetically driven gene delivery. We investigated this hypothesis by creating a family of novel biodegradable polymeric superparamagnetic nanoparticle (MNP) formulations. Polylactide MNP were formulated using a modified emulsification-solvent evaporation methodology with both the incorporation of oleate-coated iron oxide and a polyethylenimine (PEI) oleate ion-pair surface modification for DNA binding. MNP size could be controlled by varying the proportion of the tetrahydrofuran cosolvent. Magnetically driven MNP-mediated gene transfer was studied using a green fluorescent protein reporter plasmid in cultured arterial smooth muscle cells and endothelial cells. MNP-DNA internalization and trafficking were examined by confocal microscopy. Cell growth inhibition after MNP-mediated adiponectin plasmid transfection was studied as an example of a therapeutic end point. MNP-DNA complexes protected DNA from degradation and efficiently transfected quiescent cells under both low and high serum conditions after a 15 min exposure to a magnetic field (500 G). There was negligible transfection with MNP in the absence of a magnetic field. Larger sized MNP (375 nm diameter) exhibited higher transfection rates compared with 185 nm- and 240 nm-sized MNP. Internalized larger sized MNP escaped lysosomal localization and released DNA in the perinuclear zone. Adiponectin plasmid DNA delivery using MNP resulted in a dose-dependent growth inhibition of cultured arterial smooth muscle cells. It is concluded that magnetically driven plasmid DNA delivery can be achieved using biodegradable MNP containing oleate-coated magnetite and surface modified with PEI oleate ion-pair complexes that enable DNA binding.
doi:10.1096/fj.06-8070com
PMCID: PMC3378388  PMID: 17403937
GFP plasmid; magnetic nanoparticles; ion-pair complex; polyethylenimine; adiponectin
4.  Mouse lymphatic endothelial cell targeted probes: anti-LYVE-1 antibody-based magnetic nanoparticles 
Purpose
To investigate the specific targeting property of lymphatic vessel endothelial hyaluronan receptor-1 binding polyethylene glycol-coated ultrasmall superparamagnetic iron oxide (LYVE-1-PEG-USPIO) nanoparticles to mouse lymphatic endothelial cells (MLECs).
Methods
A ligand specific target to lymphatic vessels was selected by immunohistochemical staining on the sections of a Lewis subcutaneous transplanted tumor. The z-average hydrodynamic diameter (HD), zeta potential, and the relaxivity of PEG-USPIO and LYVE-1-PEG-USPIO nanoparticles were determined with a laser particle analyzer and magnetic resonance T2 spin echo sequence, respectively. Prussian blue staining and transmission electron microscopy (TEM) of nanoparticle labeled cells were performed to determine the nanoparticles’ binding form. Magnetic resonance imaging (MRI) was performed in vitro to evaluate the signal enhancement on the T2 spin echo sequence of the nanoparticle labeled cells. The iron content of the labeled cells after the Prussian blue staining and MRI scanning was determined by atomic absorption spectroscopy (AAS).
Results
The anti-LYVE-1 antibody was used as the specific ligand to synthesize the target probe to the MLECs. The mean z-average HDs of the LYVE-1-PEG-USPIO and PEG-USPIO nanoparticles were 57.42 ± 0.31 nm and 47.91 ± 0.73 nm, respectively, and the mean zeta potentials of the LYVE-1-PEG-USPIO and PEG-USPIO nanoparticles were 12.38 ± 4.87 mV and 2.57 ± 0.83 m V, respectively. The relaxivities of the LYVE-1-PEG-USPIO and PEG-USPIO nanoparticles were 185.48 mM−1s−1 and 608.32 mM−1s−1. Cells binding nanoparticles were visualized as blue granules in the Prussian blue staining. The TEM results of the labeled cells showed the specific localization of nanoparticles. The AAS results of labeled cells after the Prussian blue staining and MRI scanning showed that the LYVE-1-PEG-USPIO nanoparticles had good binding selectivity for MLECs. MRI results indicated that the PEG-USPIO and LYVE-1-PEG-USPIO nanoparticles could generate contrast on T2-weighted imaging, and the correlation between R2 and the iron content of the labeled cells was significantly positive.
Conclusion
This study demonstrated that LYVE-1-PEG-USPIO nanoparticles might potentially be used as an MRI contrast agent for targeting MLECs, and the magnetic properties of LYVE-1-PEG-USPIO nanoparticles were suitable for MRI.
doi:10.2147/IJN.S45817
PMCID: PMC3693816  PMID: 23818783
nanoparticles; lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1); ultrasmall superparamagnetic iron oxide (USPIO); mouse lymphatic endothelial cells (MLECs); magnetic resonance imaging (MRI)
5.  Hybrid magnetite nanoparticles/Rosmarinus officinalis essential oil nanobiosystem with antibiofilm activity 
Nanoscale Research Letters  2012;7(1):209.
Biofilms formed by fungal organisms are associated with drastically enhanced resistance against most antimicrobial agents, contributing to the persistence of the fungi despite antifungal therapy. The purpose of this study is to combine the unique properties of nanoparticles with the antimicrobial activity of the Rosmarinus officinalis essential oil in order to obtain a nanobiosystem that could be pelliculised on the surface of catheter pieces, in order to obtain an improved resistance to microbial colonization and biofilm development by Candida albicans and C. tropicalis clinical strains. The R. officinalis essential oils were extracted in a Neo-Clevenger type apparatus, and its chemical composition was settled by GC-MS analysis. Functionalized magnetite nanoparticles of up to 20 nm size had been synthesized by precipitation method adapted for microwave conditions, with oleic acid as surfactant. The catheter pieces were coated with suspended core/shell nanoparticles (Fe3O4/oleic acid:CHCl3), by applying a magnetic field on nanofluid, while the CHCl3 diluted essential oil was applied by adsorption in a secondary covering treatment. The fungal adherence ability was investigated in six multiwell plates, in which there have been placed catheters pieces with and without hybrid nanoparticles/essential oil nanobiosystem pellicle, by using culture-based methods and confocal laser scanning microscopy (CLSM). The R. officinalis essential oil coated nanoparticles strongly inhibited the adherence ability and biofilm development of the C. albicans and C. tropicalis tested strains to the catheter surface, as shown by viable cell counts and CLSM examination. Due to the important implications of Candida spp. in human pathogenesis, especially in prosthetic devices related infections and the emergence of antifungal tolerance/resistance, using the new core/shell/coated shell based on essential oil of R. officinalis to inhibit the fungal adherence could be of a great interest for the biomedical field, opening new directions for the design of film-coated surfaces with antibiofilm properties.
doi:10.1186/1556-276X-7-209
PMCID: PMC3368737  PMID: 22490675
6.  Impact of surface coating and particle size on the uptake of small and ultrasmall superparamagnetic iron oxide nanoparticles by macrophages 
Purpose
Magnetic resonance imaging (MRI) using contrast agents like superparamagnetic iron oxide (SPIO) is an extremely versatile technique to diagnose diseases and to monitor treatment. This study tested the relative importance of particle size and surface coating for the optimization of MRI contrast and labeling efficiency of macrophages migrating to remote inflammation sites.
Materials and methods
We tested four SPIO and ultrasmall superparamagnetic iron oxide (USPIO), alkali-treated dextran magnetite (ATDM) with particle sizes of 28 and 74 nm, and carboxymethyl dextran magnetite (CMDM) with particle sizes of 28 and 72 nm. Mouse macrophage RAW264 cells were incubated with SPIOs and USPIOs, and the labeling efficiency of the cells was determined by the percentage of Berlin blue-stained cells and by measuring T2 relaxation times with 11.7-T MRI. We used trypan blue staining to measure cell viability.
Results
Analysis of the properties of the nanoparticles revealed that ATDM-coated 74 nm particles have a lower T2 relaxation time than the others, translating into a higher ability of MRI negative contrast agent. Among the other three candidates, CMDM-coated particles showed the highest T2 relaxation time once internalized by macrophages. Regarding labeling efficiency, ATDM coating resulted in a cellular uptake higher than CMDM coating, independent of nanoparticle size. None of these particle formulations affected macrophage viability.
Conclusion
This study suggests that coating is more critical than size to optimize the SPIO labeling of macrophages. Among the formulations tested in this study, the best MRI contrast and labeling efficiency are expected with ATDM-coated 74 nm nanoparticles.
doi:10.2147/IJN.S33709
PMCID: PMC3474462  PMID: 23091384
ultrasmall superparamagnetic iron oxide; cultured mouse macrophage cells; surface coating; particle size; MRI
7.  Stability and magnetically induced heating behavior of lipid-coated Fe3O4 nanoparticles 
Nanoscale Research Letters  2013;8(1):426.
Magnetic nanoparticles that are currently explored for various biomedical applications exhibit a high propensity to minimize total surface energy through aggregation. This study introduces a unique, thermoresponsive nanocomposite design demonstrating substantial colloidal stability of superparamagnetic Fe3O4 nanoparticles (SPIONs) due to a surface-immobilized lipid layer. Lipid coating was accomplished in different buffer systems, pH 7.4, using an equimolar mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and l-α-dipalmitoylphosphatidyl glycerol (DPPG). Particle size and zeta potential were measured by dynamic laser light scattering. Heating behavior within an alternating magnetic field was compared between the commercial MFG-1000 magnetic field generator at 7 mT (1 MHz) and an experimental, laboratory-made magnetic hyperthermia system at 16.6 mT (13.7 MHz). The results revealed that product quality of lipid-coated SPIONs was significantly dependent on the colloidal stability of uncoated SPIONs during the coating process. Greatest stability was achieved at 0.02 mg/mL in citrate buffer (mean diameter = 80.0 ± 1.7 nm; zeta potential = -47.1 ± 2.6 mV). Surface immobilization of an equimolar DPPC/DPPG layer effectively reduced the impact of buffer components on particle aggregation. Most stable suspensions of lipid-coated nanoparticles were obtained at 0.02 mg/mL in citrate buffer (mean diameter = 179.3 ± 13.9 nm; zeta potential = -19.1 ± 2.3 mV). The configuration of the magnetic field generator significantly affected the heating properties of fabricated SPIONs. Heating rates of uncoated nanoparticles were substantially dependent on buffer composition but less influenced by particle concentration. In contrast, thermal behavior of lipid-coated nanoparticles within an alternating magnetic field was less influenced by suspension vehicle but dramatically more sensitive to particle concentration. These results underline the advantages of lipid-coated SPIONs on colloidal stability without compromising magnetically induced hyperthermia properties. Since phospholipids are biocompatible, these unique lipid-coated Fe3O4 nanoparticles offer exciting opportunities as thermoresponsive drug delivery carriers for targeted, stimulus-induced therapeutic interventions.
PACS
7550Mw; 7575Cd; 8185Qr
doi:10.1186/1556-276X-8-426
PMCID: PMC3853621  PMID: 24134544
SPION; Magnetic field generator; Hyperthermia; Phospholipid; Thermoresponsive; Colloid
8.  Monodisperse Magnetite Nanoparticles Coupled with Nuclear Localization Signal Peptide for Cell-Nucleus Targeting 
Chemistry, an Asian journal  2008;3(3):548-552.
Functionalization of monodisperse superparamagnetic magnetite (Fe3O4) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe3O4 nanoparticles functionalized with protein and nuclear localization signal (NLS) peptide. These NLS-coated nanoparticles were introduced into the HeLa cell cytoplasm and nucleus, where the particles were monodispersed and non-aggregated. The success of labeling was examined and identified by fluorescence microscopy and MRI. The work demonstrates that monodisperse magnetic nanoparticles can be readily functionalized and stabilized for potential diagnostic and therapeutic applications.
doi:10.1002/asia.200700301
PMCID: PMC2692425  PMID: 18080259
cell imaging; magnetic resonance imaging; magnetite; nanoparticles
9.  Biocompatible Polysiloxane-Containing Diblock Copolymer PEO-b-PγMPS for Coating Magnetic Nanoparticles 
ACS applied materials & interfaces  2009;1(10):2134-2140.
We report a biocompatible polysiloxane containing amphiphilic diblock copolymer, poly(ethylene oxide)-block-poly(γ-methacryloxypropyltrimethoxysilane) (PEO-b-PγMPS), for coating and stabilizing nanoparticles for biomedical applications. Such amphiphilic diblock copolymer which comprises both a hydrophobic segment with “surface anchoring moiety” (silane group) and a hydrophilic segment with PEO (Mn=5000 g/mol) was obtained by the reversible addition fragmentation chain transfer (RAFT) polymerization using the PEO macromolecular chain transfer agent. When used for coating paramagnetic iron oxide nanoparticles (IONPs), copolymers were mixed with hydrophobic oleic acid coated core size uniformed IONPs (D=13 nm) in co-solvent tetrahydrofuran. After being aged over a period of time, resulting monodispersed IONPs can be transferred into aqueous medium. With proper PγMPS block length (Mn=10,000 g/mol), polysiloxane containing diblock copolymers formed a thin layer of coating (~3 nm) around monocrystalline nanoparticles as measured by transmission electron microscopy (TEM). Magnetic resonance imaging (MRI) experiments showed excellent T2 weighted contrast effect from coated IONPs with a transverse relaxivity r2=98.6 mM−1s−1 (at 1.5 Tesla). Such thin coating layer has little effect on the relaxivity when compared to that of IONPs coated with conventional amphiphilic copolymer. Polysiloxane containing diblock copolymer coated IONPs are stable without aggregation or binding to proteins in serum when incubated for 24 h in culture medium containing 10% serum. Furthermore, much lower level of intracellular uptake by macrophage cells was observed with polysiloxane containing diblock copolymers coated IONPs, suggesting the reduction of non-specific cell uptakes and antibiofouling effect.
doi:10.1021/am900262j
PMCID: PMC2799899  PMID: 20161520
diblock copolymer; silanes; coating; nanoparticle; magnetic resonance imaging
10.  Alpha chymotrypsin coated clusters of Fe3O4 nanoparticles for biocatalysis in low water media 
Background
Enzymes in low water containing non aqueous media are useful for organic synthesis. For example, hydrolases in such media can be used for synthetic purposes. Initial work in this area was carried out with lyophilized powders of enzymes. These were found to have poor activity. Drying (removing bulk water) by precipitation turned out to be a better approach. As enzymes in such media are heterogeneous catalysts, spreading these precipitates over a large surface gave even better results. In this context, nanoparticles with their better surface to volume ratio provide obvious advantage. Magnetic nanoparticles have an added advantage of easy separation after the reaction. Keeping this in view, alpha chymotrypsin solution in water was precipitated over a stirred population of Fe3O4 nanoparticles in n-propanol. This led to alpha chymotrypsin activity coated over clusters of Fe3O4 nanoparticles. These preparations were found to have quite high transesterification activity in low water containing n-octane.
Results
Precipitation of alpha chymotrypsin over a stirred suspension of Fe3O4 nanoparticles (3.6 nm diameter) led to the formation of enzyme coated clusters of nanoparticles (ECCNs). These clusters were also magnetic and their hydrodynamic diameter ranged from 1.2- 2.6 microns (as measured by dynamic light scattering). Transmission electron microscopy (TEM), showed that these clusters had highly irregular shapes. Transesterification assay of various clusters in anhydrous n-octane led to optimization of concentration of nanoparticles in suspension during precipitation. Optimized design of enzyme coated magnetic clusters of nanoparticles (ECCN 3) showed the highest initial rate of 465 nmol min-1 mg-1protein which was about 9 times higher as compared to the simple precipitates with an initial rate of 52 nmol min-1 mg-1 protein.
Circular Dichroism (CD)(with a spinning cell accessory) showed that secondary structure content of the alpha Chymotrypsin in ECCN 3 [15% α-helix, 37% β-sheet and 48% random coil] was identical to the simple precipitates of alpha chymotrypsin.
Conclusion
A strategy for obtaining a high activity preparation of alpha chymotrypsin for application in low water media is described. Such high activity biocatalysts are useful in organic synthesis.
doi:10.1186/1752-153X-6-133
PMCID: PMC3505189  PMID: 23137100
Enzyme immobilization; Enzymes in low water media; Magnetic nanoparticles; Nanoclusters; Protein precipitation; Fe3O4 nanoparticles
11.  Preparation and Characterization of Ferrofluid Stabilized with Biocompatible Chitosan and Dextran Sulfate Hybrid Biopolymer as a Potential Magnetic Resonance Imaging (MRI) T2 Contrast Agent 
Marine Drugs  2012;10(11):2403-2414.
Chitosan is the deacetylated form of chitin and used in numerous applications. Because it is a good dispersant for metal and/or oxide nanoparticle synthesis, chitosan and its derivatives have been utilized as coating agents for magnetic nanoparticles synthesis, including superparamagnetic iron oxide nanoparticles (SPIONs). Herein, we demonstrate the water-soluble SPIONs encapsulated with a hybrid polymer composed of polyelectrolyte complexes (PECs) from chitosan, the positively charged polymer, and dextran sulfate, the negatively charged polymer. The as-prepared hybrid ferrofluid, in which iron chloride salts (Fe3+ and Fe2+) were directly coprecipitated inside the hybrid polymeric matrices, was physic-chemically characterized. Its features include the z-average diameter of 114.3 nm, polydispersity index of 0.174, zeta potential of −41.5 mV and iron concentration of 8.44 mg Fe/mL. Moreover, based on the polymer chain persistence lengths, the anionic surface of the nanoparticles as well as the high R2/R1 ratio of 13.5, we depict the morphology of SPIONs as a cluster because chitosan chains are chemisorbed onto the anionic magnetite surfaces by tangling of the dextran sulfate. Finally, the cellular uptake and biocompatibility assays indicate that the hybrid polymer encapsulating the SPIONs exhibited great potential as a magnetic resonance imaging T2 contrast agent for cell tracking.
doi:10.3390/md10112403
PMCID: PMC3509525  PMID: 23203267
biocompatible polymer; chitosan; superparamagnetic iron oxide nanoparticle; nanomaterials
12.  Synthesis, characterization, and antimicrobial activity of an ampicillin-conjugated magnetic nanoantibiotic for medical applications 
Because of their magnetic properties, magnetic nanoparticles (MNPs) have numerous diverse biomedical applications. In addition, because of their ability to penetrate bacteria and biofilms, nanoantimicrobial agents have become increasingly popular for the control of infectious diseases. Here, MNPs were prepared through an iron salt coprecipitation method in an alkaline medium, followed by a chitosan coating step (CS-coated MNPs); finally, the MNPs were loaded with ampicillin (amp) to form an amp-CS-MNP nanocomposite. Both the MNPs and amp-CS-MNPs were subsequently characterized and evaluated for their antibacterial activity. X-ray diffraction results showed that the MNPs and nanocomposites were composed of pure magnetite. Fourier transform infrared spectra and thermogravimetric data for the MNPs, CS-coated MNPs, and amp-CS-MNP nanocomposite were compared, which confirmed the CS coating on the MNPs and the amp-loaded nanocomposite. Magnetization curves showed that both the MNPs and the amp-CS-MNP nanocomposites were superparamagnetic, with saturation magnetizations at 80.1 and 26.6 emu g−1, respectively. Amp was loaded at 8.3%. Drug release was also studied, and the total release equilibrium for amp from the amp-CS-MNPs was 100% over 400 minutes. In addition, the antimicrobial activity of the amp-CS-MNP nanocomposite was determined using agar diffusion and growth inhibition assays against Gram-positive bacteria and Gram-negative bacteria, as well as Candida albicans. The minimum inhibitory concentration of the amp-CS-MNP nanocomposite was determined against bacteria including Mycobacterium tuberculosis. The synthesized nanocomposites exhibited antibacterial and antifungal properties, as well as antimycobacterial effects. Thus, this study introduces a novel β-lactam antibacterial-based nanocomposite that can decrease fungus activity on demand for numerous medical applications.
doi:10.2147/IJN.S61143
PMCID: PMC4134181  PMID: 25143729
iron oxide nanoparticles; chitosan; coating material; antibacterial activity; β-lactam; and nanoantibiotics
13.  Immobilization of Lipases on Alkyl Silane Modified Magnetic Nanoparticles: Effect of Alkyl Chain Length on Enzyme Activity 
PLoS ONE  2012;7(8):e43478.
Background
Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification.
Methodology/Principal Findings
Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18) modified Fe3O4 were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining.
Conclusions/Significance
The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for enzyme immobilization enabling efficient enzyme recovery and recycling.
doi:10.1371/journal.pone.0043478
PMCID: PMC3431390  PMID: 22952688
14.  Preparation and characterization of magnetic nanoparticles containing Fe3O4-dextran-anti-β-human chorionic gonadotropin, a new generation choriocarcinoma-specific gene vector 
Objective:
To evaluate the feasibility of using magnetic iron oxide (Fe3O4)-dextran-anti-β-human chorionic gonadotropin (HCG) nanoparticles as a gene vector for cellular transfections.
Study design:
Fe3O4-dextran-anti-β-HCG nanoparticles were synthesized by chemical coprecipitation. The configuration, diameter, and iron content of the nanoparticles were detected by transmission electron microscopy (TEM), light scatter, and atomic absorption spectrophotometry. A3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay was used to evaluate the cytotoxicity of Fe3O4-dextran-anti-β-HCG nanoparticles. Enzyme-linked immunosorbent assay and indirect immunofluorescence were used to evaluate immunoreactivity. The efficiency of absorbing DNA and resisting deoxyribonuclease I (DNase I) digestion when bound to Fe3O4-dextran-anti-β-HCG nanoparticles was examined by agarose gel electrophoresis. The ability of Fe3O4-dextran-anti-β-HCG nanoparticles to absorb heparanase antisense oligodeoxynucleotides (AS-ODN) nanoparticles in different cell lines was evaluated by flow cytometry. The tissue distribution of heparanase AS-ODN magnetic nanoparticles in choriocarcinoma tumors transplanted in nude mice was detected by atomic absorption spectrophotometry.
Results:
TEM demonstrated that the shape of nanoparticles is irregular. Light scatter revealed nanoparticles with a mean diameter of 75.5 nm and an iron content of 37.5 μg/mL. No cytotoxicity was observed when the concentration of Fe3O4-dextran-anti-β-HCG nanoparticles was <37.5 μg/mL. Fe3O4-dextran nanoparticles have a satisfactory potential to combine with β-HCG antibody. Agarose gel electrophoresis analysis of binding experiments showed that after treatment with sodium periodate, Fe3O4-dextran-anti-β-HCG nanoparticles have a satisfactory potential to absorb DNA, and the protection experiment showed that nanoparticles can effectively protect DNA from DNase I digestion. Aldehyde Fe3O4-dextran-anti-β-HCG nanoparticles can transfect reporter genes, and the transfection efficiency of these nanoparticles is greater than that of liposomes (P < 0.05). Fe3O4-dextran-anti-β-HCG nanoparticles can concentrate in choriocarcinoma cells and in transplanted choriocarcinoma tumors.
Conclusions:
The results confirm that Fe3O4-dextran-anti-β-HCG nanoparticles have potential as a secure, effective, and choriocarcinoma-specific targeting gene vector.
doi:10.2147/IJN.S13410
PMCID: PMC3044181  PMID: 21383853
magnetic nanoparticles; Fe3O4-dextran-anti-β-HCG; choriocarcinoma; targeting vector; gene vector
15.  Chitosan-Coated Magnetic Nanoparticles Prepared in One Step by Reverse Microemulsion Precipitation 
Chitosan-coated magnetic nanoparticles (CMNP) were obtained at 70 °C and 80 °C in a one-step method, which comprises precipitation in reverse microemulsion in the presence of low chitosan concentration in the aqueous phase. X-ray diffractometry showed that CMNP obtained at both temperatures contain a mixture of magnetite and maghemite nanoparticles with ≈4.5 nm in average diameter, determined by electron microscopy, which suggests that precipitation temperature does not affect the particle size. The chitosan coating on nanoparticles was inferred from Fourier transform infrared spectrometry measurements; furthermore, the carbon concentration in the nanoparticles allowed an estimation of chitosan content in CMNP of 6%–7%. CMNP exhibit a superparamagnetic behavior with relatively high final magnetization values (≈49–53 emu/g) at 20 kOe and room temperature, probably due to a higher magnetite content in the mixture of magnetic nanoparticles. In addition, a slight direct effect of precipitation temperature on magnetization was identified, which was ascribed to a possible higher degree of nanoparticles crystallinity as temperature at which they are obtained increases. Tested for Pb2+ removal from a Pb(NO3)2 aqueous solution, CMNP showed a recovery efficacy of 100%, which makes them attractive for using in heavy metals ion removal from waste water.
doi:10.3390/ijms141019636
PMCID: PMC3821577  PMID: 24084716
chitosan magnetic nanoparticles; one-step microemulsion precipitation; chitosan low content
16.  Novel kojic acid-polymer-based magnetic nanocomposites for medical applications 
Iron oxide magnetic nanoparticles (MNPs) were synthesized by the coprecipitation of iron salts in sodium hydroxide followed by coating separately with chitosan (CS) and polyethylene glycol (PEG) to form CS-MNPs and PEG-MNPs nanoparticles, respectively. They were then loaded with kojic acid (KA), a pharmacologically bioactive natural compound, to form KA-CS-MNPs and KA-PEG-MNPs nanocomposites, respectively. The MNPs and their nanocomposites were characterized using powder X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric analysis, vibrating sample magnetometry, and scanning electron microscopy. The powder X-ray diffraction data suggest that all formulations consisted of highly crystalline, pure magnetite Fe3O4. The Fourier transform infrared spectroscopy and thermogravimetric analysis confirmed the presence of both polymers and KA in the nanocomposites. Magnetization curves showed that both nanocomposites (KA-CS-MNPs and KA-PEG-MNPs) were superparamagnetic with saturation magnetizations of 8.1 emu/g and 26.4 emu/g, respectively. The KA drug loading was estimated using ultraviolet–visible spectroscopy, which gave a loading of 12.2% and 8.3% for the KA-CS-MNPs and KA-PEG-MNPs nanocomposites, respectively. The release profile of the KA from the nanocomposites followed a pseudo second-order kinetic model. The agar diffusion test was performed to evaluate the antimicrobial activity for both KA-CS-MNPs and KA-PEG-MNPs nanocomposites against a number of microorganisms using two Gram-positive (methicillin-resistant Staphylococcus aureus and Bacillus subtilis) and one Gram-negative (Salmonella enterica) species, and showed some antibacterial activity, which could be enhanced in future studies by optimizing drug loading. This study provided evidence for the promise for the further investigation of the possible beneficial biological activities of KA and both KA-CS-MNPs and KA-PEG-MNPs nanocomposites in nanopharmaceutical applications.
doi:10.2147/IJN.S53847
PMCID: PMC3890966  PMID: 24453486
chitosan; polyethylene glycol; magnetic nanoparticle; kojic acid; controlled release; biological activity
17.  Biocompatibility of magnetic Fe3O4 nanoparticles and their cytotoxic effect on MCF-7 cells 
Background
The objective of this study was to evaluate the synthesis and biocompatibility of Fe3O4 nanoparticles and investigate their therapeutic effects when combined with magnetic fluid hyperthermia on cultured MCF-7 cancer cells.
Methods
Magnetic Fe3O4 nanoparticles were prepared using a coprecipitation method. The appearance, structure, phase composition, functional groups, surface charge, magnetic susceptibility, and release in vitro were characterized by transmission electron microscopy, x-ray diffraction, scanning electron microscopy-energy dispersive x-ray spectroscopy, and a vibrating sample magnetometer. Blood toxicity, in vitro toxicity, and genotoxicity were investigated. Therapeutic effects were evaluated by MTT [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] and flow cytometry assays.
Results
Transmission electron microscopy revealed that the shapes of the Fe3O4 nanoparticles were approximately spherical, with diameters of about 26.1 ± 5.2 nm. Only the spinel phase was indicated in a comparison of the x-ray diffraction data with Joint Corporation of Powder Diffraction Standards (JCPDS) X-ray powder diffraction files. The O-to-Fe ratio of the Fe3O4 was determined by scanning electron microscopy-energy dispersive x-ray spectroscopy elemental analysis, and approximated pure Fe3O4. The vibrating sample magnetometer hysteresis loop suggested that the Fe3O4 nanoparticles were superparamagnetic at room temperature. MTT experiments showed that the toxicity of the material in mouse fibroblast (L-929) cell lines was between Grade 0 to Grade 1, and that the material lacked hemolysis activity. The acute toxicity (LD50) was 8.39 g/kg. Micronucleus testing showed no genotoxic effects. Pathomorphology and blood biochemistry testing demonstrated that the Fe3O4 nanoparticles had no effect on the main organs and blood biochemistry in a rabbit model. MTT and flow cytometry assays revealed that Fe3O4 nano magnetofluid thermotherapy inhibited MCF-7 cell proliferation, and its inhibitory effect was dose-dependent according to the Fe3O4 nano magnetofluid concentration.
Conclusion
The Fe3O4 nanoparticles prepared in this study have good biocompatibility and are suitable for further application in tumor hyperthermia.
doi:10.2147/IJN.S35140
PMCID: PMC3446860  PMID: 23028225
characterization; biocompatibility; Fe3O4; magnetic nanoparticles; hyperthermia
18.  An efficient magnetically modified microbial cell biocomposite for carbazole biodegradation 
Nanoscale Research Letters  2013;8(1):522.
Magnetic modification of microbial cells enables to prepare smart biocomposites in bioremediation. In this study, we constructed an efficient biocomposite by assembling Fe3O4 nanoparticles onto the surface of Sphingomonas sp. XLDN2-5 cells. The average particle size of Fe3O4 nanoparticles was about 20 nm with 45.5 emu g-1 saturation magnetization. The morphology of Sphingomonas sp. XLDN2-5 cells before and after Fe3O4 nanoparticle loading was verified by scanning electron microscopy and transmission electronic microscopy. Compared with free cells, the microbial cell/Fe3O4 biocomposite had the same biodegradation activity but exhibited remarkable reusability. The degradation activity of the microbial cell/Fe3O4 biocomposite increased gradually during recycling processes. Additionally, the microbial cell/Fe3O4 biocomposite could be easily separated and recycled by an external magnetic field due to the super-paramagnetic properties of Fe3O4 nanoparticle coating. These results indicated that magnetically modified microbial cells provide a promising technique for improving biocatalysts used in the biodegradation of hazardous compounds.
doi:10.1186/1556-276X-8-522
PMCID: PMC3874645  PMID: 24330511
Carbazole; Immobilization; Nanoparticles; Biodegradation; Reusability
19.  Degradation of Carbazole by Microbial Cells Immobilized in Magnetic Gellan Gum Gel Beads▿  
Applied and Environmental Microbiology  2007;73(20):6421-6428.
Polycyclic aromatic heterocycles, such as carbazole, are environmental contaminants suspected of posing human health risks. In this study, we investigated the degradation of carbazole by immobilized Sphingomonas sp. strain XLDN2-5 cells. Four kinds of polymers were evaluated as immobilization supports for Sphingomonas sp. strain XLDN2-5. After comparison with agar, alginate, and κ-carrageenan, gellan gum was selected as the optimal immobilization support. Furthermore, Fe3O4 nanoparticles were prepared by a coprecipitation method, and the average particle size was about 20 nm with 49.65-electromagnetic-unit (emu) g−1 saturation magnetization. When the mixture of gellan gel and the Fe3O4 nanoparticles served as an immobilization support, the magnetically immobilized cells were prepared by an ionotropic method. The biodegradation experiments were carried out by employing free cells, nonmagnetically immobilized cells, and magnetically immobilized cells in aqueous phase. The results showed that the magnetically immobilized cells presented higher carbazole biodegradation activity than nonmagnetically immobilized cells and free cells. The highest biodegradation activity was obtained when the concentration of Fe3O4 nanoparticles was 9 mg ml−1 and the saturation magnetization of magnetically immobilized cells was 11.08 emu g−1. Additionally, the recycling experiments demonstrated that the degradation activity of magnetically immobilized cells increased gradually during the eight recycles. These results support developing efficient biocatalysts using magnetically immobilized cells and provide a promising technique for improving biocatalysts used in the biodegradation of not only carbazole, but also other hazardous organic compounds.
doi:10.1128/AEM.01051-07
PMCID: PMC2075067  PMID: 17827304
20.  Manipulation of isolated brain nerve terminals by an external magnetic field using D-mannose-coated γ-Fe2O3 nano-sized particles and assessment of their effects on glutamate transport 
Summary
The manipulation of brain nerve terminals by an external magnetic field promises breakthroughs in nano-neurotechnology. D-Mannose-coated superparamagnetic nanoparticles were synthesized by coprecipitation of Fe(II) and Fe(III) salts followed by oxidation with sodium hypochlorite and addition of D-mannose. Effects of D-mannose-coated superparamagnetic maghemite (γ-Fe2O3) nanoparticles on key characteristics of the glutamatergic neurotransmission were analysed. Using radiolabeled L-[14C]glutamate, it was shown that D-mannose-coated γ-Fe2O3 nanoparticles did not affect high-affinity Na+-dependent uptake, tonic release and the extracellular level of L-[14C]glutamate in isolated rat brain nerve terminals (synaptosomes). Also, the membrane potential of synaptosomes and acidification of synaptic vesicles was not changed as a result of the application of D-mannose-coated γ-Fe2O3 nanoparticles. This was demonstrated with the potential-sensitive fluorescent dye rhodamine 6G and the pH-sensitive dye acridine orange. The study also focused on the analysis of the potential use of these nanoparticles for manipulation of nerve terminals by an external magnetic field. It was shown that more than 84.3 ± 5.0% of L-[14C]glutamate-loaded synaptosomes (1 mg of protein/mL) incubated for 5 min with D-mannose-coated γ-Fe2O3 nanoparticles (250 µg/mL) moved to an area, in which the magnet (250 mT, gradient 5.5 Т/m) was applied compared to 33.5 ± 3.0% of the control and 48.6 ± 3.0% of samples that were treated with uncoated nanoparticles. Therefore, isolated brain nerve terminals can be easily manipulated by an external magnetic field using D-mannose-coated γ-Fe2O3 nanoparticles, while the key characteristics of glutamatergic neurotransmission are not affected. In other words, functionally active synaptosomes labeled with D-mannose-coated γ-Fe2O3 nanoparticles were obtained.
doi:10.3762/bjnano.5.90
PMCID: PMC4077395  PMID: 24991515
extracellular level; γ-Fe2O3; glutamate uptake and release; manipulation by an external magnetic field; D-mannose; membrane potential; nanoparticles; rat brain nerve terminals; synaptic vesicle acidification
21.  Preparation and characterization of 6-mercaptopurine-coated magnetite nanoparticles as a drug delivery system 
Background
Iron oxide nanoparticles are of considerable interest because of their use in magnetic recording tape, ferrofluid, magnetic resonance imaging, drug delivery, and treatment of cancer. The specific morphology of nanoparticles confers an ability to load, carry, and release different types of drugs.
Methods and results
We synthesized superparamagnetic nanoparticles containing pure iron oxide with a cubic inverse spinal structure. Fourier transform infrared spectra confirmed that these Fe3O4 nanoparticles could be successfully coated with active drug, and thermogravimetric and differential thermogravimetric analyses showed that the thermal stability of iron oxide nanoparticles coated with chitosan and 6-mercaptopurine (FCMP) was markedly enhanced. The synthesized Fe3O4 nanoparticles and the FCMP nanocomposite were generally spherical, with an average diameter of 9 nm and 19 nm, respectively. The release of 6-mercaptopurine from the FCMP nanocomposite was found to be sustained and governed by pseudo-second order kinetics. In order to improve drug loading and release behavior, we prepared a novel nanocomposite (FCMP-D), ie, Fe3O4 nanoparticles containing the same amounts of chitosan and 6-mercaptopurine but using a different solvent for the drug. The results for FCMP-D did not demonstrate “burst release” and the maximum percentage release of 6-mercaptopurine from the FCMP-D nanocomposite reached about 97.7% and 55.4% within approximately 2,500 and 6,300 minutes when exposed to pH 4.8 and pH 7.4 solutions, respectively. By MTT assay, the FCMP nanocomposite was shown not to be toxic to a normal mouse fibroblast cell line.
Conclusion
Iron oxide coated with chitosan containing 6-mercaptopurine prepared using a coprecipitation method has the potential to be used as a controlled-release formulation. These nanoparticles may serve as an alternative drug delivery system for the treatment of cancer, with the added advantage of sparing healthy surrounding cells and tissue.
doi:10.2147/DDDT.S43035
PMCID: PMC3791920  PMID: 24106420
superparamagnetic nanoparticles; 6-mercaptopurine; controlled release; cytotoxicity; drug delivery
22.  Preparation and assessment of chitosan-coated superparamagnetic Fe3O4 nanoparticles for controlled delivery of methotrexate 
In this study, Fe3O4 superparamagnetic nanoparticles were synthesized and stabilized by chitosan. Then the nanoparticles were characterized by Fourier transform infrared spectroscopy and transmission electron microscopy (TEM). Particle size distribution and Zeta potential of the particles also was assessed using Malvern Zetasizer. The paramagnetic behaviors of the uncoated and chitosan coated nanoparticles were measured using vibrating scanning magnetometry Particles morphology and size ranges of uncoated iron oxide nanoparticles were evaluated by TEM, showing uniform and narrow size distribution about 10 nm. After coating nanoparticles with chitosan and loading of methotrexate (MTX), the change in size was assessed using Zetasizer. Considerable increase in size was observed following the coating of the particles with chitosan and loading with MTX (the average size was 152 nm). Paramagnetic properties of the uncoated and chitosan-coated particles were assessed showing significant decrease in paramagnetic behavior after coating with chitosan, but it was enough to respond to the magnetic field. Finally loading efficiency, release rate and cytotoxicity of MTX were assessed indicating slow release behavior with the same levels of cell toxicity in SK-BR-3 cell lines, suggesting this formulation as a good candidate for the controlled delivery of MTX.
PMCID: PMC3895297  PMID: 24459473
Superparamagnetic; Fe3O4; Nanoparticles; Chitosan; Magnetic targeting drug delivery; Methotrexate
23.  Probing the Chemical Stability of Mixed Ferrites: Implications for MR Contrast Agent Design 
Nanomaterials with mixed composition, in particular magnetic spinel ferrites, are emerging as efficient contrast agents for magnetic resonance imaging (MRI). Many factors, including size, composition, atomic structure, and surface properties are crucial in the design of such nanoparticle-based probes due to their influence on the magnetic properties. Silica-coated iron oxide (IO-SiO2) and cobalt ferrite (CoIO-SiO2) nanoparticles were synthesized using standard high temperature thermal decomposition and base-catalyzed water-in-oil microemulsion techniques. Under neutral aqueous conditions, it was found that 50–75% of the cobalt content in the CoIO-SiO2 nanoparticles leached out of the core structure. Leaching caused a 7.2-fold increase in longitudinal relaxivity and an increase in the saturation magnetization from ~48 emu/g core to ~65 emu/g core. X-ray absorption fine structure studies confirmed that the atomic structure of the ferrite core was altered following leaching, while TEM and DLS confirmed that the morphology and size of the nanoparticle remained unchanged. The CoIO-SiO2 nanoparticles converted from a partially inverted spinel cation arrangement (unleached state) to an inverse spinel arrangement (leached state). The control IO-SiO2 nanoparticles remained stable with no change in structure and negligible changes in magnetic behavior. This detailed analysis highlights how important understanding the properties of nanomaterials is in the development of reliable agents for diagnostic and therapeutic applications.
doi:10.1021/cm200509g
PMCID: PMC3097046  PMID: 21603070
MRI; ferrite; nanoparticle; leaching; magnetic properties
24.  Optimized Anti-pathogenic Agents Based on Core/Shell Nanostructures and 2-((4-Ethylphenoxy)ethyl)-N-(substituted-phenylcarbamothioyl)-benzamides 
The purpose of this study was to design a new nanosystem for catheter surface functionalization with an improved resistance to Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 colonization and subsequent biofilm development. New 2-((4-ethylphenoxy)methyl)-N-(substituted-phenylcarbamothioyl)-benzamides were synthesized and used for coating a core/shell nanostructure. Their chemical structures were elucidated by NMR, IR and elemental analysis, being in agreement with the proposed ones. Fe3O4/C12 of up to 5 nm size had been synthesized with lauric acid as a coating agent and characterized by XRD, FT-IR, TGA, TEM and biological assays. The catheter pieces were coated with the fabricated nanofluid in magnetic field. The microbial adherence ability was investigated in 6 multiwell plates by using culture based methods and Scanning Electron Microscopy (SEM). The nanoparticles coated with the obtained compounds 1a–c inhibited the adherence and biofilm development ability of the S. aureus and P. aeruginosa tested strains on the catheter functionalized surface, as shown by the reduction of viable cell counts and SEM examination of the biofilm architecture. Using the novel core/shell/adsorption-shell to inhibit the microbial adherence could be of a great interest for the biomedical field, opening new directions for the design of film-coated surfaces with improved anti-biofilm properties.
doi:10.3390/ijms131012584
PMCID: PMC3497289  PMID: 23202915
benzamides; thiourea derivatives; core/shell nanostructure; magnetite; anti-biofilm; biointerface application
25.  Single-Step Production of a Recyclable Nanobiocatalyst for Organophosphate Pesticides Biodegradation Using Functionalized Bacterial Magnetosomes 
PLoS ONE  2011;6(6):e21442.
Enzymes are versatile catalysts in laboratories and on an industrial scale; improving their immobilization would be beneficial to broadening their applicability and ensuring their (re)use. Lipid-coated nano-magnets produced by magnetotactic bacteria are suitable for a universally applicable single-step method of enzyme immobilization. By genetically functionalizing the membrane surrounding these magnetite particles with a phosphohydrolase, we engineered an easy-to-purify, robust and recyclable biocatalyst to degrade ethyl-paraoxon, a commonly used pesticide. For this, we genetically fused the opd gene from Flavobacterium sp. ATCC 27551 encoding a paraoxonase to mamC, an abundant protein of the magnetosome membrane in Magnetospirillum magneticum AMB-1. The MamC protein acts as an anchor for the paraoxonase to the magnetosome surface, thus producing magnetic nanoparticles displaying phosphohydrolase activity. Magnetosomes functionalized with Opd were easily recovered from genetically modified AMB-1 cells: after cellular disruption with a French press, the magnetic nanoparticles are purified using a commercially available magnetic separation system. The catalytic properties of the immobilized Opd were measured on ethyl-paraoxon hydrolysis: they are comparable with the purified enzyme, with Km (and kcat) values of 58 µM (and 178 s−1) and 43 µM (and 314 s−1) for the immobilized and purified enzyme respectively. The Opd, a metalloenzyme requiring a zinc cofactor, is thus properly matured in AMB-1. The recycling of the functionalized magnetosomes was investigated and their catalytic activity proved to be stable over repeated use for pesticide degradation. In this study, we demonstrate the easy production of functionalized magnetic nanoparticles with suitably genetically modified magnetotactic bacteria that are efficient as a reusable nanobiocatalyst for pesticides bioremediation in contaminated effluents.
doi:10.1371/journal.pone.0021442
PMCID: PMC3125186  PMID: 21738665

Results 1-25 (1363550)