Molecular Elucidation of Biological Response to Mesoporous Silica Nanoparticles in Vitro and in Vivo.

Biomedical applications of mesoporous silica nanoparticles (MSNs) require efficient cellular uptake and low toxicity. The purpose of this study is to investigate the cellular uptake and toxicity of MSNs with different sizes and charges (50, 100, and 250 nm with a positive surface charge and 100 nm with a negative surface charge) exposed to human monocyte-derived macrophages, lung epithelium BEAS-2B cells, and mice using genome-wide gene expression analysis and cellular/animal-level end point tests. We found that MSNs can be taken up into cells through endocytosis in a charge- and size-dependent manner, with positively charged and larger MSNs being more easily taken up into the cells by recruiting more types of endocytotic pathways for more cellular uptake. Moreover, the cytotoxicity of MSNs could be correlated with the amount of MSNs taken up by cells, which positively correlates to the particle size and dosage. Therefore, only positively charged and larger MSNs (≥100 nm) during higher treatment doses (≥500 µg mL-1) resulted in a sufficient accumulation of internalized MSNs in cells to induce significant release of reactive oxygen species (ROS) and oxidative stress, inflammatory gene upregulation through NF-κB and AP-1, and eventually autophagy-mediated necrotic cell death. Furthermore, genome-wide gene expression analysis could reflect the above in vitro cellular damages and corresponding in vivo injuries in mice, indicating that specific gene expression footprints may be used for assessing the safety of nanoparticles. The present finding provides some insights into the rational design of effective MSN-based drug/gene delivery systems and biomedical applications.

Local pH tracking in living cells.

Continuous and simultaneous 3D single-particle movement and local pH detection in HeLa cells were demonstrated for the first time by combining fluorescent mesoporous silica nanoparticles (FMSNs) and a single-particle tracking (SPT) technique with a precision of ∼10 nm. FMSNs, synthesized by the co-condensation of both pH-sensitive and reference dyes with a silica/surfactant source, allow long-term reliable ratiometric pH measurements with a precision better than 0.3 pH unit because of their excellent brightness and stability. pH variation in the surrounding area of FMSNs during endocytosis was monitored in real-time. Acidification and low mobility of FMSNs were observed at the early endocytic stage, whereas basification and high mobility of FMSNs were observed at the late stage. Our results indicate that it is possible to monitor local pH changes in the environments surrounding nanoparticles during the cellular uptake process of FMSNs, which provides much needed information for designing an efficient drug delivery nanosystem.

Enzyme encapsulated hollow silica nanospheres for intracellular biocatalysis.

Hollow silica nanospheres (HSN) with low densities, large interior spaces and permeable silica shells are suitable for loading enzymes in the cavity to carry out intracellular biocatalysis. The porous shell can protect the encapsulated enzymes against proteolysis and attenuate immunological response. We developed a microemulsion-templating method for confining horseradish peroxidase (HRP) in the cavity of HSN. This simple one-pot enzyme encapsulation method allows entrapping of the enzyme, which retains high catalytic activity. Compared with HRP supported on solid silica spheres, HRP@HSN with thin porous silica shells displayed better enzyme activity. The small HRP@HSN (∼50 nm in diameter), giving satisfactory catalytic activity, can act as an intracellular catalyst for the oxidation of the prodrug indole-3-acetic acid to produce toxic free radicals for killing cancer cells. We envision this kind of hollow nanosystem could encapsulate multiple enzymes or other synergistic drugs and function as therapeutic nanoreactors.

Enhanced non-endocytotic uptake of mesoporous silica nanoparticles by shortening the peptide transporter arginine side chain.

Mesoporous silica nanoparticles (MSNs) are multifunctional nanocarriers with potential biomedical applications. However, MSNs are frequently trapped in endosomes upon cellular uptake through endocytosis, requiring endosomal escape. Herein, enhanced nonendocytosis was observed for 300 nm MSNs by conjugating peptides with noncanonical arginine analogs.

Nonviral cell labeling and differentiation agent for induced pluripotent stem cells based on mesoporous silica nanoparticles.

The generation of induced pluripotent stem cells (iPSCs) is an innovative personalized-regenerative technology, which can transform own-self somatic cells into embryonic stem (ES)-like cells, which have the potential to differentiate into all cell types of three dermal lineages. However, how to quickly, efficiently, and safely produce specific-lineage differentiation from pluripotent-state cells and iPSCs is still an open question. The objective of the present study was to develop a platform of a nonviral gene delivery system of mesoporous silica nanoparticles (MSNs) to rapidly generate iPSC-derived definitive-lineage cells, including endodermal-differentiated cells. We also evaluated the feasibility and efficiency of FITC-conjugated MSNs (FMSNs) for labeling of iPSCs and utilized the multifunctional properties of FMSNs for a suitable carrier for biomolecule delivery. We showed that FMSNs of various surface charges could be efficiently internalized by iPSCs without causing cytotoxicity. The levels of reactive oxygen species and pluripotent status, including in vitro stemness signatures and in vivo teratoma formation, remained unaltered. Notably, positive-charged FMSN enhanced cellular uptake efficiency and retention time. Moreover, when using positive-charged FMSN to deliver hepatocyte nuclear factor 3ß (HNF3ß) plasmid DNA (pDNA), the treated iPSCs exhibited significantly improved definitive endoderm formation and further quickly differentiated into hepatocyte-like cells with mature functions (low-density lipoprotein uptake and glycogen storage) within 2 weeks in vitro. Double delivery of pHNF3ß further improved mRNA expression levels of liver-specific genes. These findings reveal the multiple advantages of FMSNs to serve as ideal vectors not only for stem cell labeling but also for safe gene delivery to promote the production of hepatocyte-like cells from iPSCs.

Manganese-enhanced MRI of rat brain based on slow cerebral delivery of manganese(II) with silica-encapsulated Mn x Fe(1-x) O nanoparticles.

In this work, we report a monodisperse bifunctional nanoparticle system, MIO@SiO2 -RITC, as an MRI contrast agent [core, manganese iron oxide (MIO); shell, amorphous silica conjugated with rhodamine B isothiocyanate (RITC)]. It was prepared by thermal decomposition and modified microemulsion methods. The nanoparticles with varying iron to manganese ratios displayed different saturated magnetizations and relaxivities. In vivo MRI of rats injected intravenously with MIO@SiO2-RITC nanoparticles exhibited enhancement of the T1 contrast in brain tissue, in particular a time-delayed enhancement in the hippocampus, pituitary gland, striatum and cerebellum. This is attributable to the gradual degradation of MIO@SiO2-RITC nanoparticles in the liver, resulting in the slow release of manganese(II) [Mn(II)] into the blood pool and, subsequently, accumulation in the brain tissue. Thus, T1-weighted contrast enhancement was clearly detected in the anatomic structure of the brain as time progressed. In addition, T2*-weighted images of the liver showed a gradual darkening effect. Here, we demonstrate the concept of the slow release of Mn(II) for neuroimaging. This new nanoparticle-based manganese contrast agent allows one simple intravenous injection (rather than multiple infusions) of Mn(II) precursor, and results in delineation of the detailed anatomic neuroarchitecture in MRI; hence, this provides the advantage of the long-term study of neural function.

A new strategy for intracellular delivery of enzyme using mesoporous silica nanoparticles: superoxide dismutase.

We developed mesoporous silica nanoparticle (MSN) as a multifunctional vehicle for enzyme delivery. Enhanced transmembrane delivery of a superoxide dismutase (SOD) enzyme embedded in MSN was demonstrated. Conjugation of the cell-penetrating peptide derived from the human immunodeficiency virus 1 (HIV) transactivator protein (TAT) to mesoporous silica nanoparticle is shown to be an effective way to enhance transmembrane delivery of nanoparticles for intracellular and molecular therapy. Cu,Zn-superoxide dismutase (SOD) is a key antioxidant enzyme that detoxifies intracellular reactive oxygen species, ROS, thereby protecting cells from oxidative damage. In this study, we fused a human Cu,Zn-SOD gene with TAT in a bacterial expression vector to produce a genetic in-frame His-tagged TAT-SOD fusion protein. The His-tagged TAT-SOD fusion protein was expressed in E. coli using IPTG induction and purified using FMSN-Ni-NTA. The purified TAT-SOD was conjugated to FITC-MSN forming FMSN-TAT-SOD. The effectiveness of FMSN-TAT-SOD as an agent against ROS was investigated, which included the level of ROS and apoptosis after free radicals induction and functional recovery after ROS damage. Confocal microscopy on live unfixed cells and flow cytometry analysis showed characteristic nonendosomal distribution of FMSN-TAT-SOD. Results suggested that FMSN-TAT-SOD may provide a strategy for the therapeutic delivery of antioxidant enzymes that protect cells from ROS damage.

High payload Gd(iii) encapsulated in hollow silica nanospheres for high resolution magnetic resonance imaging.

For clear MR imaging of blood vessels, a long blood circulation time of effective T1 contrast agents is necessary. Nanoparticulate MR contrast agents are much more effective owing to their enhanced relaxivity, a result of reduced tumbling rates, and large payloads of active magnetic species. PEGylated yolk-shell silica nanospheres containing high payloads of Gd(iii) with cross-linking ligands are synthesized and evaluated as a blood-pool magnetic resonance contrast agent. The hydrophilic PEG coating and the microporous silica shell allow water exchange while keeping the multi-nuclear Gd species from leaching out. These Gd(iii)-containing yolk-shell silica nanoparticles with PEGylated surfaces give excellent resolution and contrast in magnetic resonance angiography images of vasculature in rat brains.

A broad range fluorescent pH sensor based on hollow mesoporous silica nanoparticles, utilising the surface curvature effect.

In this study, a broad range pH sensor was synthesized by loading the pH sensitive dye, fluorescein isothiocyanate (FITC), and a reference dye, rhodamine B isothiocyanate (RITC), into the mesostructure of hollow mesoporous silica nanoparticles (HMSNs) synthesized using a co-condensation method. Compared to a pH sensor based on the same pair of dyes on conventional mesoporous silica nanoparticles (MSNs), this dual-labeled pH sensor based on HMSNs shows a larger pH sensitive range, between 4.5 and 8.5, because of its broad surface curvature distribution which has a strong effect on the apparent pKa values of the FITC dye. The hollow mesoporous silica-dye nanoparticles were used to monitor intracellular pH via a ratiometric fluorescence method. The confocal images demonstrated the capacity of this broad range sensor which can differentiate simultaneously the local pH between various environments, for example, in the medium (pH = 7.2), cytosol (pH ∼ 7) and the endosome-lysosome pathway (pH = 4-5.5).

A simple plant gene delivery system using mesoporous silica nanoparticles as carriers.

A facile DNA delivery method would greatly facilitate studies of plant functional genomics. However, plant cell walls limit the utilization of nanoparticles on plant research. Here, we employed functionalized mesoporous silica nanoparticles (MSNs) to develop a MSN-mediated plant transient gene expression system. In this system, MSNs served as carriers to deliver foreign DNA into intact Arabidopsis thaliana roots without the aid of mechanical force. Gene expression was detected in the epidermal layer and in the more inner cortical and endodermal root tissues by both fluorescence and antibody labeling. This is a novel alternative to the conventional gene-gun or ultrasonic methods. In addition, the parameters that affect the MSN uptake and the mechanism and subcellular distribution of particles were also analyzed. The present study may provide valuable information on the manipulation of functional nanoparticles in plants and have significant impact on plant biotechnology.

Integrated nanophotonic hubs based on ZnO-Tb(OH)3/SiO2 nanocomposites.

Optical integration is essential for practical application, but it remains unexplored for nanoscale devices. A newly designed nanocomposite based on ZnO semiconductor nanowires and Tb(OH)3/SiO2 core/shell nanospheres has been synthesized and studied. The unique sea urchin-type morphology, bright and sharply visible emission bands of lanthanide, and large aspect ratio of ZnO crystalline nanotips make this novel composite an excellent signal receiver, waveguide, and emitter. The multifunctional composite of ZnO nanotips and Tb(OH)3/SiO2 nanoparticles therefore can serve as an integrated nanophotonics hub. Moreover, the composite of ZnO nanotips deposited on a Tb(OH)3/SiO2 photonic crystal can act as a directional light fountain, in which the confined radiation from Tb ions inside the photonic crystal can be well guided and escape through the ZnO nanotips. Therefore, the output emission arising from Tb ions is truly directional, and its intensity can be greatly enhanced. With highly enhanced lasing emissions in ZnO-Tb(OH)3/SiO2 as well as SnO2-Tb(OH)3/SiO2 nanocomposites, we demonstrate that our approach is extremely beneficial for the creation of low threshold and high-power nanolaser.

PEGylated silica nanoparticles encapsulating multiple magnetite nanocrystals for high-performance microscopic magnetic resonance angiography.

A novel magnetic resonance (MR) angiographic method, 3DΔR2-mMRA (three dimensional and ΔR2 based microscopy magnetic resonance angiography), is developed as a clinical diagnosis for depicting the function and structure of cerebral small vessels. However, the visibility of microvasculatures and the precision of cerebral blood volume calculation greatly rely on the transverse relaxivity and intravascular half-life of contrast agent, respectively. In this work, we report a blood pool contrast agent named H-Fe3O4@SiO2-PEG where multiple Fe3O4 nanocrystals are encapsulated in a thin silica shell to enhance the T2-relaxivity (r2 = 342.8 mM⁻¹ s⁻¹) and poly(ethylene glycol) (PEG) is employed to reduce opsonization and prolong circulation time of nanoparticles. Utilization of the newly developed H-Fe3O4@SiO2-PEG with a novel MR angiographic methodology, a high-resolution MR image of rat cerebral microvasculatures is successfully obtained.

Mesoporous silica nanoparticles as nanocarriers.

Modern nanomedicine aims at delivering drugs or cells specifically to defective cells; therefore, this calls for developing multifunctional nanocarriers for drug delivery and cell-tracking. Mesoporous silica nanoparticles (MSNs) are well suited for this task. In this feature article, we highlight the strategies in the synthesis and functionalization of small, uniform and colloidal stable MSNs. We then discuss cell uptake of MSNs and tracking cells, as both aspects are closely related to the efficacy of drug delivery and theranostics. Some examples of stimulated drug delivery are described. For application considerations, toxicity and pharmacokinetics are critical issues and in vivo studies are summarized.

Uniform mesoporous silica hexagon and its two-dimensional colloidal crystal.

Well-ordered mesoporous silica nanoparticles with uniform hexagonal disk shapes are synthesized under dilute alkaline conditions with a two-step process, separating the nucleation and growth process. The resulting monodisperse hexagons can be arranged in a 2-dimensional (2D) ordered periodical super-structure. The hexagonal symmetry is similar in both scales. A statistical mechanical cell model is applied to analyze consequences of the interesting packing structure, including osmotic bulk modulus, phase separation and defects.

Synthesis of hollow silica nanospheres with a microemulsion as the template.

We demonstrate a sol-gel approach, using a water-in-oil microemulsion as the template, for the synthesis of hollow and yolk/shell silica nanospheres, which can encapsulate pre-formed hydrophobic nanoparticles, and we then explore these multifunctional hollow nanospheres in cell-labeling applications.

Mesoporous silica nanoparticles as a delivery system of gadolinium for effective human stem cell tracking.

The progress of using gadolinium (Gd)-based nanoparticles in cellular tracking lags behind that of superparamagnetic iron oxide (SPIO) nanoparticles in magnetic resonance imaging (MRI). Here, dual functional Gd-fluorescein isothiocyanate mesoporous silica nanoparticles (Gd-Dye@MSN) that possess green fluorescence and paramagnetism are developed in order to evaluate their potential as effective T1-enhancing trackers for human mesenchymal stem cells (hMSCs). hMSCs are labeled efficiently with Gd-Dye@MSN via endocytosis. Labeled hMSCs are unaffected in their viability, proliferation, and differentiation capacities into adipocytes, osteocytes, and chondrocytes, which can still be readily MRI detected. Imaging, with a clinical 1.5-T MRI system and a low incubation dosage of Gd, low detection cell numbers, and short incubation times is demonstrated on both loaded cells and hMSC-injected mouse brains. This study shows that the advantages of biocompatibility, durability, high internalizing efficiency, and pore architecture make MSNs an ideal vector of T1-agent for stem-cell tracking with MRI.

Internalization of mesoporous silica nanoparticles induces transient but not sufficient osteogenic signals in human mesenchymal stem cells.

The biocompatibility of nanoparticles is the prerequisite for their applications in biomedicine but can be misleading due to the absence of criteria for evaluating the safety and toxicity of those nanomaterials. Recent studies indicate that mesoporous silica nanoparticles (MSNs) can easily internalize into human mesenchymal stem cells (hMSCs) without apparent deleterious effects on cellular growth or differentiation, and hence are emerging as an ideal stem cell labeling agent. The objective of this study was to thoroughly investigate the effect of MSNs on osteogenesis induction and to examine their biocompatibility in hMSCs. Uptake of MSNs into hMSCs did not affect the cell viability, proliferation and regular osteogenic differentiation of the cells. However, the internalization of MSNs indeed induced actin polymerization and activated the small GTP-bound protein RhoA. The MSN-induced cellular protein responses as believed to cause osteogenesis of hMSCs did not result in promotion of regular osteogenic differentiation as analyzed by cytochemical stain and protein activity assay of alkaline phosphatase (ALP). When the effect of MSNs on ALP gene expression was further examined by reverse transcriptase polymerase chain reaction, MSN-treated hMSCs were shown to have significantly higher mRNA expression than control cells after 1-hour osteogenic induction. The induction of ALP gene expression by MSNs, however, was absent in cells after 1-day incubation with osteogenic differentiation. Together our results show that the internalization of MSNs had a significant effect on the transient protein response and osteogenic signal in hMSCs, thereby suggesting that the effects of nanoparticles on diverse aspects of cellular activities should be carefully evaluated even though the nanoparticles are generally considered as biocompatible at present.

Mesoporous silica nanoparticles improve magnetic labeling efficiency in human stem cells.

Tumblerlike magnetic/fluorescein isothiocyanate (FITC)-labeled mesoporous silica nanoparticles, Mag-Dye@MSNs, have been developed, which are composed of silica-coated core-shell superparamagnetic iron oxide (SPIO@SiO(2)) nanoparticles co-condensed with FITC-incorporated mesoporous silica. Mag-Dye@MSNs can label human mesenchymal stem cells (hMSCs) through endocytosis efficiently for magnetic resonance imaging (MRI) in vitro and in vivo, as manifested by using a clinical 1.5-T MRI system with requirements of simultaneous low incubation dosage of iron, low detection cell numbers, and short incubation time. Labeled hMSCs are unaffected in their viability, proliferation, and differentiation capacities into adipocytes and osteocytes, which can still be readily detected by MRI. Moreover, a higher MRI signal intensity decrease is observed in Mag-Dye@MSN-treated cells than in SPIO@SiO(2)-treated cells. This is the first report that MCM-41-type MSNs are advantageous to cellular uptake, as manifested by a higher labeling efficiency of Mag-Dye@MSNs than SPIO@SiO(2).