Quantitative assessment of the comparative nanoparticle-uptake efficiency of a range of cell lines

The mechanism(s) of nanoparticle-cell interactions are still not understood. At present there is little knowledge of the relevant length- and timescales for nanoparticle intracellular entry and localization within cells, or the cell-specificity of nanoparticle uptake and localisation. Here, the effect of particle size on the in-vitro intracellular uptake of model fluorescent carboxyl-modified polystyrene nanoparticles is investigated in various cell lines. A range of micro- and nanoparticles of defined sizes (40 nm to 2 μm) are incubated with a series of cell types, including HeLa and A549 epithelial cells, 1321N1 astrocytes, HCMEC D3 endothelial cells, and murine RAW 264.7 macrophages. Techniques such as confocal microscopy and flow cytometry are used to study particle uptake and subcellular localisation, making significant efforts to ensure reproducibility in a semiquantitative approach. The results indicate that internalization of (nano)particles is highly size-dependent for all cell lines studied, and the kinetics of uptake for the same type of nanoparticle varies in the different cell types. Interestingly, even cells not specialized for phagocytosis are able to internalize the larger nanoparticles. Intracellular uptake of all sizes of particles is observed to be highest in RAW 264.7 cells (a specialized phagocytic cell line) and the lowest in the HeLa cells. These results suggest that (nano)particle uptake might not follow commonly defined size limits for uptake processes, and highlight the variability of uptake kinetics for the same material in different cell types. These conclusions have important implications for the assessment of the safety of nanomaterials and for the potential biomedical applications of nanoparticles.     

Mammalian Cells Exhibit a Range of Sensitivities to Silver Nanoparticles that are Partially Explicable by Variations in Antioxidant Defense and Metallothionein Expression

While it is well known that there are interspecies differences in Ag sensitivity, differences in the cytotoxic responses of mammalian cells to silver nanoparticles (Ag NPs) are also observed. In order to explore these response outcomes, six cell lines, including epithelial cells (Caco‐2, NHBE, RLE‐6TN, and BEAS‐2B) and macrophages (RAW 264.7 and THP‐1) of human and rodent origin, are exposed to 20 nm citrate‐ and PVP‐coated Ag NPs with Au cores, as well as 20 nm citrate‐coated particles without cores. An MTS assay shows that while Caco‐2 and NHBE cells are resistant to particles over a 0.1–50 μg mL^?1 dose range, RAW 264.7, THP‐1, RLE‐6TN, and BEAS‐2B cells are more susceptible. While there are small differences in dissolution rates, there are no major differences in the cytotoxic potential of the different particles. However, differences in anti‐oxidant defense and metallothionein expression among different cell types are observed, which can partially explain differential Ag NP sensitivity. So, it is important to consider these differences in understanding the potential heterogeneous effects of nano Ag on mammalian biological systems.     

Immobilized-OPG-Fc on a titanium surface inhibits RANKL-dependent osteoclast differentiation in vitro

The purpose of the present study was to examine the effect of osteoprotegerin (OPG)-Fc fusion protein immobilized on a titanium surface on the initial differentiation of osteoclast precursor RAW264.7 cells. These cells were cultured on titanium specimens over which OPG-Fc was immobilized. The enhancement of tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expression in RAW264.7 cells exposed to receptor activator of NF-κB ligand (RANKL) stimulation on OPG-Fc-coated titanium was significantly lower than that in RAW264.7 cells exposed to RANKL on titanium specimens without immobilized OPG-Fc (ANOVA, P < 0.01). Preincubation of OPG-Fc-coated titanium, in a medium supplemented with 10% fetal bovine serum at 37°C for two days before the cells were seeded, had no significant effect on the decrease in mRNA expression (ANOVA, P < 0.01). Taken together, these results indicate that OPG-Fc immobilized on a titanium surface blocks the differentiation of RAW264.7 cells induced by RANKL stimulation.     

Antibiofilm,anti cancer and ecotoxicity properties of collagen based ZnO nanoparticles

The use of natural biopolymers in the synthesis of nanomaterials can have a low cost and eco-friendly approach. ZnO nanoparticles synthesized through biological method has been reported to have biomedical applications to control pathogenic microbes as it is cost effective compared to commonly used physical and chemical methods. In this work we would like to report the “bioinspired” synthesis of ZnO nanopowders (ZnO-NPs) using type 1 collagen. Collagen based ZnO NPs (Cl-ZnO NPs) were bio-physically characterized by UV–vis Spectroscopy, XRD, FTIR, HR-TEM, EDX and Zeta potential analysis. HR-TEM recorded the presence of hexagonal wurtzite structure of Cl-ZnO NPs with particle size ranged between 20–50?nm. Further, Cl-ZnO NPs exhibited antibacterial and antibiofilm activity against Gram positive Streptococcus mutans, Gram negative Proteus vulgaris and fungi Candida albicans at 75?μg/ml. Moreover, the cytotoxicity assay demonstrated that the Cl-ZnO NPs was not toxic to murine (RAW 264.7) macrophage cells up to 75?μg/ml. However, it exhibited cytotoxicity against human liver cancer (HepG2) cells at 75?μg/ml. The HepG2 cell viability was significantly reduced at 75?μg/ml. In addition, the ecotoxicity of Cl-ZnO NPs on the freshwater micro crustacean Daphnia longicephala showed no mortality up to 250?μg/ml. The current study clearly demonstrated that the Cl-ZnO NPs had greater potential for antimicrobial and anticancer activities.     

Investigation of polymeric amphiphilic nanoparticles as antitumor drug carriers

In this paper, polymeric amphiphilic nanoparticles based on oleoyl–chitosan (OCH) with different degrees of substitution (DS, 5%, 11% and 27%) were prepared by Oil/Water emulsification method. Mean diameters of the nanoparticles were 327.4 nm, 255.3 nm and 192.6 nm, respectively. Doxorubicin (DOX) was efficiently loaded into OCH nanoparticles and provided a sustained released after a burst release in PBS. These nanoparticles showed no cytotoxicity to mouse embryo fibroblasts (MEF) and low hemolysis rates (<5%). The results of SDS-PAGE indicated that bovine calf serum (BCS) adsorption on OCH nanoparticles was inhibited by smaller particle size. Cellular uptake was evaluated by incubating fluorescence labeled OCH nanoparticles with human lung carcinoma cells (A549) and mouse macrophages (RAW264.7). Cellular uptake of OCH nanoparticles was time––and concentration––dependent. Finding the appropriate incubation time and concentration of OCH nanoparticles used as drug carriers might decrease phagocytic uptake, increase cancer cell uptake and ultimately improve therapeutic efficiency of antitumor therapeutic agents.     

Small-sized carbon nanohorns enabling cellular uptake control

Carbon nanotubes perform well in preclinical tests for drug delivery and diagnostic imaging, but controlling the size at less than 100 nm to avoid nonspecific uptake by reticuloendothelial systems while targeting delivery to cells of interest via receptor-mediated endocytosis is difficult, which currently limits their widespread use. Herein, 20-50-nm graphene tubules, small-sized single-walled carbon nanohorns (S-SWNHs), are obtained with a yield of 20% or higher by an oxidative exfoliation of 100 nm pristine SWNH aggregates. S-SWNHs are highly hydrophilic and remarkably resistant to cellular uptake by macrophages (RAW 264.7 cells), tumor cells (HeLa or KB), or normal cells (FHs 173We). The nonstimulatory property to cell membranes therefore makes cellular uptake control of S-SWNHs by functionalization easy. By attaching phospholipid polyethylene glycol, the cellular internalization of S-SWNHs is almost completely inhibited in RAW 264.7 macrophages. When functionalized with tumor-targeting folic acid (FA), FA-S-SWNHs are taken up by FA receptor-overexpressing KB cells but not by normal human embryonic cells (FHs 173We), which do not express the FA receptor. With a high rate of stealth and targeting in vitro, S-SWNHs are one of the most promising nanoparticles for medical use.     

Effect of the surface texture and crystallinity of ZnO nanoparticles on their toxicity

We have investigated the correlation between the structural properties of ZnO nanoparticles (NPs) and their toxicity to mesenchymal stem cells (C2C12 cell line) and macrophage-derived cells (RAW 264.7 cell line). Nanopowders of grain size ranging between 5 nm and 50 nm were prepared by chemical route. Their structural properties were characterized extensively by X-ray Diffraction (XRD) and High Resolution Transmission Electron Microscopy (HRTEM). The XRD spectra showed that 50 nm sized NPs are well crystallized and present a preferential orientation along the direction normal to the (001) plane while the HREM observations revealed that most of the large size (50 nm) crystallized nanoparticles have polygonal shape which is consistent with a texture of along [001] direction. The toxicity tests showed that [001] large textured NPs have higher toxicity to inflammatory cells than nanoparticles of low crystallinity and much smaller size (5 nm). In addition, NPs have cytotoxic effects on inflammatory cells at concentration as low as 0.05 mM while ten times higher concentrations did not have significant cytotoxic effects on cells representing mesenchymal tissues. These observations are explained by the enhanced generation of Reactive Oxygen Species (ROS) at the (0001) polar surface of ZnO NP. These results provide a direct evidence of the correlation between the toxicity and the surface texture of the oxide nanoparticles. Similar correlation has been reported for the photocatalytic properties of ZnO nanoparticles.     

Multi-walled carbon nanotubes induce apoptosis in RAW 264.7 cell-derived osteoclasts through mitochondria-mediated death pathway

Carbon nanotubes (CNTs) have attracted great interest with respect to biomaterials, particularly for use as an implant material in bone-tissue engineering. Accordingly, the bone-tissue compatibility of CNTs and their influence on new bone formation are important issues. In the present study, we examined the effects of multi-wall carbon nanotubes (MWCNTs) on the receptor activator of nuclear factor kappaB ligand (RANKL)-supported osteoclastogenesis using a murine monocytic cell line RAW 264.7. MWCNTs significantly suppressed the differentiation of RAW 264.7 cells into osteoclasts. Treatment with MWCNTs induced apoptosis in osteoclasts as characterized by nuclear condensation, DNA fragmentation, caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, but did not decrease the cell viability of the osteoblast-like cell line MC3T3-E1. MWCNTs also induced loss of the mitochondrial membrane potential (deltapsim) by regulating expression of Bcl-2 family proteins and caused release of cytochrome c from mitochondria to cytosol. MWCNTs-induced apoptosis in osteoclasts was inhibited both by cyclosporin A, a blocker of the mitochondrial permeability transition pore, and by DEVD-CHO, a cell-permeable inhibitor of caspase-3. The present study suggests that MWCNTs suppresse osteoclastogenesis via the inhibition of osteoclast differentiation and the induction of apoptosis in osteoclasts, rendering them promising candidate for the treatment of osteoclast-related diseases.     

Pure titanium particle loaded nanocomposites: study on the polymer/filler interface and hMSC biocompatibility

The integration of inorganic nanoparticles into polymer matrices allows for the modification of physical properties as well as the implementation of new features for unexplored application fields. Here, we propose the study of a new metal/polymer nanocomposite fabricated by dispersing pure Ti nanoparticles into a poly(methylmetacrilate) matrix via solvent casting process, to investigate its potential use as new biomaterial for biomedical applications. We demonstrated that Ti nanoparticles embedded in the poly(methylmetacrilate) matrix can act as reinforcing agent, not negatively influencing the biological response of human mesenchymal stem cell in terms of cytotoxicity and cell viability. As a function of relative amount and surface treatment, Ti nanoparticles may enhance mechanical strength of the composite—ranging from 31.1?±?2.5 to 43.7?±?0.7?MPa—also contributing to biological response in terms of adhesion and proliferation mechanisms. In particular, for 1?wt% Ti, treated Ti nanoparticles improve cell materials recognition, as confirmed by higher cell spreading-quantified in terms of cell area via image analysis—locally promoting stronger interactions at cell matrix interface. At this stage, these preliminary results suggest a promising use of pure Ti nanoparticles as filler in polymer composites for biomedical applications.     

Comparison of cytotoxicity of pristine and covalently functionalized multi-walled carbon nanotubes in RAW 264.7 macrophages

Carbon nanotubes may be applied in different fields including biomedicine and mechanical engineering. It is important to understand the potential hazards of carbon nanotubes. In the present study, the toxicological effects of the pristine multi-walled carbon nanotubes (p-MWCNTs) and taurine functionalized multi-walled carbon nanotubes (tau-MWCNTs) were assessed on RAW 264.7 macrophages. We tested cell viability, GSH/GSSG ratio, apoptosis, intracellular calcium concentration, ultrastructural changes of cell morphology, and the release of IL-8. We observed the loss of cell viability, decline in the cellular GSH/GSSG ratio, increase of IL-8, and the increase of intracellular calcium concentration in RAW 264.7 macrophages when exposed to p-MWCNTs at high dosage. Additionally, exposure to p-MWCNTs resulted in ultrastructural and morphological changes in RAW 264.7 macrophages. In contrast, the RAW 264.7 macrophages exposed to the tau-MWCNTs did not exhibit altered morphology. Our results conclude that the tau-MWCNTs show lower toxicity than that of p-MWCNTs.     

Differential internalization of superparamagnetic iron oxide nanoparticles in different types of cells

Superparamagnetic iron oxide nanoparticles (SPION) have attracted great attention for nanomedical applications, but the mechanisms underlying the transmembrane transport of SPION in variant cells has not been fully defined. The present study investigated the internalization of SPION in three cell models with different phagocytic capacity using transmission electron microscopy (TEM) and energy dispersive spectrometer (EDS) analyses. The EDS study aimed to further confirm if the suspected internalized particles were iron-containing SPION. SPION could be taken up quickly by macrophage-like cell line RAW264.7 (with strong phagocytic capacity) and slowly by the 3T3-L1 cells (with weak phagocytic capacity), but not by red blood cells (with no phagocytic capacity). The internalized SPION were mainly found in the cytoplasmic vesicles, with no localization in the endoplasmic reticulum, mitochondria and nucleus. We conclude that the internalization of SPION in the three types of mammalian cells was mediated by phagocytosis, not by direct membrane penetration.     

Nanoparticle-mediated cellular response is size-dependent

Nanostructures of different sizes, shapes and material properties have many applications in biomedical imaging, clinical diagnostics and therapeutics. In spite of what has been achieved so far, a complete understanding of how cells interact with nanostructures of well-defined sizes, at the molecular level, remains poorly understood. Here we show that gold and silver nanoparticles coated with antibodies can regulate the process of membrane receptor internalization. The binding and activation of membrane receptors and subsequent protein expression strongly depend on nanoparticle size. Although all nanoparticles within the 2-100 nm size range were found to alter signalling processes essential for basic cell functions (including cell death), 40- and 50-nm nanoparticles demonstrated the greatest effect. These results show that nanoparticles should no longer be viewed as simple carriers for biomedical applications, but can also play an active role in mediating biological effects. The findings presented here may assist in the design of nanoscale delivery and therapeutic systems and provide insights into nanotoxicity.     

A superhydrophilic titanium implant functionalized by ozone gas modulates bone marrow cell and macrophage responses

Bone-forming cells and M? play key roles in bone tissue repair. In this study, we prepared a superhydrophilic titanium implant functionalized by ozone gas to modulate osteoconductivity and inhibit inflammatory response towards titanium implants. After 24 h of ozone gas treatment, the water contact angle of the titanium surface became zero. XPS analysis revealed that hydroxyl groups were greatly increased, but carbon contaminants were largely decreased 24 h after ozone gas functionalization. Also, ozone gas functionalization did not alter titanium surface topography. Superhydrophilic titanium (O₃–Ti) largely increased the aspect ratio, size and perimeter of cells when compared with untreated titanium (unTi). In addition, O₃–Ti facilitated rat bone marrow derived MSCs differentiation and mineralization evidenced by greater ALP activity and bone-like nodule formation. Interestingly, O₃–Ti did not affect RAW264.7 M? proliferation. However, naive RAW264.7 M? cultured on unTi produced a two-fold larger amount of TNFα than that on O₃–Ti. Furthermore, O₃–Ti greatly mitigated proinflammatory cytokine production, including TNFα and IL-6 from LSP-stimulated RAW264.7 M?. These results demonstrated that a superhydrophilic titanium prepared by simple ozone gas functionalization successfully increased MSCs proliferation and differentiation, and mitigated proinflammatory cytokine production from both naive and LPS-stimulated M?. This superhydrophilic surface would be useful as an endosseous implantable biomaterials and as a biomaterial for implantation into other tissues.     

Cytotoxicity and TNF-alpha secretion in RAW264.7 macrophages exposed to different fullerene derivatives

Fullerene derivatives have been reported as potential nanomedicines, however the role of surface chemical modification on the biological effects remains unclear. In this study, five kinds of water soluble C60 derivatives with different surface chemical modification, C60-(OH)20 (HFD), C60-(beta-Ala)10.1 (AFD), C60-(Lys)8.7 (KFD), C60-(Arg)8.6 (RFD) and C60-(NH(CH2)2NH2)8.8 (NFD) were synthesized. Their cytotoxicity as well as TNF-alpha secretions were evaluated in RAW264.7 macrophage cell line. The results show that no significant cytotoxicity can be observed upon 24 h exposure to C60 derivatives at less than 50 microg/mL. However, higher concentration (> 100 microg/mL) of these C60 derivatives decreases the proliferation of RAW264.7. The cytotoxicity of these fullerene derivatives is probably through the apoptosis pathway, while the extent of cytotoxicity varies with the different surface charges. Higher celluar uptake of HFD was observed in RAW264.7 cells than AFD, which correlates with the more toxic effect of HFD over AFD. The secretion of cytokine tumor necrosis factor alpha (TNF-alpha) was determined to evaluate the immunostimulating activity of these fullerene derivatives. The data show that the fullerene derivatives with negative surface charges secrete more TNF-alpha, whereas derivatives with positive charges show insignificant effect. The possible influence of various surface charge property on the observed biological effects is discussed.     

Biological and Magnetic Contrast Evaluation of Shape-Selective Mn–Fe Nanowires

One-dimensional composite Mn–Fe oxide nanostructures of different sizes (nanoneedles, nanorods, and nanowires) were prepared by a linker-induced organization of manganese-doped iron oxide nanoparticles. The nanostructures were obtained by the treatment of $hbox{MnFe}_{2}hbox{O}_{4}$ nanoparticles in the presence of cystamine. The average lengths of nanoneedle, nanorod, and nanowire are approximately 400, 800, and 1000 nm, respectively. High-resolution transmission electron microscopy (HR-TEM), energy-dispersive X-ray (EDX) spectroscopy, and inductively coupled plasma-optical emission spectroscopy (ICP–OES) were employed to characterize the morphologies and the elemental contents of the nanostructures. As an example of their potential applications, these nanostructures were explored as the cell-labeling agents for magnetic resonance imaging (MRI). The magnetic contrast properties of the nanostructures were characterized by a 1.5 T (Tesla) whole body MR system. 10 $mu,$g/mL of the nanostructures caused substantial negative contrast. After in vitro incubation, the nanostructures could be effectively incorporated into the cells of a monocyte/macrophage cell line (RAW264.7). These cells’ viability and proliferation potential were not affected when the labeling concentration was less than 50 $mu,$g/mL.      

A Review on Iron Oxide Nanoparticles and Their Biomedical Applications

The iron oxide nanoparticles have a great attraction in biomedical applications due to their non-toxic role in the biological systems. The iron oxide nanoparticles have both magnetic behaviour and semiconductor property which lead to multifunctional biomedical applications. The iron oxide nanoparticles used in biomedical fields such as antibacterial, antifungal and anticancer were reviewed. The uses of hematite (α-Fe₂O₃), maghemite (γ-Fe₂O₃) and magnetite (Fe₃O₄) nanoparticles, for an inhibition time in biological activities, are listed in this work. Also, this review explains the use of iron oxide nanoparticles in the biomedical fields with particular attention to the application of hematite and superparamagnetic iron oxide nanoparticles. In this review, analysis reveals that the role of iron oxide in biological activity is good due to its biocompatibility, biodegradability, ease of synthesis and different magnetic behaviours. The change of properties of iron oxide nanoparticles such as particle size, morphology, surface, agglomeration and electronic properties has specific impact in biomedical application. The review mainly focused in and discussed about antibacterial, anticancer, bone marrow and cell labelling activities. From this review work, the iron oxide nanoparticle may be specialised in particular bacterial and cancer treatments. Also discussed are the iron oxide nanoparticle-specific biomedical applications like human placenta, insulin and retinal locus treatments.     

Multifunctional nanoparticles, nanocages and degradable polymers as a potential novel generation of non-invasive molecular and cellular imaging systems

In recent years, polymeric scaffolds have been used in several biomedical applications for delivery of drugs or other biologically relevant molecules. Polymeric nanostructures display different (and in some cases more powerful) properties respect to bulk materials. This, lead academic researchers and industry to cooperate in developing pioneering nanostructured materials for industrial and biomedical applications. Moreover, the preparation and use of systems with multiple (multifunctional) properties (i.e., bioconjugation with superparamagnetic, fluorescent or targeting molecules) is positioned to become a viable and innovative tool for application in several clinical fields. Other nanostructured systems like nanocages and degradable nanoparticles, are emerging as potential innovative systems that could be exploited as multifunctional delivery vectors. This brief critical review is aimed at collecting and discussing some recent patents dealing with the preparation and use of multifunctional nanoparticles, nanocages and degradable nanoparticles in biomedicine and non-invasive bioimaging applications. Perspectives for a potential use of these multifunctional nanosystems in pediatries have been also discussed.     

Characterization of magnetite-organic complex nanoparticles by metal-reducing bacteria

Magnetite nanoparticles exhibit clear technological potential for biomedical applications. The objectives of this study were to synthesize magnetite-organic complex nanoparticles through the use of metal-reducing bacteria and characterize the mineralogical and surface chemical properties of these nanoparticles as well as to test their potential applications in biomedical technology with regards to their protein immobilization capacity. The microbially formed magnetite nanoparticles had a size of around 10 nm with a spherical shape and were coated with organics containing an abundance of reactive carboxyl groups without any chemical process for functionalizing them. These microbial processes may lead to a simple preparation of functional magnetite-organic complex nanoparticles which have benefits for biomedical applications.     

The translocation of fullerenic nanoparticles into lysosome via the pathway of clathrin-mediated endocytosis

Manufactured fullerene nanoparticles easily enter into cells and hence have been rapidly developed for biomedical uses. However, it is generally unknown which route the nanoparticles undergo when crossing cell membranes and where they localize to the intracellular compartments. Herein we have used both microscopic imaging and biological techniques to explore the processes of [C(60)(C(COOH)(2))(2)](n) nanoparticles across cellular membranes and their intracellular translocation in 3T3 L1 and RH-35 living cells. The fullerene nanoparticles are quickly internalized by the cells and then routed to the cytoplasm with punctate localization. Upon entering the cell, they are synchronized to lysosome-like vesicles. The [C(60)(C(COOH)(2))(2)](n) nanoparticles entering cells are mainly via endocytosis with time-, temperature-?and energy-dependent manners. The cellular uptake of [C(60)(C(COOH)(2))(2)](n) nanoparticles was found to be clathrin-mediated but not caveolae-mediated endocytosis. The endocytosis mechanism and the subcellular target location provide key information for the better understanding and predicting of the biomedical function of fullerene nanoparticles inside?cells.     

Self‐Assembly of Poly‐Adenine‐Tailed CpG Oligonucleotide‐Gold Nanoparticle Nanoconjugates with Immunostimulatory Activity

Synthetic unmethylated cytosine–guanine (CpG) oligodeoxynucleotides (CpG ODNs) possess high immunostimulatory activity and have been widely used as a therapeutic tool for various diseases including infection, allergies, and cancer. A variety of nanocarriers have been developed for intracellular delivery of CpG ODNs that are otherwise nonpermeable through the cellular membrane. For example, previous studies showed that gold nanoparticles (AuNPs) could efficiently deliver synthetic thiolated CpG ODNs into cultured cells and induce expression of proinflammatory cytokines. Nevertheless, the necessity of using thiolated CpG ODNs for the modification of AuNPs inevitably complicates the synthesis of the nanoconjugates and increases the cost. A new approach is demonstrated for facile assembly of AuNP‐CpG nanoconjugates for cost‐effective drug delivery. It is found that non‐thiolated, diblock ODNs containing a CpG motif and a poly‐adenine (polyA) tail can readily self‐assemble on the surface of AuNPs with controllable and tunable density. Such nanoconjugates are efficiently delivered into RAW264.7 cells and induce immune response in a Toll‐like receptor 9 (TLR9)‐dependent manner. Under optimal conditions, polyA‐CpG‐AuNPs show significantly higher immunostimulatory activity than their thiolated counterpart. In addition, the immunostimulatory activity of CpG‐AuNPs can be modulated by varying the length of the polyA tail. In vivo induction of immune responses in mice is demonstrated by using polyA‐tailed CpG‐AuNP nanoconjugates.