The functional dissection of the plasma corona of SiO2-NPs spots histidine rich glycoprotein as a major player able to hamper nanoparticle capture by macrophages.

A coat of strongly-bound host proteins, or hard corona, may influence the biological and pharmacological features of nanotheranostics by altering their cell-interaction selectivity and macrophage clearance. With the goal of identifying specific corona-effectors, we investigated how the capture of amorphous silica nanoparticles (SiO2-NPs; Ø = 26 nm; zeta potential = -18.3 mV) by human lymphocytes, monocytes and macrophages is modulated by the prominent proteins of their plasma corona. LC MS/MS analysis, western blotting and quantitative SDS-PAGE densitometry show that Histidine Rich Glycoprotein (HRG) is the most abundant component of the SiO2-NP hard corona in excess plasma from humans (HP) and mice (MP), together with minor amounts of the homologous Kininogen-1 (Kin-1), while it is remarkably absent in their Foetal Calf Serum (FCS)-derived corona. HRG binds with high affinity to SiO2-NPs (HRG Kd ∼2 nM) and competes with other plasma proteins for the NP surface, so forming a stable and quite homogeneous corona inhibiting nanoparticles binding to the macrophage membrane and their subsequent uptake. Conversely, in the case of lymphocytes and monocytes not only HRG but also several common plasma proteins can interchange in this inhibitory activity. The depletion of HRG and Kin-1 from HP or their plasma exhaustion by increasing NP concentration (>40 µg ml(-1) in 10% HP) lead to a heterogeneous hard corona, mostly formed by fibrinogen (Fibr), HDLs, LDLs, IgGs, Kallikrein and several minor components, allowing nanoparticle binding to macrophages. Consistently, the FCS-derived SiO2-NP hard corona, mainly formed by hemoglobin, α2 macroglobulin and HDLs but lacking HRG, permits nanoparticle uptake by macrophages. Moreover, purified HRG competes with FCS proteins for the NP surface, inhibiting their recruitment in the corona and blocking NP macrophage capture. HRG, the main component of the plasma-derived SiO2-NPs' hard corona, has antiopsonin characteristics and uniquely confers to these particles the ability to evade macrophage capture.

PEGylation of ORMOSIL nanoparticles differently modulates the in vitro toxicity toward human lung cells.

ORganically MOdified SILica (ORMOSIL) nanoparticles (NPs) appear promising carriers for the delivery of drugs to target tissues but concerns on possible cytotoxic effects exist. Here, we studied the in vitro responses to ORMOSIL NPs in different types of human lung cells to determine the effects of polyethylene glycol (PEG) coating on NP cytotoxicity. Non-PEG NPs caused a concentration-dependent decrease of viability of all types of cells, while PEG NPs induced deleterious effects and death in carcinoma alveolar type II A549 cells but not in CCD-34Lu fibroblasts and NCI-H2347 adenocarcinoma cells. Reactive oxygen species were detected in cells incubated with PEG NPs, but their deactivation by superoxide dismutase and catalase did not protect A549 cells from death, suggesting that the oxidative stress was not the main determinant of cytotoxicity. Only in A549 cells PEG NPs modulated the transcription of genes involved in inflammation, signal transduction and cell death. Transmission electron microscopy evidenced a unique intracellular localization of PEG NPs in the lamellar bodies of A549 cells, which could be the most relevant factor leading to cytotoxicity by reducing the production of surfactant proteins and by interfering with the pulmonary surfactant system.

Altered gene transcription in human cells treated with Ludox® silica nanoparticles.

Silica (SiO2) nanoparticles (NPs) have found extensive applications in industrial manufacturing, biomedical and biotechnological fields. Therefore, the increasing exposure to such ultrafine particles requires studies to characterize their potential cytotoxic effects in order to provide exhaustive information to assess the impact of nanomaterials on human health. The understanding of the biological processes involved in the development and maintenance of a variety of pathologies is improved by genome-wide approaches, and in this context, gene set analysis has emerged as a fundamental tool for the interpretation of the results. In this work we show how the use of a combination of gene-by-gene and gene set analyses can enhance the interpretation of results of in vitro treatment of A549 cells with Ludox® colloidal amorphous silica nanoparticles. By gene-by-gene and gene set analyses, we evidenced a specific cell response in relation to NPs size and elapsed time after treatment, with the smaller NPs (SM30) having higher impact on inflammatory and apoptosis processes than the bigger ones. Apoptotic process appeared to be activated by the up-regulation of the initiator genes TNFa and IL1b and by ATM. Moreover, our analyses evidenced that cell treatment with LudoxÒ silica nanoparticles activated the matrix metalloproteinase genes MMP1, MMP10 and MMP9. The information derived from this study can be informative about the cytotoxicity of Ludox® and other similar colloidal amorphous silica NPs prepared by solution processes.

Variations of the corona HDL:albumin ratio determine distinct effects of amorphous SiO2 nanoparticles on monocytes and macrophages in serum.

AIM: We investigated monocyte and macrophage death and cytokine production induced by amorphous silica nanoparticles (SiO2-NPs) to clarify the role of defined serum corona proteins. MATERIALS & METHODS: The cytotoxic proinflammatory effects of SiO2-NPs on human monocytes and macrophages were characterized in no serum, in fetal calf serum and in the presence of purified corona proteins. RESULTS: In no serum and in fetal calf serum above approximately 75 µg/ml, SiO2-NPs lysed monocytes and macrophages by plasma membrane damage (necrosis). In fetal calf serum below approximately 75 µg/ml, SiO2-NPs triggered an endolysosomal acidification and caspase-1-dependent monocyte death (pyroptosis). The corona high-density lipoproteins:albumin ratio accounted for the features of the SiO2-NPs in serum. DISCUSSION: Corona high-density lipoproteins are a major determinant of the differential cytotoxic action of SiO2-NPs on monocytes and macrophages.

Targeted delivery of photosensitizers: efficacy and selectivity issues revealed by multifunctional ORMOSIL nanovectors in cellular systems.

PEGylated and non-PEGylated ORMOSIL nanoparticles prepared by microemulsion condensation of vinyltriethoxy-silane (VTES) were investigated in detail for their micro-structure and ability to deliver photoactive agents. With respect to pure silica nanoparticles, organic modification substantially changes the microstructure and the surface properties. This in turn leads to a modulation of both the photophysical properties of embedded photosensitizers and the interaction of the nanoparticles with biological entities such as serum proteins. The flexibility of the synthetic procedure allows the rapid preparation and screening of multifunctional nanosystems for photodynamic therapy (PDT). Selective targeting of model cancer cells was tested by using folate, an integrin specific RGD peptide and anti-EGFR antibodies. Data suggest the interference of the stealth-conferring layer (PEG) with small targeting agents, but not with bulky antibodies. Moreover, we showed that selective photokilling of tumour cells may be limited even in the case of efficient targeting because of intrinsic transport limitations of active cellular uptake mechanisms or suboptimum localization.

Catastrophic inflammatory death of monocytes and macrophages by overtaking of a critical dose of endocytosed synthetic amorphous silica nanoparticles/serum protein complexes.

AIMS: We tested whether phagocytic monocytes/macrophages are more susceptible than nonphagocytes to nanoparticle (NP) toxicity. MATERIALS & METHODS: We compared in vitro cell death and proinflammatory cytokine production in human monocytes, macrophages, lymphocytes and HeLa cells due to synthetic amorphous silica (SiO2)-NPs in different serum concentrations and correlated them with cellular uptake and distribution. RESULTS: Phagocytes were approximately ten-times more sensitive than nonphagocytes to SiO2-NPs and more effectively endocytosed SiO2-NP-serum protein nanoagglomerates, so determining their accumulation in acidic endocytic compartments well beyond a critical/cytotoxic threshold. Monocyte/macrophage death was paralleled by cytokine secretion. CONCLUSION: The physiological specialization of monocytes/macrophages to effectively capture NPs may expose them to the risk of catastrophic inflammatory death upon saturation of their maximal storage capacity.

The toxicity outcome of silica nanoparticles (Ludox®) is influenced by testing techniques and treatment modalities.

We analyzed the influence of the kind of cytotoxicity test and its application modality in defining the level of hazard of the in vitro exposures to nanostructures. We assessed the cytotoxicity induced by two different Ludox® silica nanoparticles (NPs), AS30 and SM30, on three human cell lines, CCD-34Lu, A549, and HT-1080. Dynamic light scattering measurements showed particle agglomeration when NPs are diluted in culture medium supplemented with fetal calf serum. We examined the impact of such particle aggregation on the cytotoxicity by exposing the cells to NPs under different treatment modalities: short incubation (2 h) in serum-free medium or long incubation (24-72 h) in serum-containing medium. Under this last modality, NP suspensions tended to form aggregates and were toxic at concentrations five- to tenfold higher than in serum-free medium. The results of cell survival varied considerably when the long-term clonogenic assay was performed to validate the data of the short-term MTS assay. Indeed, the half maximum effective concentrations (EC(50)) in all the three cell lines were four- to fivefold lower when calculated from the data of clonogenic assay than of MTS. Moreover, the mechanisms of NP toxicity were cell-type-specific, showing that CCD-34Lu are prone to the induction of plasma membrane damages and HT-1080 are prone to DNA double-strand break and apoptosis induction. Taken together, our results demonstrate that the choice of testing strategy and treatment conditions plays an important role in assessing the in vitro toxicity of NPs.

A cell-penetrating ratiometric nanoprobe for intracellular chloride.

NanoChlor, a nanoparticle-based fluorescent probe for chloride that is both ratiometric and capable of spontaneously penetrating neuronal cells at submillimolar concentrations, was designed and studied. NanoChlor is built on silica nanoparticles grafted with 6-methoxyquinolinium as the chloride-sensitive component and fluorescein as the reference dye. A Stern-Volmer constant of 50 M(-1) was measured in Ringer's buffer at pH 7.2, and the response to chemically induced chloride currents was recorded in real time in hippocampal cells.

Proinflammatory effects of bare and PEGylated ORMOSIL-, PLGA- and SUV-NPs on monocytes and PMNs and their modulation by f-MLP.

AIMS: We wanted to test the proinflammatory effects of vinyltriethoxysilane-based organically modified silica nanoparticles (ORMOSIL-NPs) in vitro on blood leukocytes. MATERIALS & METHODS: Cell selectivity, cytokines/chemokines and O(2) (-) production were analyzed using nonpolyethylene glycol (PEG)ylated and PEGylated ORMOSIL-NPs, poly(lactic-co-glycolic acid) (PLGA)-NPs and small unilamellar vesicles (SUV)-NPs. RESULTS: ORMOSIL-NPs mostly bound to monocytes while other NPs to all leukocyte types similarly. Cell capture of PEGylated-NPs decreased strongly (ORMOSIL), moderately (PLGA) and weakly (SUV). Bare ORMOSIL-NPs effectively stimulated the production of IL-1ß/IL-6/TNF-α/IL-8 by monocytes and of IL-8 by polymorphonuclear leukocytes (PMNs). NP PEGylation inhibited such effects only partially. Formyl-methionine-leucine phenylalanine (f-MLP) further increased the release of cytokines/chemokines by monocytes/PMNs primed with bare and PEGylated ORMOSIL-NPs. PEGylated SUV-NPs, bare and PEGylated ORMOSIL- and PLGA-NPs sensitize PMNs and monocytes to secrete O(2) (-) upon f-MLP stimulation. CONCLUSION: ORMOSIL-NPs are preferentially captured by circulating monocytes but stimulate both monocytes and PMNs per se or by sensitizing them to another agonist (f-MLP). PEG-coating confers stealth effects but does not completely eliminate leukocyte activation. Safe nanomedical applications require the evaluation of both intrinsic and cooperative proinflammatory potential of NPs.

Procoagulant properties of bare and highly PEGylated vinyl-modified silica nanoparticles.

AIMS: Undesired alterations of the blood clotting balance may follow the intravascular injection of nanotherapeutics/diagnostics. Here, we tested the procoagulant activity of synthetic amorphous silica (SAS) and organically modified silica (ORMOSIL) nanoparticles (NPs) and whether a high-density polyethylene glycol coating minimizes these effects. MATERIALS & METHODS: Hageman factor- and tissue factor-dependent activation of human blood/plasma coagulation, and binding to human monocytes, endothelial cells and platelets were quantified in vitro using naked and PEGylated ORMOSIL-NPs. Their effects were compared with those of SAS-NPs, present in many industrial products, and of poly(lactic-co-glycolic acid)- and small unilamellar vesicles-NPs, already approved for use in humans. RESULTS: Both SAS-NPs and ORMOSIL-NPS presented a significant procoagulant activity. However, highly PEGylated ORMOSIL-NPs were particularly averse to the interaction with the soluble factors and cellular elements that may lead to intravascular blood coagulation. CONCLUSION: Stealth, highly PEGylated ORMOSIL-NPs with a poor procoagulant activity can be used as starting blocks to design hemocompatible nanomedical-devices.

The cellular uptake of meta-tetra(hydroxyphenyl)chlorin entrapped in organically modified silica nanoparticles is mediated by serum proteins.

Nanosized objects made of various materials are gaining increasing attention as promising vehicles for the delivery of therapeutic and diagnostic agents for cancer. Photodynamic therapy (PDT) appears to offer a very attractive opportunity to implement drug delivery systems since no release of the sensitizer is needed to obtain the therapeutic effect and the design of the nanovehicle should be much easier. The aim of our study was to investigate the use of organic-modified silica nanoparticles (NPs) for the delivery of the second-generation photosensitizer meta-tetra(hydroxyphenyl)chlorin (mTHPC) to cancer cells in vitro. mTHPC was entrapped in NPs (approximately 33 nm diameter) in a monomeric form which produced singlet oxygen with a high efficiency. In aqueous media with high salt concentrations, the NPs underwent aggregation and precipitation but their stability could be preserved in the presence of foetal bovine serum. The cellular uptake, localization and phototoxic activity of mTHPC was determined comparatively in human oesophageal cancer cells after its delivery by the NPs and the standard solvent ethanol/poly(ethylene glycol) 400/water (20:30:50, by vol). The NP formulation reduced the cellular uptake of mTHPC by about 50% in comparison to standard solvent while it did not affect the concentration-dependent photokilling activity of mTHPC and its intracellular localization. Fluorescence resonance energy transfer measurements, using NPs with mTHPC physically entrapped and a cyanine covalently linked, and ultracentrifugation experiments indicated that mTHPC is transferred from NPs to serum proteins when present in the medium. However, the coating of the NP surface with poly(ethylene glycol) largely prevented the transfer to proteins. In conclusion, mTHPC is rapidly transferred from the uncoated nanoparticles to the serum proteins and then internalized by the cells as a protein complex, irrespective of its modality of delivery.

Phosphate diester and DNA hydrolysis by a multivalent, nanoparticle-based catalyst.

2-nm gold nanoclusters coated with Zn(II) complexes bearing auxiliary hydrogen bond donors act as multivalent catalysts capable of promoting the hydrolysis of model phosphate diesters with exceptional activity and inducing DNA double strand cleavage.