Poly‐L‐Arginine Grafted Silica Mesoporous Nanoparticles for Enhanced Cellular Uptake and their Application in DNA Delivery and Controlled Drug Release

Mesoporous silica nanoparticles (MSNs), that are capable of delivering gene and drugs to organisms in an effective and selective way have attracted much attention lately for its potential in the treatment of cancer. However, the successful application of MSNs for delivery of plasmid DNA or drugs requires surface modification of the silica with positively charged functional groups so that it binds to the negatively charged nucleic acids and also helps it penetrate through the cell membrane. We report for the first time the synthesis of a hybrid MSN where the cell penetrating cationic polypeptide poly‐L‐arginine synthesized by NCA polymerization is grafted onto the external surface of MSN using click chemistry. These poly‐L‐arginine grafted MSNs show low cytotoxity (85% cell viability at 100 μg/mL MSN concentration) and high cellular uptake by both HeLa and A549 (>90%). The poly‐L‐arginine grafted MSNs were used effectively to deliver mCherry DNA plasmid into cells leading to expression of the protein mCherry inside the cells (transfection efficiency 60%). In contrast, poly‐L‐arginine grafted non‐porous silica nanoparticles were unable to express the protein mCherry inside the cells although their uptake into the cells was as efficient as with poly‐L‐arginine grafted MSNs. We also show preliminary results to demonstrate that these hybrid MSNs can be used as a delivery vehicle for the anticancer drug Doxorubicin towards cancerous cells HeLa and A549. The biocompatibility of poly‐L‐arginine and its cell penetrating ability are expected to make these MSN conjugates very useful carriers for the delivery of genes and drugs into cancer cells.     

Subcellular impact of sonoporation on plant cells: Issues to be addressed in ultrasound-mediated gene transfer

Sonoporation (membrane perforation via ultrasonic cavitation) is known to be realizable in plant cells on a reversible basis. However, cell viability may concomitantly be affected over the process, and limited knowledge is now available on how such cytotoxic impact comes about. This work has investigated how sonoporation may affect plant cells at a subcellular level and in turn activate programmed cell death (PCD). Tobacco BY-2 cells were used as the plant model, and sonoporation was applied through a microbubble-mediated approach with 100:1 cell-to-bubble ratio, free-field peak rarefaction pressure of either 0.4 or 0.9 MPa, and 1 MHz ultrasound frequency (administered in pulsed standing-wave mode at 10% duty cycle, 1 kHz pulse repetition frequency, and 1 min duration). Fluoroscopy results showed that sonoporated tobacco cells may undergo plasma membrane depolarization and reactive oxygen species elevation (two cellular disruption events closely connected to PCD). It was also found that the mitochondria of sonoporated tobacco cells may lose their outer membrane potential over time (observed using confocal microscopy) and consequently release stores of cytochrome-c proteins (determined by Western Blotting) into the cytoplasm to activate PCD. These findings provide insight into the underlying mechanisms responsible for sonoporation-induced cytotoxicity in plant cells. They should be taken into account when using this membrane perforation approach for gene transfection applications in plant biotechnology.     

Comparison between the effects of ultrasound and γ-rays on the inactivation of Saccharomyces cerevisiae: Analyses of cell membrane permeability and DNA or RNA synthesis by flow cytometry

The effects of 200 kHz ultrasonic irradiation on DNA or RNA formation and membrane permeability of yeast cells were investigated by flow cytometry and compared with those of ⁶⁰Co γ-ray radiation. Colony counting analyses were also performed for comparison. It was observed that the colony-forming activity of yeast cells was not affected by small doses of ultrasonic irradiation, but was closely related to the amounts of sonolytically formed hydrogen peroxide at concentrations of more than 80 μM. On the other hand, γ-rays directly retarded colony-forming ability in addition to the effects of radiolytically formed hydrogen peroxide. The results obtained by flow cytometry also indicated that the amounts of DNA or RNA formed decreased with an increase in ultrasonic irradiation time without any threshold. These results indicated that flow cytometry can show early growth activities, but that colony counting analyses are insufficient to evaluate continuous and quantitative changes in these activities. In addition, by analyzing the amounts of DNA or RNA formed in the presence of the same amount of hydrogen peroxide, it was found that DNA or RNA formation behavior in the presence of hydrogen peroxide with no irradiation was similar to that following ultrasonic irradiation. These results suggested that similar chemical effects due to the formation of hydrogen peroxide were produced during ultrasonic irradiation. In addition, physical effects of ultrasound, such as shock wave, hardly contributed to cell inactivation and cell membrane damage, because relatively high frequency ultrasound was used here. In the case of γ-ray radiation, direct physical effects on the cells were clearly observed.     

Optimization and mechanism exploration for Escherichia coli transformed with plasmid pUC19 by the combination with ultrasound treatment and chemical method

As a basic technique of molecular cloning, bio-transformation has been successfully used in the fields of biomedicine and food processing. In this study, we established a transformation system of exogenous DNA into E. coli cells mediated by ultrasound. Under the optimal conditions (i.e. 35 °C, 40 W, 25 s, OD600 = 0.4–0.6) optimized by RSM, the transformation efficiency reached at 1.006 × 10⁷ CFU/μg DNA. The results of membrane permeability, macromolecular substance and cell structure analysis before and after ultrasound treatment showed that the damage of host cells induced by lower (40 W) ultrasound and shorter ultrasound time (25 s) was reversible, and the transformation efficiency and cell survival rate were not significantly affected under this condition. In brief, proper changes in cell membrane and cell wall were the basic conditions for host cells to uptake exogenous DNA, while, whether exogenous DNA could be replicated and expressed in cells depends on the viability of host cells.     

Biological effects of combined ultrasound and cisplatin treatment on ovarian carcinoma cells

The effects of low-power ultrasound, the anti-cancer drug cisplatin, and their combined application were studied in two lines of human ovarian carcinoma cells, A2780 and A2780cis. Four modes of treatment were used: exposure to ultrasonic field, application of cisplatin, exposure to ultrasound followed by cisplatin, and presence of cisplatin followed by exposure to application ultrasound. Ultrasound was used at intensities of 0.5 W/cm² and 1.0 W/cm² for 10 min, cisplatin was applied at concentrations of 1 μM and 6 μM per cell suspension treated in A2780 and cisplatin-resistant A2780cis cells, respectively. The results of each experimental treatment were assessed by the resultant cell viability related to the viability of control cells, using a standard MTT test. It was shown that a combined effect of ultrasound and cisplatin was more effective than that of ultrasound or cisplatin alone. It also appeared that the order of application played a role, with the cisplatin-ultrasound treatment lowering cell viability more than the ultrasound-cisplatin treatment. It can be assumed that the exposure of cells to a low-power ultrasonic field has an immediate effect on the structure of cell surfaces and, consequently, on entry of cisplatin into the cell.The study also included observations on changes in the cell cycle associated with the treatments used in both cell lines and their evaluation by flow cytometry.     

Lactobacillus plantarum-derived biosurfactant: Ultrasound-induced production and characterization

The aim of the present study was to investigate the effect of ultrasonic treatment (25 kHz) on biosurfactant production by Lactobacillus plantarum ATCC 8014. The impacts of the ultrasonication (with a frequency of 25 kHz and power of 7.4 W for 30 min time duration) were examined at different stages of the fermentation process to obtain the optimum stimulation instant(s). The optimum scenario was found to be one-time sonication at the 12^th hour of fermentation which can be beneficial from an economic point of view (compared with multiple applications of sonication). Ultrasonic treatment at this time resulted in enhancement of the productivities of biomass (4.5 g/L) and biosurfactant (2.01 g/L) which was almost 1.3 times higher than those of the non-sonicated control samples. According to our results, it was clearly observed that glucose consumption increased after ultrasonic treatment representing the improved substrate uptake and progression of the cellular metabolism. Furthermore, the transmission electron microscopic images immediately after sonication clarified the pore formation on the cell surfaces. The results also indicated the enhancement of plasma membrane permeability of the sonicated cells. Fourier transform infrared spectroscopy and scanning electron microscopy coupled with energy dispersive x-ray spectroscopy analyses also disclosed respectively no structural differences before and after ultrasonic exposure in the produced biosurfactant and bacterial cell membrane. The biosurfactant was characterized to be a mixture of carbohydrate (28%), protein (23%) and lipid (specified by gas chromatography-mass spectrometry) known as glycolipoprotein. The sustainable critical micelle concentration and the stability of the synthesized biosurfactant can feature its potential applicability in various processes in the food and pharmaceutical industries.     

Antibacterial mechanism of ultrasound against Escherichia coli: Alterations in membrane microstructures and properties

This study was aimed at providing new insights on the response of bacterial cell membranes to ultrasound exposure. Escherichia coli (E. coli) O157:H7 cells were exposed to different ultrasound treatments (power intensities of 64, 191, 372, and 573 W/cm², frequency of 20 kHz, pulsed mode of 2 sec: 2 sec) and the dynamic changes in cell viability within 27 min were assessed. With an increase in ultrasonic intensity and prolonged duration, a 0.76–3.52 log CFU/mL reduction in E. coli populations was attained. The alterations in the sensitivity of ultrasound-treated cells to antimicrobial compounds were evaluated by exposure to thyme essential oil nanoemulsion (TEON). The treatment reduced the E. coli population by 2.16–7.10 log CFU/mL, indicating the effects of ultrasonic field on facilitating the antibacterial efficacy of TEON. Ultrasonic-treated E. coli cells also displayed remarkable morphological and ultrastructural damages with destroyed membrane integrity and misshaped cell structures, which was observed by electron microscopy analysis. Significant increase in outer and inner membrane permeability, along with the cytoplasmic leakage and membrane depolarization were assessed utilizing spectrophotometry. For the first time, significant reduction in the membrane fluidity in response to ultrasound exposure were investigated. Additional efforts in exploring the effect of ultrasonic field on some bacterial membrane compositions were performed with infrared spectroscopy. In this study, multiple lines of evidence effectively served to elucidate the alterations on cellular membrane structure and property during exposure to sonication that could extend our understanding of the antimicrobial molecular mechanisms of ultrasound.     

Sonophotocatalytic inactivation of E. coli using ZnO nanofluids and its mechanism

The present study evaluated inactivation efficiency of a sonophotocatalytic process using ZnO nanofluids including ultrasonic parameters such as power density, frequency and time. The result showed that inactivation efficiency was increased by 20% when ultrasonic irradiation was combined with photocatalytic process in the presence of natural light. Comparison of inactivation efficiency in photocatalytic, ultrasonic and sonocatalytic processes using Escherichia coli as a model bacteria identified that inactivation efficiencies are shown in the following order: ultrasonic irradiation < sonocatalysis < photocatalysis < sonophotocatalysis. Furthermore, inactivation mechanism of sonophotocatalysis was proposed. Studies of reactive oxygen species (ROS) and zinc ions (Zn²⁺) release evaluation revealed that ROS play a key role in bacterial inactivation rather than Zn²⁺. Permeability of outer membrane (OM) and inner membrane (IM) of E. coli bacterial cells were studied and exhibited that sonophotocatalysis increased the permeability of OM and IM significantly. The enhanced bacterial inactivation effect in sonophotocatalytic process contributed to acoustic cavitation, sonocatalysis of ZnO and sonoporation phenomenon.     

Targeted microbubbles for ultrasound mediated gene transfection and apoptosis induction in ovarian cancer cells

Ultrasound-targeted microbubble destruction (UTMD) technique can be potentially used for non-viral delivery of gene therapy. Targeting wild-type p53 (wtp53) tumor suppressor gene may provide a clinically promising treatment for patients with ovarian cancer. However, UTMD mediated gene therapy typically uses non-targeted microbubbles with suboptimal gene transfection efficiency. We synthesized a targeted microbubble agent for UTMD mediated wtp53 gene therapy in ovarian cancer cells. Lipid microbubbles were conjugated with a Luteinizing Hormone–Releasing Hormone analog (LHRHa) via an avidin–biotin linkage to target the ovarian cancer A2780/DDP cells that express LHRH receptors. The microbubbles were mixed with the pEGFP-N1-wtp53 plasmid. Upon exposure to 1 MHz pulsed ultrasound beam (0.5 W/cm²) for 30 s, the wtp53 gene was transfected to the ovarian cancer cells. The transfection efficiency was (43.90 ± 6.19)%. The expression of wtp53 mRNA after transfection was (97.08 ± 12.18)%. The cell apoptosis rate after gene therapy was (39.67 ± 5.95)%. In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher transfection efficiency and higher cell apoptosis rate (p < 0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance the gene transfection efficiency in ovarian cancer cells.     

Variation of dissolved organic nitrogen concentration during the ultrasonic pretreatment to Microcystis aeruginosa

Algae cells were the main sources of dissolved organic nitrogen (DON) in raw water with plenty of algae, and ultrasonic pretreatment was one of the algae-controlling methods through the damage of algae cells. However, the variation of DON concentration during the ultrasonic treatment process was not confirmed. Variation of DON concentration during the processes of low frequency ultrasound treatment of Microcystis aeruginosa was investigated. In addition, the effect of sonication on the metabolite concentration, algae cellar activity and the subsequent coagulation performance were discussed. The results showed that after a long duration of ultrasonic (60 s), nearly 90% of the algal cells were damaged and the maximum concentration of DON attained more than 3 mg/L. In order to control the leakage extent of DON, the sonication time should be less than 30 s with power intensity of more than 1.0 W/cm³. In the mean time, ultrasonic treatment could inhibit the reactivation and the proliferation of algal, keep the algae cell wall integrity and enhance coagulation effectively under the same condition. However, ultrasound frequency had little effect on DON at the frequency range used in this study (20–150 kHz).     

The effect of ultrasonic irradiation on doxorubicin-induced cytotoxicity in three human bladder cancer cell lines

Although ultrasonic irradiation has been proven to increase membrane permeability and enhance chemotherapeutic cytotoxicity in a number of cell lines, this effect has never been demonstrated in bladder cancer cells. Bladder cancer may offer a unique setting for ultrasound enhancement of chemotherapy, since intravesicular rather than intravenous administration of chemotherapy is used in superficial cases. The aim of this study was to investigate whether a non-toxic dose of ultrasound could increase membrane permeability, and potentiate the cytotoxicity of doxorubicin to three human bladder carcinoma cell lines (TCC-SUP, T24, and RT4) in vitro. An EuTDA-Efflux assay, which measures the amount of a chemical that is allowed to seep out of labeled cells, was used to analyze membrane permeability, and an MTS assay, which directly measures cell viability, was used to determine the effect of chemotherapy on cells after they were treated with a variety of doxorubicin concentrations and ultrasonic exposures. Ultrasound treatment for 5 min and 10 min at an intensity of approximately 0.3 W/cm² resulted in a significant increase in EuTDA efflux in all three cell lines. However, no ultrasonic enhancement of doxorubicin growth inhibition in these human bladder carcinoma cells was observed. This suggests that either ultrasound does not increase doxorubicin uptake by the cell or that doxorubicin uptake is increased but in insufficient amounts to affect growth inhibition. Further investigation should focus on explaining these results.     

Garlic essential oil in water nanoemulsion prepared by high-power ultrasound: Properties,stability and its antibacterial mechanism against MRSA isolated from pork

Food-borne methicillin-resistance Staphylococcus aureus (MRSA) has caused significant health threats and economic loss in livestock and poultry products. Garlic essential oil (GEO) is an effective antibacterial agent but presents strong instability and hydrophobicity. In this study, GEO in water nanoemulsion (GEON) with good stability was produced by emulsification technique of high-power ultrasound. Its antibacterial activity and underlying mechanism against MRSA isolated from retailed pork were investigated. Results showed that ultrasonic treatment significantly reduced the particle size of GENO from 820.3 to 215.0 nm as time increased from 0 to 10 min. Comparatively, GEON of 10 min ultrasound was more stable than other GEONs (0, 1, 5 min) during 30 d storage. It also displayed good thermal stability and relatively good ion stability (NaCl, MgCl₂, and glucose). Antibacterial analysis showed that GEON (10 min) exhibited the best anti-MRSA activity among all GEONs, and the minimum inhibitory concentration of GEO in this nanoemulsion was 0.125 % (1.25 mg/mL). Treatment of GEON (10 min) significantly suppressed the cell proliferation of MRSA, which was mainly achieved by damaging the cell membrane as evidenced by membrane depolarization and considerable leakage of intracellular nucleic acids and protein. Laser scanning confocal microscope and scanning electron microscopy showed that treatment of GEON (10 min) significantly altered the membrane integrity and severely damaged the cellular membrane and structure. The present work illustrated that GEON produced by ultrasonic emulsification is a promising alternative to inhibit the contamination and spread of MRSA in livestock and poultry products.     

Stable silencing of SNAP-25 in PC12 cells by RNA interference

Background  

SNAP-25 is a synaptic protein known to be involved in exocytosis of synaptic vesicles in neurons and of large dense-core vesicles in neuroendocrine cells. Its role in exocytosis has been studied in SNAP-25 knockout mice, in lysed synaptosomes lacking functional SNAP-25 and in cells after treatment with botulinum toxins A or E that specifically cleave SNAP-25. These studies have shown that SNAP-25 appears to be required for most but not all evoked secretion. In order to further study the role of SNAP-25 in catecholamine secretion from PC12 cells we have used the recently developed technique of RNA interference to generate PC12 cell lines with virtually undetectable levels of SNAP-25. RNA interference is the sequence-specific silencing or knockdown of gene expression triggered by the introduction of double-stranded RNA into a cell. RNA interference can be elicited in mammalian cells in a number of ways, one of which is by the expression of small hairpin RNAs from a transfected plasmid. Selection of stably transfected cell lines expressing a small hairpin RNA allows one-time characterization of the degree and specificity of gene silencing and affords a continuing source of well-characterized knockdown cells for experimentation.     

Scale-up disaggregation of palygorskite crystal bundles via ultrasonic process for using as potential drilling fluid

High-efficient disaggregation of palygorskite (PAL) crystal aggregates into individual nanorods is the key to exploiting its nanometer properties, which remains a challenge at present. The sonochemical cavitation effects have been successfully employed for the intensification of physical and chemical processing applications, but it still lacks the relevant study on the scale-up disaggregation of PAL crystal bundles. Here, the energy-efficient, scale-up ultrasonic process was developed to disaggregate PAL aggregates in batches, and the effects of ultrasonic treatment time, temperature, and power on physicochemical features of PAL were systematically investigated. The results showed that the single dispersed PAL nanorods could be continuously produced by sonicating 15 wt% of PAL suspension at 20 kHz, 2000 W and 30 °C for 5 min retaining the original nanorod length and layered-chain structure. It also greatly improved the dispersion of nanorod crystal, specific surface area and suspension stability of PAL. The ultrasonically disaggregated PAL has a higher pulping rate in water (14.96 m³/t) and saturated NaCl system (14.45 m³/t), which is significantly better than that of natural PAL in water (14.72 m³/t) and saturated NaCl solution (12.37 m³/t). It suggests that the disaggregated PAL exhibits excellent potential and adaptability as a viscosity enhancer for drilling fluid. Therefore, this work provides a feasible and efficient ultrasonic process for large-scale industrialized disaggregation of PAL crystal bundles, laying a foundation for the high-value utilization of natural PAL as one-dimensional nanomaterials.     

Ultrasound-intensified laccase production from Trametes versicolor

An efficient intermittent ultrasonic treatment strategy was developed to improve laccase production from Trametes versicolor mycelia cultures. The optimized strategy consisted of exposing 2-day-old mycelia cultures to 5-min ultrasonic treatments for two times with a 12-h interval at the fixed ultrasonic power and frequency (120 W, 40 kHz). After 5 days of culture, this strategy produced the highest extracellular laccase activity of 588.9 U/L among all treatments tested which was 1.8-fold greater than the control without ultrasound treatment. The ultrasonic treatment resulted in a higher pellet porosity that facilitated the mass transfer of nutrients and metabolites from the pellets to the surrounding liquid. Furthermore, the ultrasonic treatment induced the expression of the laccase gene (lcc), which correlated with a sharp increase in both extracellular and intracellular laccase activity. This is the first study to find positive effects of ultrasound on gene expression in fungal cells. These results provide a basis for understanding the stimulation of metabolite production and process intensification by ultrasonic treatment in filamentous fungal culture.     

Ultrasonic stimulation of the brain to enhance the release of dopamine – A potential novel treatment for Parkinson’s disease

Parkinson’s disease (PD) is characterized by the decrease of dopamine (DA) production and release in the substantia nigra and striatum regions of the brain. Transcranial ultrasound has been exploited recently for neuromodulation of the brain in a number of fields. We have stimulated DA release in PC12 cells using low-intensity continuous ultrasound (0.1 W/cm² − 0.3 W/cm², 1 MHz), 12 h after exposure at 0.2 W/cm², 40 s, the amount of DA content eventually increased 78.5% (p = 0.004). After 10-day ultrasonic treatment (0.3 W/cm², 5 min/d), the DA content in the striatum of PD mice model restored to 81.07% of the control (vs 43.42% in the untreated PD mice model). In addition to this the locomotion activity was restored to the normal level after treatment. We suggest that the low intensity ultrasound-induced DA release can be attributed to a combination of neuron regeneration and improved membrane permeability produced by the mechanical force of ultrasound. Our study indicates that the application of transcranial ultrasound applied below FDA limits, could provide a candidate for relatively safe and noninvasive PD therapy through an amplification of DA levels and the stimulation of dopaminergic neuron regeneration without contrast agents.     

Transfection using hydroxyapatite nanoparticles in the inner ear via an intact round window membrane in chinchilla

Hydroxyapatite nanoparticles (nHAT) are known to have excellent biocompatibility, and have attracted increasing attention as new candidates of non-viral vectors for gene therapy. In our previous studies, nHAT carrying a therapeutic gene and a reporter gene were successfully transfected into the spiral ganglion neurons in the inner ear of guinea pigs in vivo as well as in the cultured cell lines, although the transfection efficiencies were never higher than 30%. In this study, the surface modification of nHAT with polyethylenimine (PEI) was made (PEI–nHAT, diameter = 73.09 ± 27.32 nm) and a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) gene and neurotrophin-3 (NT-3) gene was constructed as pEGFPC2–NT3. The PEI modified nHAT and the recombinant plasmid was then connected to form the nHAT-based vector–gene complex (PEI–nHAT–pEGFPC2–NT3). This complex was then placed onto the intact round window membranes of the chinchillas for inner ear transfection. Auditory brainstem response (ABR) was tested to evaluate auditory function. Green fluorescence of EGFP was observed using confocal microscopy 48 h after administering vector–gene complexes. There was no significant threshold shift in tone burst-evoked ABR at any tested frequency. Abundant, condensed green fluorescence was found in dark cells on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells, some Schwann cells in the cochlear spiral ganglion region, some outer pillar cells in the organ of Corti, and a few cells in the stria vascularis. The density of green fluorescence-marked cells was obviously higher in the vestibular dark cell area than in other areas of the inner ear, suggesting that vestibular dark cells may have the ability to actively engulf the nHAT-based vector–gene complexes. Considering the high transfection efficiency in the vestibular system, PEI–nHAT may be a potential vector for gene therapy of inner ear diseases, especially vestibular disorders, and deserves further study.     

Gene delivery process in a single animal cell after femtosecond laser microinjection

Microinjection of extracellular molecules into a single animal cell was performed by an amplified femtosecond laser irradiation. When a single-shot laser pulse was focused on the plasma membrane of a single fibroblast from the mouse cell line NIH3T3 with a high-numerical aperture objective lens, a transient hole with a diameter of 1 μm was formed. The delivery process of extracellular molecules immediately after the hole formation was monitored by a fluorescence staining with fluoresceinisothiocyanate-dextran (FITC-dextran). Then the gene expression was confirmed using a DNA plasmid of an enhanced green fluorescent protein (EGFP). The gene expression was observed when the laser pulse was focused first on the cellular membrane and then on the nuclear membrane, while the gene was not expressed when the laser was focused only on the cellular membrane. On the basis of these results, the efficiency of gene delivery by the femtosecond laser microinjection and the subsequent gene expression were clarified.     

Analysis of 4-dimethylaminopyridine (DMAP)-gold nanoparticles behaviour in solution and of their interaction with calf thymus DNA and living cells

4-(Dimethylamino)pyridine-coated gold nanoparticles (DMAP-Au NPs) were synthesized, characterised and their interaction with DNA and living cells was analysed. Concerning the interaction of the DMAP-Au NPs with DNA, absorbance titrations indicate that a non-covalent interaction between DNA and the external surface of the NPs does take place. The binding constant was evaluated to be (2.8 ± 0.8) × 10⁵ M⁻¹. Exposure of cultured cells to NPs revealed a dose-dependent effect on cell proliferation which was increased or reduced in dependence of DMAP-Au NPs concentrations. Subcellular localisation by transmission electron microscopy showed mitochondrial and nuclear localisations of NPs, thus suggesting their direct involvement in the mitochondrial alterations observed and a possible direct interaction with cell DNA. These findings clearly indicate that DMAP-Au NPs can strongly interact with living cells and confirm the importance of systematic evaluations of NPs properties, also in the perspective of their arising diagnostic and therapeutic applications.