Logic-embedded vectors for intracellular partitioning, endosomal escape, and exocytosis of nanoparticles

A new generation of nanocarriers, logic-embedded vectors (LEVs), is endowed with the ability to localize components at multiple intracellular sites, thus creating an opportunity for synergistic control of redundant or dual-hit pathways. LEV encoding elements include size, shape, charge, and surface chemistry. In this study, LEVs consist of porous silicon nanocarriers, programmed for cellular uptake and trafficking along the endosomal pathway, and surface-tailored iron oxide nanoparticles, programmed for endosomal sorting and partitioning of particles into unique cellular locations. In the presence of persistent endosomal localization of silicon nanocarriers, amine-functionalized nanoparticles are sorted into multiple vesicular bodies that form novel membrane-bound compartments compatible with cellular secretion, while chitosan-coated nanoparticles escape from endosomes and enter the cytosol. Encapsulation within the porous silicon matrix protects these nanoparticle surface-tailored properties, and enhances endosomal escape of chitosan-coated nanoparticles. Thus, LEVs provide a mechanism for shielded transport of nanoparticles to the lesion, cellular manipulation at multiple levels, and a means for targeting both within and between cells.     

Endosomal escape of protein nanoparticles engineered through humanized histidine-rich peptides

Poly-histidine peptides such as H6(HHHHHH)are used in protein biotechnologies as purification tags,protein-assembling agents and endosomal-escape entities.The pleiotropic properties of such peptides make them appealing to design protein-based smart materials or nanoparticles for imaging or drug delivery to be produced in form of recombinant proteins.However,the clinical applicability of H6-tagged proteins is restricted by the potential immunogenicity of these segments.In this study,we have explored several humanized histidine-rich peptides in tumor-targeted modular proteins,which can specifically bind and be internalized by the target cells through the tumoral marker CXCR4.We were particularly interested in exploring how protein purification,self-assembling and endosomal escape perform in proteins containing the variant histidine-rich tags.Among the tested candidates,the peptide H5 E(HEHEHEHEH)is promising as a good promoter of endosomal escape of the associated fulllength protein upon endosomal internalization.The numerical modelling of cell penetration and endosomal escape of the tested proteins has revealed a negative relationship between the amount of protein internalized into target cells and the efficiency of cytoplasmic release.This fact demonstrates that the His-mediated,proton sponge-based endosomal escape saturates at moderate amounts of internalized protein,a fact that might be critical for the design of protein materials for cytosolic molecular delivery.     

Drug delivery: Logic‐Embedded Vectors for Intracellular Partitioning,Endosomal Escape,and Exocytosis of Nanoparticles (Small 23/2010)

A new generation of nanocarriers, logic‐embedded vectors (LEVs), is endowed with the ability to localize components at multiple intracellular sites, thus creating an opportunity for synergistic control of redundant or dual‐hit pathways. LEV encoding elements include size, shape, charge, and surface chemistry. In this study, LEVs consist of porous silicon nanocarriers, programmed for cellular uptake and trafficking along the endosomal pathway, and surface‐tailored iron oxide nanoparticles, programmed for endosomal sorting and partitioning of particles into unique cellular locations. In the presence of persistent endosomal localization of silicon nanocarriers, amine‐functionalized nanoparticles are sorted into multiple vesicular bodies that form novel membrane‐bound compartments compatible with cellular secretion, while chitosan‐coated nanoparticles escape from endosomes and enter the cytosol. Encapsulation within the porous silicon matrix protects these nanoparticle surface‐tailored properties, and enhances endosomal escape of chitosan‐coated nanoparticles. Thus, LEVs provide a mechanism for shielded transport of nanoparticles to the lesion, cellular manipulation at multiple levels, and a means for targeting both within and between cells.     

Inflicting controlled nonthermal damage to subcellular structures by laser-activated gold nanoparticles

We show that low-intensity laser irradiation of cancer cells containing endosomal gold nanoparticles leads to endosome rupture and escape of the nanoparticles into the cytosol without affecting the cells' viability. The low light intensity of our experiments allows us to rule out photothermal effects as the underlying mechanism, and we present results that suggest photoinduced radicals as the photogenerated active species. This nonthermal mechanism may also be important in the context of cell death at higher laser intensities, which had been reported previously.     

Protein-conjugated quantum dots effectively delivered into living cells by a cationic nanogel

Quantum dots (QDs) have attracted attention for their potential as a cell imaging regent. However, the development of effective intracellular delivery system for QDs is needed to apply various cell lines without affecting cellular function. We reported here new QDs delivery system by using cationic nanogel consisting of cholesterol-bearing pullulan modified with an amino group (CHPNH2). The uptake of hybrid nanoparticles into HeLa cells was followed by flow cytometry, and confocal laser scanning fluorescence microscopy. Protein-conjugated QDs were effectively internalized into cells by the nanogel compared with a cationic liposome system. The hybrid nanoparticle was used to stain rabbit mesenchymal stem cells (MSCs) so as to evaluate their effect on cell function. CHPNH2-QD hybrid nanoparticles remained detectable inside MSCs for at least 2 weeks of culture and had little effect on the in vitro chondrogenic ability of MSCs. The hybrid nanoparticles are a promising candidate as a cell tracer in tissue engineering.     

An Experimental and Computational Approach to the Development of ZnO Nanoparticles that are Safe by Design

Zinc oxide nanoparticles have found wide application due to their unique optoelectronic and photocatalytic characteristics. However, their safety aspects remain of critical concern, prompting the use of physicochemical modifications of pristine ZnO to reduce any potential toxicity. However, the relationships between these modifications and their effects on biology are complex and still relatively unexplored. To address this knowledge gap, a library of 45 types of ZnO nanoparticles with varying particle size, aspect ratio, doping type, doping concentration, and surface coating is synthesized, and their biological effects measured. Three biological assays measuring cell damage or stress are used to study the responses of human umbilical vein endothelial cells (HUVECs) or human hepatocellular liver carcinoma cells (HepG2) to the nanoparticles. These experimental data are used to develop quantitative and predictive computational models linking nanoparticle properties to cell viability, membrane integrity, and oxidative stress. It is found that the concentration of nanoparticles the cells are exposed to, the type of surface coating, the nature and extent of doping, and the aspect ratio of the particles make significant contributions to the cell toxicity of the nanoparticles tested. Our study shows that it is feasible to generate models that could be used to design or optimize nanoparticles with commercially useful properties that are also safe to humans and the environment.     

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.     

Nanoparticles for cellular drug delivery: mechanisms and factors influencing delivery

Polymeric nanoparticles have demonstrated enormous potential as cellular drug delivery vehicles. Nanoparticles improve drug's stability as well as its availability and retention at the target intracellular site of action. Therapeutic efficacy of nanoparticles can be further enhanced by conjugating specific ligands to nanoparticle surface. Ligand conjugation can also be used to favorably modify the intracellular disposition of nanoparticles. A number of ligands are available for this purpose; use of a specific ligand depends on the target cell, the material used for nanoparticle formulation, and the chemistry available for ligand-nanoparticle conjugation. Cellular drug delivery using nanoparticles is also affected by clearance through the reticuloendothelial system. In this paper, we review the recent progress on our understanding of physicochemical factors that affect the cellular uptake of nanoparticles and the different cellular processes that could be exploited to enhance nanoparticle uptake into cells.     

Cytocompatible and multifunctional polymeric nanoparticles for transportation of bioactive molecules into and within cells

Multifunctional polymeric nanoparticles are materials with great potential for a wide range of biomedical applications. For progression in this area of research, unfavorable interactions of these nanoparticles with proteins and cells must be avoided in biological environments, for example, through treatment of the nanoparticle surfaces. Construction of an artificial cell membrane structure based on polymers bearing the zwitterionic phosphorylcholine group can prevent biological reactions at the surface effectively. In addition, certain bioactive molecules can be immobilized on the surface of the polymer to generate enough affinity to capture target biomolecules. Furthermore, entrapment of inorganic nanoparticles inside polymeric matrices enhances the nanoparticle functionality significantly. This review summarizes the preparation and characterization of cytocompatible and multifunctional polymeric nanoparticles; it analyzes the efficiency of their fluorescence function, the nature of the artificial cell membrane structure, and their performance as in-cell devices; and finally, it evaluates both their chemical reactivity and effects in cells.     

Role of cell cycle on the cellular uptake and dilution of nanoparticles in a cell population

Nanoparticles are considered a primary vehicle for targeted therapies because they can pass biological barriers and enter and distribute within cells by energy-dependent pathways. So far, most studies have shown that nanoparticle properties, such as size and surface, can influence how cells internalize nanoparticles. Here, we show that uptake of nanoparticles by cells is also influenced by their cell cycle phase. Although cells in different phases of the cell cycle were found to internalize nanoparticles at similar rates, after 24 h the concentration of nanoparticles in the cells could be ranked according to the different phases: G2/M > S > G0/G1. Nanoparticles that are internalized by cells are not exported from cells but are split between daughter cells when the parent cell divides. Our results suggest that future studies on nanoparticle uptake should consider the cell cycle, because, in a cell population, the dose of internalized nanoparticles in each cell varies as the cell advances through the cell cycle.     

Synthesis, stability, and cellular internalization of gold nanoparticles containing mixed peptide-poly(ethylene glycol) monolayers

Gold nanoparticles have shown great promise as therapeutics, therapeutic delivery vectors, and intracellular imaging agents. For many biomedical applications, selective cell and nuclear targeting are desirable, and these remain a significant practical challenge in the use of nanoparticles in vivo. This challenge is being addressed by the incorporation of cell-targeting peptides or antibodies onto the nanoparticle surface, modifications that frequently compromise nanoparticle stability in high ionic strength biological media. We describe herein the assembly of poly(ethylene glycol) (PEG) and mixed peptide/PEG monolayers on gold nanoparticle surfaces. The stability of the resulting bioconjugates in high ionic strength media was characterized as a function of nanoparticle size, PEG length, and monolayer composition. In total, three different thiol-modified PEGs (average molecular weight (MW), 900, 1500, and 5000 g mol-1), four particle diameters (10, 20, 30, and 60 nm), and two cell-targeting peptides were explored. We found that nanoparticle stability increased with increasing PEG length, decreasing nanoparticle diameter, and increasing PEG mole fraction. The order of assembly also played a role in nanoparticle stability. Mixed monolayers prepared via the sequential addition of PEG followed by peptide were more stable than particles prepared via simultaneous co-adsorption. Finally, the ability of nanoparticles modified with mixed PEG/RME (RME = receptor-mediated endocytosis) peptide monolayers to target the cytoplasm of HeLa cells was quantified using inductively coupled plasma optical emission spectrometry (ICP-OES). Although it was anticipated that the MW 5000 g mol-1 PEG would sterically block peptides from access to the cell membrane compared to the MW 900 PEG, nanoparticles modified with mixed peptide/PEG 5000 monolayers were internalized as efficiently as nanoparticles containing mixed peptide/PEG 900 monolayers. These studies can provide useful cues in the assembly of stable peptide/gold nanoparticle bioconjugates capable of being internalized into cells.     

Erythrocyte–Platelet Hybrid Membrane Coating for Enhanced Nanoparticle Functionalization

Cell‐membrane‐coated nanoparticles have recently been studied extensively for their biological compatibility, retention of cellular properties, and adaptability to a variety of therapeutic and imaging applications. This class of nanoparticles, which has been fabricated with a variety of cell membrane coatings, including those derived from red blood cells (RBCs), platelets, white blood cells, cancer cells, and bacteria, exhibit properties that are characteristic of the source cell. In this study, a new type of biological coating is created by fusing membrane material from two different cells, providing a facile method for further enhancing nanoparticle functionality. As a proof of concept, the development of dual‐membrane‐coated nanoparticles from the fused RBC membrane and platelet membrane is demonstrated. The resulting particles, termed RBC–platelet hybrid membrane‐coated nanoparticles ([RBC‐P]NPs), are thoroughly characterized, and it is shown that they carry properties of both source cells. Further, the [RBC‐P]NP platform exhibits long circulation and suitability for further in vivo exploration. The reported strategy opens the door for the creation of biocompatible, custom‐tailored biomimetic nanoparticles with varying hybrid functionalities, which may be used to overcome the limitations of current nanoparticle‐based therapeutic and imaging platforms.     

Flow Rate Affects Nanoparticle Uptake into Endothelial Cells

Nanoparticles are commonly administered through systemic injection, which exposes them to the dynamic environment of the bloodstream. Injected nanoparticles travel within the blood and experience a wide range of flow velocities that induce varying shear rates to the blood vessels. Endothelial cells line these vessels, and have been shown to uptake nanoparticles during circulation, but it is difficult to characterize the flow-dependence of this interaction in vivo. Here, a microfluidic system is developed to control the flow rates of nanoparticles as they interact with endothelial cells. Gold nanoparticle uptake into endothelial cells is quantified at varying flow rates, and it is found that increased flow rates lead to decreased nanoparticle uptake. Endothelial cells respond to increased flow shear with decreased ability to uptake the nanoparticles. If cells are sheared the same way, nanoparticle uptake decreases as their flow velocity increases. Modifying nanoparticle surfaces with endothelial-cell-binding ligands partially restores uptake to nonflow levels, suggesting that functionalizing nanoparticles to bind to endothelial cells enables nanoparticles to resist flow effects. In the future, this microfluidic system can be used to test other nanoparticle–endothelial cell interactions under flow. The results of these studies can guide the engineering of nanoparticles for in vivo medical applications.     

Can silver nanoparticles be useful as potential biological labels?

Silver (Ag) nanoparticles have unique plasmon-resonant optical scattering properties that are finding use in nanomedical applications such as signal enhancers, optical sensors, and biomarkers. In this study, we examined the chemical and biological properties of Ag nanoparticles of similar sizes, but that differed primarily in their surface chemistry (hydrocarbon versus polysaccharide), in neuroblastoma cells for their potential use as biological labels. We observed strong optical labeling of the cells in a high illumination light microscopy system after 24?h of incubation due to the excitation of plasmon resonance by both types of Ag nanoparticle. Surface binding of both types of Ag nanoparticle to the plasma membrane of the cells was verified with scanning electron microscopy as well as the internalization and localization of the Ag nanoparticles into intracellular vacuoles in thin cell sections with transmission electron microscopy. However, the induction of reactive oxygen species (ROS), degradation of mitochondrial membrane integrity, disruption of the actin cytoskeleton, and reduction in proliferation after stimulation with nerve growth factor were found after incubation with Ag nanoparticles at concentrations of 25?μg?ml(-1) or greater, with a more pronounced effect produced by the hydrocarbon-based Ag nanoparticles in most cases. Therefore, the use of Ag nanoparticles as potential biological labels, even if the surface is chemically modified with a biocompatible material, should be approached with caution.     

Uptake and Intracellular Fate of Engineered Nanoparticles in Mammalian Cells: Capabilities and Limitations of Transmission Electron Microscopy—Polymer‐Based Nanoparticles

In order to elucidate mechanisms of nanoparticle (NP)–cell interactions, a detailed knowledge about membrane–particle interactions, intracellular distributions, and nucleus penetration capabilities, etc. becomes indispensable. The utilization of NPs as additives in many consumer products, as well as the increasing interest of tailor‐made nanoobjects as novel therapeutic and diagnostic platforms, makes it essential to gain deeper insights about their biological effects. Transmission electron microscopy (TEM) represents an outstanding method to study the uptake and intracellular fate of NPs, since this technique provides a resolution far better than the particle size. Additionally, its capability to highlight ultrastructural details of the cellular interior as well as membrane features is unmatched by other approaches. Here, a summary is provided on studies utilizing TEM to investigate the uptake and mode‐of‐action of tailor‐made polymer nanoparticles in mammalian cells. For this purpose, the capabilities as well as limitations of TEM investigations are discussed to provide a detailed overview on uptake studies of common nanoparticle systems supported by TEM investigations. Furthermore, methodologies that can, in particular, address low‐contrast materials in electron microscopy, i.e., polymeric and polymer‐modified nanoparticles, are highlighted.     

Silica nanoparticle induces oxidative stress and provokes inflammation in human lung cells

Amorphous silica has been used in a wide variety of industrial and consumer applications. Nanosized silica is being considered a potentially ideal nanomaterial for biomedical and biotechnological applications. In the present study, hierarchical oxidative stress paradigms of nanoparticle toxicity were assumed to evaluate the toxicity of amorphous silica nanoparticle in human healthy lung cells, L-132. The cells were exposed to varied concentrations viz. 0, 100, 200, 300, 400 and 500 μg/ml of silica nanoparticle for 24 h. Different parameters of cytotoxicity, oxidative stress and pro-inflammatory markers were assessed. Silica nanoparticle exposure showed concentration-dependent decrease in cell viability, increase in reactive oxygen species with depletion of antioxidant enzyme activity, induction of inteleukin-8 and cyclooxygenase-2 expression and subsequent DNA damage in L-132 cells. The results indicated that amorphous silica nanoparticle followed hierarchical oxidative stress model and generated oxidative stress which provoked the pro-inflammatory response and caused necrosis in human lung cells.     

In vitro safety toxicology data for evaluation of gold nanoparticles-chronic cytotoxicity, genotoxicity and uptake

Safety and toxic effects of nanoparticles are still largely unexplored due to the multiple aspects that influence their behaviour toward biological systems. Here, we focus the attention on 12 nm spherical gold nanoparticle coated or not with hyaluronic acid compared to its precursor counterpart salt. Results ranging from the effects of a 10-days exposure in an in vitro model with BALB/c 3T3 fibroblast cells show how 12 nm spherical gold nanoparticles are internalized from 3T3 cells by endo-lysosomal pathway by an indirect measurement technique; and how gold nanoparticles, though not being a severe cytotoxicant, induce DNA damage probably through an indirect mechanism due to oxidative stress. While coating them with hyaluronic acid reduces gold nanoparticles cytotoxicity and slows their cell internalization. These results will be of great interest to medicine, since they indicate that gold nanoparticles (with or without coating) are suitable for therapeutic applications due to their tunable cell uptake and low toxicity.     

SiRNA drug delivery by biodegradable polymeric nanoparticles

RNA interference (RNAi) is an emerging technology in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. It offers a broad spectrum of applications in both biological and medical research. Small interference RNA (siRNA) was recently explored for its therapeutical potential. However, the drug delivery of siRNA oligos is very novel and is in great need of future research. To this end, a biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanoparticle drug carrier system was prepared to load siRNA oligos with desired physicochemical properties. The nanoparticles were characterized by scanning electron microscopy and laser diffraction particle sizer. The delivery of siRNA into the targeted 293T cells was observed using fluorescent-labeled double-stranded Cy3-oligos. The model siRNA oligos, si-GFP-RNA, were also successfully loaded into PLGA nanoparticles and delivered in 293T cells. The gene silencing effect and the inhibition of GFP expression were investigated using fluorescent microscopy. Both positive and negative controls were used to compare with the new siRNA nanoparticle delivery system. It was found that nanoparticles offered both effective delivery of siRNA and prominent GFP gene silencing effect. Compared to conventional carrier systems, the new biodegradable polymeric nanoparticle system may also offer improved formulation stability, which is practically beneficial and may be used in the future clinical studies of siRNA therapeutics.     

Detection of Circulating Cancer Cells Using Electrocatalytic Gold Nanoparticles

A rapid cancer cell detection and quantification assay, based on the electrocatalytic properties of gold nanoparticles towards the hydrogen evolution reaction, is described. The selective labeling of cancer cells is performed in suspension, allowing a fast interaction between the gold nanoparticle labels and the target proteins expressed at the cell membrane. The subsequent electrochemical detection is accomplished with small volumes of sample and user‐friendly equipment through a simple electrochemical method that generates a fast electrochemical response used for the quantification of nanoparticle‐labeled cancer cells. The system establishes a selective cell‐detection assay capable of detecting 4 × 10³ cancer cells in suspension that can be extended to several other cells detection scenarios.