Controlled Tyrosine Kinase Inhibitor Delivery to Liver Cancer Cells by Gate-Capped Mesoporous Silica Nanoparticles.

Hepatocellular carcinoma is the most common type of primary malignancy in the liver and one of the most common types of cancer worldwide. Its readily increasing mortality rate highlights the urgent need for the development of efficient therapeutic strategies. Tyrosine kinase inhibitors (TKIs) such as sorafenib and sunitinib are used as efficient angiogenesis inhibitors for this purpose. However, despite their pharmacological effects, their transfer into clinical practice is characterized by their poor aqueous solubility and accumulation in off-target tissues, resulting in unfavorable side effects. Here, we report a nanocomposite made of amine-functionalized mesoporous silica nanocomposites (MSNs) that are surface-coated with cerium oxide nanoparticles (CNPs) for the controlled delivery and release of TKIs. Amine-functionalized MSNs were prepared using a sol-gel method and loaded with TKIs. To trap drug molecules into the mesoporous structure, CNPs were covalently conjugated to the surface of MSNs. The synthesis and functionalization steps were controlled using different characterization methods, confirming the desired morphology and structure, the identity of functional groups on the surface, successful coating, and appropriate loading efficiency. Under physiological conditions, CNP-capped MSNs demonstrated a sustained drug release over time as a result of CNPs' gatekeeping effect on the payloads. Strong cellular interactions with different liver cancer cells and enhanced cellular uptake were also observed in vitro for the gate-capped MSNs. Internalization of nanocomposites induced cell death via the production of reactive oxygen species, and subsequent activation of apoptosis pathways. This study demonstrates that gate-capped MSNs are promising chemotherapeutic vehicles characterized by a sustained drug release profile and high cellular internalization.

Targeting Somatostatin Receptors By Functionalized Mesoporous Silica Nanoparticles - Are We Striking Home?

The concept of delivering nanoformulations to desired tissues by means of targeting membrane receptors of high local abundance by ligands anchored to the nanocarrier has gained a lot of attention over the last decade. Currently, there is no unanimous opinion on whether surface functionalization of nanocarriers by targeting ligands translates into any real benefit in terms of pharmacokinetics or treatment outcomes. Having examined the published nanocarriers designed to engage with somatostatin receptors, we realized that in the majority of cases targetability claims were not supported by solid evidence of targeting ligand-targeted receptor coupling, which is the very crux of a targetability concept. Here, we present an approach to characterize targetability of mesoporous silica-based nanocarriers functionalized with ligands of somatostatin receptors. The targetability proof in our case comes from a functional assay based on a genetically-encoded cAMP probe, which allows for real-time capture of receptor activation in living cells, triggered by targeting ligands on nanoparticles. We elaborate on the development and validation of the assay, highlighting the power of proper functional tests in the characterization pipeline of targeted nanoformulations.

Mesoporous silica materials: From physico-chemical properties to enhanced dissolution of poorly water-soluble drugs.

New approaches in pharmaceutical chemistry have resulted in more complex drug molecules in the quest to achieve higher affinity to their targets. However, these 'highly active' drugs can also suffer from poor water solubility. Hence, poorly water soluble drugs became a major challenge in drug formulation, and this problem is increasing, as currently about 40 of the marketed drugs and 90% of drug candidates are classified as poorly water soluble. Various approaches exist to circumvent poor water solubility and poor dissolution rate in aqueous environment, however, each having disadvantages and certain limitations. Recently, mesoporous silica materials (MSMs) have been proposed to be used as matrices for enhancing the apparent solubility and dissolution rate of different drug molecules. MSMs are ideal candidates for this purpose, as silica is a "generally regarded as safe" (GRAS) material, is biodegradable, and can be readily surface-modified in order to optimize drug loading and subsequent release in the human body. The major advantage of mesoporous silica as drug delivery systems (DDSs) for poorly water soluble drugs lies in their pore size, pore morphology, and versatility in alteration of the surface groups, which can result in optimized interactions between a drug candidate and MSM carrier by modifying the pore surfaces. Furthermore, the drug of interest can be loaded into these pores in a preferably amorphous state, which can increase the drug dissolution properties dramatically. The highlights of this review include a critical discussion about the modification of the physico-chemical properties of MSMs and how these physico-chemical modifications influence the drug loading and the subsequent dissolution of poorly water soluble drugs. It aims to further promote the use of MSMs as alternative strategy to common methods like solubility enhancement by cyclodextrins, micronization, or microemulsion techniques. This review can provide guidance on how to tailor MSMs to achieve optimized drug loading and drug dissolution.

Interactions between silica nanoparticles and phospholipid membranes.

UNLABELLED: Silica nanoparticles (SNPs) are widely used for biomedical applications. However, their parenteral administration may induce hemolysis. Molecular mechanisms leading to this effect are still controversially discussed. We therefore used a combination of biophysical techniques to investigate the interaction of hemolytic and non-hemolytic SNPs with model phospholipid membranes. METHODS: Interaction of SNPs with membranes was studied using a dye-leakage assay, dynamic light scattering (DLS), isothermal titration calorimetry, and solid state nuclear magnetic resonance. RESULTS AND DISCUSSION: The dye leakage assay revealed that only hemolytic, negatively charged SNPs, but not non-hemolytic positively charged SNPs, destabilized POPC based phospholipid bilayers. Interaction of SNPs with lipid vesicles leading to particle agglomeration was demonstrated by DLS. Isothermal titration calorimetry confirmed the interaction between negatively charged SNPs and phospholipids, which is characterized by an exothermic reaction enthalpy ΔH(0)SNP of -0.04cal/g at 25°C. Calorimetric titrations at different temperatures revealed a molar heat capacity change of zero. This finding excluded a contribution of electrostatic interactions. Mechanistic insight was provided by solid state phosphorus-31 NMR and deuterium NMR measurements. CONCLUSIONS: Our results demonstrate that electrostatic interaction between hemolytic SNPs and model phospholipid membranes is negligible. SNPs induce membrane destabilization and adsorptive processes induced by agglomeration of phospholipid vesicles. The interaction is driven by van der Waals forces at the level of the hydration layer on the vesicles surface.

Comparative safety evaluation of silica-based particles.

PURPOSE: Silica nanoparticles (SNPs) are increasingly used as drug delivery systems (DDS) and for biomedical imaging. Therapeutic and diagnostic agents can be incorporated into the silica matrix to improve the stability and dissolution of drug substances in biological systems. However, the safety of SNPs as drug carriers remains controversial. To date, no validated and accepted nano-specific tests exist to predict the potentially harmful impact of these materials on the human body. METHODS: We synthesized by a systematic approach 12 different types of SNPs with varying size, surface topology (porous vs non-porous), and surface modifications. We characterized these particles in terms of dry state and hydrodynamic diameter, specific surface area, and net surface charge (ζ-potential). For cellular studies, we exposed non-phagocytic (HepG2) cells, phagocytic (THP-1) cells, and erythrocytes to SNPs. Cellular uptake and stability of fluorescently labeled SNPs were analyzed by confocal microscopy and flow cytometry. RESULTS: SNPs with a porous surface and negative net surface charge had the strongest impact on cell viability. This is in contrast to non-porous SNPs. None of the studied particles induced oxidative stress in either cell lines. Particles with a negative surface charge induced hemolysis in a concentration-dependent manner. CONCLUSIONS: Physico-chemical properties promoting cytotoxicity and hemolysis were investigated. Our study revealed potential hazards of spherical amorphous SNPs.

ATP-induced cellular stress and mitochondrial toxicity in cells expressing purinergic P2X7 receptor.

Under pathological conditions, the purinergic P2X7 receptor is activated by elevated concentrations of extracellular ATP. Thereby, the receptor forms a slowly dilating pore, allowing cations and, upon prolonged stimulation, large molecules to enter the cell. This process has a strong impact on cell signaling, metabolism, and viability. This study aimed to establish a link between gradual P2X7 activation and pharmacological endpoints including oxidative stress, hydrogen peroxide generation, and cytotoxicity. Mechanisms of cellular stress and cytotoxicity were studied in P2X7-transfected HEK293 cells. We performed real-time monitoring of metabolic and respiratory activity of cells expressing the P2X7-receptor protein using a cytosensor system. Agonistic effects were monitored using exogenously applied ATP or the stable analogue BzATP. Oxidative stress induced by ATP or BzATP in target cells was monitored by hydrogen peroxide release in human mononuclear blood cells. P2X7-receptor activation was studied by patch-clamp experiments using a primary mouse microglia cell line. Stimulation of the P2X7 receptor leads to ion influx, metabolic activation of target cells, and ultimately cytotoxicity. Conversion of the P2X7 receptor from a small cation channel to a large pore occurring under prolonged stimulation can be monitored in real time covering a time frame of milliseconds to hours. Selectivity of the effects can be demonstrated using the selective P2X7-receptor antagonist AZD9056. Our findings established a direct link between P2X7-receptor activation by extracellular ATP or BzATP and cellular events culminating in cytotoxicity. Mechanisms of toxicity include metabolic and oxidative stress, increase in intracellular calcium concentration and disturbance of mitochondrial membrane potential. Mitochondrial toxicity is suggested to be a key event leading to cell death.

Polymersomes containing quantum dots for cellular imaging.

Quantum dots (QDs) are highly fluorescent and stable probes for cellular and molecular imaging. However, poor intracellular delivery, stability, and toxicity of QDs in biological compartments hamper their use in cellular imaging. To overcome these limitations, we developed a simple and effective method to load QDs into polymersomes (Ps) made of poly(dimethylsiloxane)-poly(2-methyloxazoline) (PDMS-PMOXA) diblock copolymers without compromising the characteristics of the QDs. These Ps showed no cellular toxicity and QDs were successfully incorporated into the aqueous compartment of the Ps as confirmed by transmission electron microscopy, fluorescence spectroscopy, and fluorescence correlation spectroscopy. Ps containing QDs showed colloidal stability over a period of 6 weeks if stored in phosphate-buffered saline (PBS) at physiological pH (7.4). Efficient intracellular delivery of Ps containing QDs was achieved in human liver carcinoma cells (HepG2) and was visualized by confocal laser scanning microscopy (CLSM). Ps containing QDs showed a time- and concentration-dependent uptake in HepG2 cells and exhibited better intracellular stability than liposomes. Our results suggest that Ps containing QDs can be used as nanoprobes for cellular imaging.

Engineered nanomaterial uptake and tissue distribution: from cell to organism.

Improved understanding of interactions between nanoparticles and biological systems is needed to develop safety standards and to design new generations of nanomaterials. This article reviews the molecular mechanisms of cellular uptake of engineered nanoparticles, their intracellular fate, and their distribution within an organism. We have reviewed the available literature on the uptake and disposition of engineered nanoparticles. Special emphasis was placed on the analysis of experimental systems and their limitations with respect to their usefulness to predict the in vivo situation. The available literature confirms the need to study particle characteristics in an environment that simulates the situation encountered in biological systems. Phenomena such as protein binding and opsonization are of prime importance since they may have a strong impact on cellular internalization, biodistribution, and immunogenicity of nanoparticles in vitro and in vivo. Extrapolation from in vitro results to the in vivo situation in the whole organism remains a challenge. However, improved understanding of physicochemical properties of engineered nanoparticles and their influence on biological systems facilitates the design of nanomaterials that are safe, well tolerated, and suitable for diagnostic or therapeutic use in humans.

A cell-based, multiparametric sensor approach characterises drug-induced cytotoxicity in human liver HepG2 cells.

Drug-induced toxicity is of considerable concern in drug discovery and development, placing emphasis on the need for predictive in vitro technologies that identify potential cytotoxic side effects of drugs. A label-free, real-time, multiparametric cytosensor system has therefore been established for in vitro assessment of drug-induced toxicity. The system is based on monitoring cellular oxygen consumption, acidification and impedance of human hepatocarcinoma-derived HepG2 cells. The read-out derived from the multiparametric cytosensor system has been optimised and permits sensitive, reliable, and simultaneous recording of cell physiological signals, such as metabolic activity, cellular respiration and morphological changes and cell adhesion upon exposure to a drug. Analysis of eight prototypic reference drugs revealed distinct patterns of drug-induced physiological signals. Effects proved to be rigidly concentration-dependent. Based on signal patterns and reversibility of the observed effects, compounds could be classified based as triggering mechanisms of respiratory or metabolic stress or conditions leading to cell death (necrosis-like and apoptosis-like). A test-flag-risk mitigation strategy is proposed to address potential risks for drug-induced cytotoxicity.