Carboxymethyl dextran-trimethyl chitosan coated superparamagnetic iron oxide nanoparticles: An effective siRNA delivery system for HIV-1 Nef

Gene therapy, including small interfering RNA (siRNA) technology, is one of the leading strategies that help to improve the outcomes of the current therapeutic systems against HIV-1 infection. The successful therapeutic application of siRNAs requires their safe and efficient delivery to specific cells. Here, we introduce a superparamagnetic iron oxide nanoparticle (SPION) for delivering siRNA against HIV-1 nef (anti-nef siRNA) into two cell lines, HEK293 and macrophage RAW 264.7. SPIONs were coated with trimethyl chitosan (TMC), and thereafter, different concentrations of SPION–TMC were coated with different ratios of a carboxymethyl dextran (CMD) to modify the physicochemical properties and improve the biological properties of the nanocarriers. The nanoparticles exhibited a spherical shape with an average size of 112 nm. The obtained results showed that the designed delivery route enhanced the uptake of siRNA into both HEK293 and RAW 264.7 cells compared with control groups. Moreover, CMD–TMC–SPIONs containing anti-nef siRNA significantly reduced the expression of HIV-1 nef in HEK293 stable cells. The modified siRNA-loaded SPIONs also displayed no toxicity or apoptosis-inducing effects on the cells. The CMD–TMC–SPIONs are suggested as potential nanocarriers for siRNA delivery in gene therapy of HIV-1 infection.     

Alpha-Fe2O3 elicits diameter-dependent effects during exposure to an in vitro model of the human placenta

Iron oxide nanoparticles offer unique possibilities due to the change in their physico-chemical parameters when synthesized on the nanoscale (10^?9 m) compared to their bulk forms. While novel uses exist for these materials when synthesized as nanoparticles, their unintended effects on the human body and specifically during pregnancy remain ill defined. In this study, an iron oxide nanoparticle, α-Fe₂O₃, was employed and the potential toxicity due to exposure was assessed in the widely used model human placental cell line BeWo b30. These cells were grown as epithelia, and subsequently assessed for their epithelial integrity, reactive oxygen species production and cellular viability, ultrastructural and morphological disruption, and genotoxicity as a result of exposure to α-Fe₂O₃ nanoparticles. Transepithelial electrical resistance indicated that exposure to the large (50 and 78 nm), but not small (15 nm) diameter particles of α-Fe₂O₃ nanomaterial resulted in leakiness of the epithelium. Exposure to the large diameters of 50 and 78 nm resulted in increases in cell death and reactive oxygen species. Disruption of junctional integrity as monitored by immunolocalization of the tight junction protein ZO-1 was found to occur as a consequence of exposure to large diameter NPs. It was found that there was reduction in the number of microvilli responsible for increased surface area for nutrient absorption after exposing the epithelia to large diameter NPs. Finally, genotoxicity as assessed by DNA microarray and confirmed by QPCR indicated that the large diameter particles (78 nm) induce apoptosis in these cells. These data indicate that large (50 and 78 nm), but not small (15 nm) α-Fe₂O₃ nanoparticles disrupt the barrier function of this epithelium as assessed by in vitro analysis.     

A Simple and Highly Sensitive Method for Magnetic Nanoparticle Quantitation Using H-NMR Spectroscopy

Iron oxide superparamagnetic nanoparticles (SPIONs) have drawn significant attention because of their potential impact on medical diagnosis and therapy. However, the difficulty of achieving reliable and standardized quantification of these nanoparticles has limited the uniform study of nanoparticle systems. Current measurement techniques have limited sensitivity, and are sophisticated and subject to individual instrumental settings. Here, a characterization method using proton nuclear magnetic resonance (¹H-NMR) spectroscopy is presented that can quantify SPIONs regardless of surface modification. In addition to routine quantification of SPIONs during nanoparticle development, the method can also be used with in vitro nanoparticle assays and potentially with tissue samples for biodistribution studies. Specifically, measurement of water relaxivity shifts (R₁ or R₂) of dissolved SPION samples is correlated with nanoparticle concentration. Unmodified and dextran- and poly(ethylene glycol)-coated SPIONs gave linear correlations between SPION concentration and R₁ and R₂ relaxivities over five orders of magnitude, to below 10 ppb iron. Quantification of SPION concentration was also demonstrated in the presence of RAW 264.7 macrophage cells. A linear correlation between the SPION concentration and relaxivities was observed to <10 ng Fe/mL. This method is a rapid and inexpensive approach for quantitation of SPIONs and exhibits a number of advantages over many of the current methods for quantitative SPION analysis.     

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe₃O₄) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.     

Incorporation of SPION-casein core-shells into silk-fibroin nanofibers for cardiac tissue engineering

Mimicking the structure of extracellular matrix (ECM) of myocardium is necessary for fabrication of functional cardiac tissue. The superparamagnetic iron oxide nanoparticles (SPIONs, Fe₃O₄), as new generation of magnetic nanoparticles (NPs), are highly intended in biomedical studies. Here, SPION NPs (1 wt%) were synthesized and incorporated into silk-fibroin (SF) electrospun nanofibers to enhance mechanical properties and topography of the scaffolds. Then, the mouse embryonic cardiac cells (ECCs) were seeded on the scaffolds for in vitro studies. The SPION NPs were studied by scanning electron microscope (SEM), X-ray diffraction (XRD), and transmission electron microscope (TEM). SF nanofibers were characterized after incorporation of SPIONs by SEM, TEM, water contact angle measurement, and tensile test. Furthermore, cytocompatibility of scaffolds was confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. SEM images showed that ECCs attached to the scaffolds with elongated morphologies. Also, the real-time PCR and immunostaining studies approved upregulation of cardiac functional genes in ECCs seeded on the SF/SPION-casein scaffolds including GATA-4, cardiac troponin T, Nkx 2.5, and alpha-myosin heavy chain, compared with the ones in SF. In conclusion, incorporation of core-shells in SF supports cardiac differentiation, while has no negative impact on ECCs' proliferation and self-renewal capacity.     

Effect of ZnO and TiO2 nanoparticles preilluminated with UVA and UVB light on Escherichia coli and Bacillus subtilis

We evaluated the effects of zinc oxide (ZnO) and titanium dioxide (TiO₂) nanoparticles (NPs) preilluminated with ultraviolet light on Escherichia coli and Bacillus subtilis. The experiments were conducted using three different types of light: visible, Ultraviolet A (UVA, 315–400 nm), and Ultraviolet B (UVB, 280–315 nm). The bacteria were exposed to NPs, either as liquid suspensions for growth inhibition assays or on agar plates for colony forming unit (CFU) assays. We found that the ZnO NPs were more toxic when preilluminated with UVA or UVB light than with visible light in both growth inhibition and CFU assays. TiO₂ NPs were not toxic to the bacteria under UVA or UVB preillumination conditions. The photo-dissolution of ZnO NPs increased with UV preillumination, which could explain the observed toxicity of ZnO NPs. We detected oxidative stress elicited by photoactive nanoparticles by measuring superoxide dismutase activity. The results of this study show that the toxicity of photoactive nanoparticles can be increased by UV preillumination by dissolution of toxic ions, which suggests the potential for preillumination-dependent toxicity of nanoparticles on soil environments in low light or darkness.     

Preparation of superparamagnetic iron oxide/doxorubicin loaded chitosan nanoparticles as a promising glioblastoma theranostic tool

Theranostic nanoparticles (NPs) are promising for opening new windows toward personalized disease management. Using a single particle capable of both diagnosis and drug delivery, is the major benefit of such particles. In the present study, chitosan NPs were used as a dual action carrier for doxorubicin (DOX; chemotherapeutic agent) and superparamagnetic iron oxide nanoparticles (SPIONs; imaging agent). SPIONs and DOX were loaded at different concentrations within poly-l -arginine-chitosan-triphosphate matrix (ACSD) using the ionic gelation method. NPs’ size were in the range of 184.33 ± 4.4 nm. Drug release analysis of DOX loaded NPs (NP-DOX) showed burst release at pH 5.5 (as in tumor environment) and slow release at pH 7.4 (physiological condition), demonstrating pH-sensitive drug release profile. NP-DOX internalization was confirmed by flowcytometry and fluorescent microscopy. Uptake process results were corroborated by accumulation of drug in the intracellular space. Iron content was evaluated by inductively coupled plasma and prussian blue staining. In vitro magnetic resonance imaging (MRI) showed a decline in T ₂ relaxation times by increasing iron concentration. MRI analysis also confirmed uptake of NPs at the optimum concentration in C6 glioma cells. In conclusion, ACSD NPs could be utilized as a promising theranostic formulation for both diagnosis and treatment of glioblastoma.     

Relating the Surface Properties of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) to Their Bactericidal Effect towards a Biofilm of Streptococcus mutans

This study was designed to determine the effects of superparamagnetic iron oxide nanoparticles (SPIONs) on the biological activity of a bacterial biofilm (Streptococcus mutans). Our hypothesis was that the diffusion of the SPIONs into biofilms would depend on their surface properties, which in turn would largely be determined by their surface functionality. Bare, positively charged and negatively charged SPIONs, with hydrodynamic diameters of 14.6 ± 1.4 nm, 20.4 ± 1.3 nm and 21.2 ± 1.6 nm were evaluated. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) and electrophoretic mobility (EPM) measurements were used to confirm that carboxylic functional groups predominated on the negatively charged SPIONS, whereas amine functional groups predominated on the positively charged particles. Transmission electron microscopy (TEM) showed the morphology and sizes of SPIONs. Scanning electron microscopy (SEM) and EPM measurements indicated that the surfaces of the SPIONs were covered with biomolecules following their incubation with the biofilm. Bare SPIONs killed bacteria less than the positively charged SPIONs at the highest exposure concentrations, but the toxicity of the bare and positively charged SPIONs was the same for lower SPION concentrations. The positively charged SPIONs were more effective in killing bacteria than the negatively charged ones. Nonetheless, electrophoretic mobilities of all three SPIONs (negative, bare and positively charged) became more negative following incubation with the (negatively-charged) biofilm. Therefore, while the surface charge of SPIONS was important in determining their biological activity, the initial surface charge was not constant in the presence of the biofilm, leading eventually to SPIONS with fairly similar surface charges in situ. The study nonetheless suggests that the surface characteristics of the SPIONS is an important parameter controlling the efficiency of antimicrobial agents. The analysis of the CFU/mL values shows that the SPIONs have the same toxicity on bacteria in solution in comparison with that on the biofilm.     

Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI

Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs.     

Circulation and distribution of gold nanoparticles and induced alterations of tissue morphology at intravenous particle delivery

Kinetics, biodistribution, and histological studies were performed to evaluate the particle‐size effects on the distribution of 15 nm and 50 nm PEG‐coated colloidal gold (CG) particles and 160 nm silica/gold nanoshells (NSs) in rats and rabbits. The above nanoparticles (NPs) were used as a model because of their importance for current biomedical applications such as photothermal therapy, optical coherence tomography, and resonance‐scattering imaging. The dynamics of NPs circulation in vivo was evaluated after intravenous administration of 15 nm CG NPs to rabbit, and the maximal concentrations of gold were observed 15–30 min after injection. Rats were injected in the tail vein with PEG‐coated NPs (about 0.3 mg Au/kg rats). 24 h after injection, the accumulation of gold in different organs and blood was determined by atomic absorption spectroscopy. In accordance with the published reports, we observed 15 nm particles in all organs with rather smooth distribution over liver, spleen and blood. By contrast, the larger NSs were accumulated mainly in the liver and spleen. For rabbits, the biodistribution was similar (72 h after intravenous injection). We report also preliminary data on the light microscopy and TEM histological examination that allows evaluation of the changes in biotissues after gold NPs treatment. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The influence of H2 partial pressure on biogenic palladium nanoparticle production assessed by single-cell ICP-mass spectrometry

The production of biogenic palladium nanoparticles (bio-Pd NPs) is widely studied due to their high catalytic activity, which depends on the size of nanoparticles (NPs). Smaller NPs (here defined as <100 nm) are more efficient due to their higher surface/volume ratio. In this work, inductively coupled plasma-mass spectrometry (ICP-MS), flow cytometry (FCM) and transmission electron microscopy (TEM) were combined to obtain insight into the formation of these bio-Pd NPs. The precipitation of bio-Pd NPs was evaluated on a cell-per-cell basis using single-cell ICP-MS (SC-ICP-MS) combined with TEM images to assess how homogenously the particles were distributed over the cells. The results provided by SC-ICP-MS were consistent with those provided by “bulk” ICP-MS analysis and FCM. It was observed that heterogeneity in the distribution of palladium over an entire cell population is strongly dependent on the Pd²⁺ concentration, biomass and partial H₂ pressure. The latter three parameters affected the particle size, ranging from 15.6 to 560 nm, and exerted a significant impact on the production of the bio-Pd NPs. The TEM combined with SC-ICP-MS revealed that the mass distribution for bacteria with high Pd content (144 fg Pd cell⁻¹) indicated the presence of a large number of very small NPs (D50 = 15.6 nm). These results were obtained at high cell density (1 × 10⁵ ± 3 × 10⁴ cells μl⁻¹) and H₂ partial pressure (180 ml H₂). In contrast, very large particles (D50 = 560 nm) were observed at low cell density (3 × 10⁴ ± 10 × 10² cells μl⁻¹) and H₂ partial pressure (10–100 ml H₂). The influence of the H₂ partial pressure on the nanoparticle size and the possibility of size-tuned nanoparticles are presented.     

PEG-PLA nanoparticles decorated with small-molecule PSMA ligand for targeted delivery of galbanic acid and docetaxel to prostate cancer cells

Prostate cancer (PCa) is one of the most prevalent non-drug delivery system cutaneous malignancies. Undoubtedly, introducing novel treatment options to achieve higher therapeutic index will be worthwhile. In this study, we report for the first time, a novel targeted self-assembled based on PEG-PLA nanoparticles (PEG-PLA NPs) containing galbanic acid (GBA) and docetaxel, which was targeted using ((S)-2-(3-((S)-5-amino-1-carboxypentyl) ureido) pentanedioic acid (ACUPA), a small molecule inhibitor targeting prostate-specific membrane antigen (PSMA), in prostate cancer cell line. The prepared NPs were characterized by different analytical methods. The MTT assay was used to compare the anti-proliferation of drugs-loaded PEG-PLA NPs and ACUPA-PEG-PLA against LNCaP (PSMA⁺) and PC3 (PSMA⁻) cells. PEG-PLA NPs with an average size of 130–140 nm had an enhanced release of GBA and docetaxel at pH 5.5 compared with pH 7.5. Spectrofluorometric analysis suggested that ACUPA-modified PEG-PLA could effectively enhance the drug uptake in PSMA⁺ prostate cancer cells. Cytotoxicity studies showed that the targeted NPs loaded with different concentrations of GBA and fixed concentration of docetaxel (4 nM) have shown higher toxicity (IC₅₀ 30 ± 3 µM) than both free GBA (80 ± 4.5 µM) and nontargeted NPs (IC₅₀ 40 ± 4.6 µM) in LNCaP cells. Collectively, these findings suggest that ACUPA-conjugated PEG-PLA nanosystem containing GBA and docetaxel is a viable delivery carrier for various cancer-targeting PSMA that suffer from short circulation half-life and limited therapeutic efficacy.     

Assessment of UVB-photoprotective and antioxidative activities of carrageenan in keratinocytes

Ultraviolet B (UVB) (290–320 nm) is the foremost cause of photoaging, sunburn, wrinkles and skin cancer. Photoprotection against harmful UVB radiation is essential through various means including the use of skincare products. The seaweed polysaccharide carrageenan is widely used as an excipient in cosmetics and skincare products. However, its effects on normal skin keratinocytes or potential use as a photoprotective agent have yet to be established. The primary aim of this study was to assess the cytotoxic, photoprotective and antioxdative effects of carrageenan in UVB-induced immortalised normal human keratinocytes (HaCaT cells). Results showed that the percentage of cell viability decreased linearly with increasing UVB doses from 10, 50, 100, 222 to 1,000 mJ cm^?2. Four isomers of carrageenan, namely iota 2 [ι (??)], iota 5 [ι (V)], lambda (λ) and kappa (κ) carrageenan were used in this study. Vitamin E was used as a positive control. In terms of cytotoxicity, the CD₅₀ of kappa carrageenan was ~200 μg mL^?1 while for the other isomers, the values ranged from 122 to 162 μg mL^?1. Carrageenan showed significant protection against detrimental effects of UVB-induced cell killing and reactive oxygen species (ROS) release based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) assays, respectively. Carrageenan was also able to quench 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. The ability to protect against UVB suggests that carrageenan has potential application as a photoprotective agent in addition to just being used as an excipient.     

Cytotoxicity and Genotoxicity in Human Embryonic Kidney Cells Exposed to Surface Modify Chitosan Nanoparticles Loaded with Curcumin

The rapid progress in the development and scientific investments of modified nanoparticles are due to their owed activity to various diseased conditions for which they are prepared. But the toxicity which they cause cannot be overlooked. The present study demonstrates the development of phosphatidylserine (PS)-coated chitosan (CS) nanoparticles (NPs) loaded with curcumin (CU), which was then investigated against human embryonic kidney cells (HEK 293) for its cytotoxic and genotoxic effect in rats. The CU-loaded CNPs (CNPs-CU) have been prepared by ionic gelation method, later which were grafted with PS. CNPs-CU and PS-CNPs-CU have been evaluated for their size, poly dispersity index, amount of drug entrapped, and in vitro CU release. CNPs-CU has an average size 167.6?±?3.53 nm and polydispersity index (PDI) 0.115?±?0.014, whereas PS-CNPs-CU shows average size 220?±?3.67 nm and PDI 0.148?±?0.019. Surface morphology of prepared NPs was confirmed by high-resolution transmission electron microscopy (HR-TEM). There was no major difference in cell viability between PS-CNPs-CU and CNPs-CU when they were exposed to HEK 293 cells at all equivalent concentrations. A series of genotoxic studies were conducted, which revealed the non-genotoxicity potential of the developed complexes. These results demonstrated that PS-CNPs-CU may be useful as potential delivery system.     

Inactivation of Pseudomonas aeruginosa PA01 biofilms by hyperthermia using superparamagnetic nanoparticles

The primary goal of this study was to develop a new strategy to inactivate bacterial biofilms using the thermal stress derived from superparamagnetic iron oxide nanoparticles (SPIONs) in an alternating current (AC) magnetic field. A large number of studies have examined the inactivation of bacterial biofilms using antimicrobial agents; however, there have been no attempts to inactivate biofilms by hyperthermia using SPIONs. In this study, a SPION solution was added to Pseudomonas aeruginosa (P. aeruginosa) PA01 biofilm, and heat was generated by placing the nanoparticle-containing biofilm in an AC magnetic field. The heating temperature was dependent on the concentration of the added SPION solution. More than 4 log inactivation of the PA01 biofilm was obtained using a 60 mg mL⁻¹ SPION solution in 8 min, and this resulted in a dramatic disintegration of the bacterial cell membrane in the biofilm. This inactivation was largely due to the thermal effect. Local heating of a specific area is also possible using this method, and the heating temperature can be easily adjusted by controlling the concentration of the SPION solution. Therefore, hyperthermia using magnetic nanoparticles holds promise as an effective tool for inactivating the bacterial biofilm.     

Effect of Zinc Oxide Nanoparticles on the Function of MC3T3-E1 Osteoblastic Cells

Zinc oxide nanoparticles (ZnO NPs) can be ingested directly when used in food, food packaging, drug delivery, and cosmetics. This study evaluated the cellular effects of ZnO NPs (50 and 100 nm diameter particle sizes) on the function of osteoblastic MC3T3-E1 cells. ZnO NPs showed cytotoxicity at concentrations of above 50 μg/ml, and there was no significant effect of the size on the cytotoxicity of ZnO NPs. Within the testing concentrations of 0.01～1 μg/ml, which did not cause a marked drop in cell viability, ZnO NPs (0.1 μg/ml) caused a significant elevation of alkaline phosphatase activity, collagen synthesis, mineralization, and osteocalcin content in the cells (P?<?0.05). Moreover, pretreatment with ZnO NPs (0.01～1 μg/ml) significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, and ATP loss. Measurement of reactive oxygen species (ROS) indicated decrease in ROS level upon exposure to ZnO nanoparticles (0.01 μg/ml). Hence, our study indicated that ZnO nanoparticles can have protective effects on osteoblasts at low concentrations where there are little or no observable cytotoxic effects.     

Cytotoxicity evaluation of silica nanoparticles using fish cell lines

Nanoparticles (NPs) have extensive industrial, biotechnological, and biomedical/pharmaceutical applications, leading to concerns over health risks to humans and biota. Among various types of nanoparticles, silica nanoparticles (SiO₂ NPs) have become popular as nanostructuring, drug delivery, and optical imaging agents. SiO₂ NPs are highly stable and could bioaccumulate in the environment. Although toxicity studies of SiO₂ NPs to human and mammalian cells have been reported, their effects on aquatic biota, especially fish, have not been significantly studied. Twelve adherent fish cell lines derived from six species (rainbow trout, fathead minnow, zebrafish, goldfish, haddock, and American eel) were used to comparatively evaluate viability of cells by measuring metabolic impairment using Alamar Blue. Toxicity of SiO₂ NPs appeared to be size-, time-, temperature-, and dose-dependent as well as tissue-specific. However, dosages greater than 100 μg/mL were needed to achieve 24 h EC₅₀ values (effective concentrations needed to reduce cell viability by 50%). Smaller SiO₂ NPs (16 nm) were relatively more toxic than larger sized ones (24 and 44 nm) and external lining epithelial tissue (skin, gills)-derived cells were more sensitive than cells derived from internal tissues (liver, brain, intestine, gonads) or embryos. Higher EC₅₀ values were achieved when toxicity assessment was performed at higher incubation temperatures. These findings are in overall agreement with similar human and mouse cell studies reported to date. Thus, fish cell lines could be valuable for screening emerging contaminants in aquatic environments including NPs through rapid high-throughput cytotoxicity bioassays.     

Glutaric acid as a spacer facilitates improved intracellular uptake of LHRH-SPION into human breast cancer cells

Superparamagnetic iron oxide nanoparticles (SPIONs) bound directly to luteinizing hormone releasing hormone (LHRH) have shown high efficiency for intracellular uptake to breast cancer cells, MDA-MB-435S.luc. We demonstrate in this communication that inclusion of a small spacer molecule such as glutaric acid (Glu) in between SPION and LHRH increases further receptor mediated intracellular uptake. LHRH-bound SPIONs with and without the spacer molecule were nontoxic.     

Transfer of Sensitivity to Tumor Promoters by Transfection of DNA from Sensitive into Insensitive Mouse JB6 Epidermal Cells

Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P⁺) and nonpromotable (P⁻) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12-o-tetradecanoyl-phorbol-13-acetate (TPA). Cellular DNA isolated from one of two P⁺ lines, JB6 Cl 41 or JB6 Cl 22, was CaPO₄ precipitated and used to transfect the P⁻ cell line JB6 Cl 30. At 7 days posttransfection, the cells were suspended in agar with or without TPA at 1.6 × 10⁻⁸ M and assayed 10 days later for TPA-dependent colony formation. Untreated or Cl 30 DNA-treated P⁻ JB6 Cl 30 cells yielded 40 to 50 colonies per 10⁵ cells. In contrast, transfection of Cl 30 cells with “P⁺ DNA” derived from either Cl 41 or Cl 22 yielded 200 to 500 TPA-induced colonies per 10⁵ cells, or a five- to eightfold enhancement of promotability. The enhanced promotability obtained after transfection with P⁺ DNA was stable, as judged by the retention of promotability for at least eight passages in cell lines derived from TPA-induced agar colonies. Other transfectants showed irreversible transformation by TPA, as observed in the parental P⁺ lines. When NIH 3T3 cells instead of the putative preneoplastic JB6 Cl 30 cells were used as recipients for transfection of P⁺ DNA, no evidence for acquisition of promotability was obtained. P⁻ JB6 Cl 25, like Cl 30, also permitted expression of transfected P⁺ DNA. These results suggest that sensitivity to phorbol ester promotion of transformation in JB6 cells is determined by DNA sequence(s) present in the P⁺ DNA and requires recipient cells of the appropriate phenotype for expression.     

A new chemical complex can rapidly concentrate lentivirus and significantly enhance gene transduction

In this study, we developed a new purification method using chondroitin sulfate C (CSC) and protamine sulfate (PS) to concentrate lentivirus. To evaluate the efficiency of this new method, we compared it with several previously described purification protocols, including virus concentrated by ultracentrifugation (Ultra), precipitated by polyethylene glycol (PEG), and sedimented by CSC combined with polybrene (PB). After using the different methods to purify and concentrate equivalent amounts of lentivirus supernatant, the virus pellets precipitated by the different methods were resuspended using the equivalent volumes of DMEM. Subsequently, 10 μl of each lentivirus stock carrying EGFP gene was used to transduce two types of cells, human embryonic kidney 293T (HEK293T) cells and mouse mesenchymal stem cells (mMSC). It was obvious that HEK293T and mMSC appeared much intensiver green fluorescence through virus transduction from PS method than from other methods. To quantitate the transduction efficiency of the viruses, we examined virus titer in the cells after transduction using a real-time PCR-based analysis. Accordingly, we verified that PS precipitation could generate virus with a higher titer (4.39 × 10⁸ IU/ml) than PB (2.43 × 10⁸ IU/ml), Ultra (1.16 × 10⁸ IU/ml), and PEG (0.56 × 10⁸ IU/ml) in HEK293T cells. As for HEK293T cells in mMSC, the PS method also generated virus with a higher titer (4.66 × 10⁸ IU/ml) than the Ultra method (2.36 × 10⁸ IU/ml), and a much higher titer than those of the other chemical-based precipitation methods using PB (4.82 × 10⁶ IU/ml) and PEG (8.98 × 10⁴ IU/ml). Furthermore, the HEK293T cells and mMSC transduced by PS(1X)-virus appeared to have higher cell growth ratios, respectively, than the HEK293T cells and mMSC transduced by lentivirus using the other methods. We conclude that our new method for purifying lentivirus is cost-effective, time-saving, and highly efficient, and that lentivirus purification by this means could possibly be used to transduce a variety of cells, including stem cells.