Common gene therapy viral vectors do not efficiently penetrate sputum from cystic fibrosis patients

Norwalk virus and human papilloma virus, two viruses that infect humans at mucosal surfaces, have been found capable of rapidly penetrating human mucus secretions. Viral vectors for gene therapy of Cystic Fibrosis (CF) must similarly penetrate purulent lung airway mucus (sputum) to deliver DNA to airway epithelial cells. However, surprisingly little is known about the rates at which gene delivery vehicles penetrate sputum, including viral vectors used in clinical trials for CF gene therapy. We find that sputum spontaneously expectorated by CF patients efficiently traps two viral vectors commonly used in CF gene therapy trials, adenovirus (d～80 nm) and adeno-associated virus (AAV serotype 5; d～20 nm), leading to average effective diffusivities that are ～3,000-fold and 12,000-fold slower than their theoretical speeds in water, respectively. Both viral vectors are slowed by adhesion, as engineered muco-inert nanoparticles with diameters as large as 200 nm penetrate the same sputum samples at rates only ～40-fold reduced compared to in pure water. A limited fraction of AAV exhibit sufficiently fast mobility to penetrate physiologically thick sputum layers, likely because of the lower viscous drag and smaller surface area for adhesion to sputum constituents. Nevertheless, poor penetration of CF sputum is likely a major contributor to the ineffectiveness of viral vector based gene therapy in the lungs of CF patients observed to date.     

Immobilization of Pseudorabies Virus in Porcine Tracheal Respiratory Mucus Revealed by Single Particle Tracking

Pseudorabies virus (PRV) initially replicates in the porcine upper respiratory tract. It easily invades the mucosae and submucosae for subsequent spread throughout the body via blood vessels and nervous system. In this context, PRV developed ingenious processes to overcome different barriers such as epithelial cells and the basement membrane. Another important but often overlooked barrier is the substantial mucus layer which coats the mucosae. However, little is known about how PRV particles interact with porcine respiratory mucus. We therefore measured the barrier properties of porcine tracheal respiratory mucus, and investigated the mobility of nanoparticles including PRV in this mucus. We developed an in vitro model utilizing single particle tracking microscopy. Firstly, the mucus pore size was evaluated with polyethylene glycol coupled (PEGylated) nanoparticles and atomic force microscope. Secondly, the mobility of PRV in porcine tracheal respiratory mucus was examined and compared with that of negative, positive and PEGylated nanoparticles. The pore size of porcine tracheal respiratory mucus ranged from 80 to 1500 nm, with an average diameter of 455±240 nm. PRV (zeta potential: −31.8±1.5 mV) experienced a severe obstruction in porcine tracheal respiratory mucus, diffusing 59-fold more slowly than in water. Similarly, the highly negatively (−49.8±0.6 mV) and positively (36.7±1.1 mV) charged nanoparticles were significantly trapped. In contrast, the nearly neutral, hydrophilic PEGylated nanoparticles (−9.6±0.8 mV) diffused rapidly, with the majority of particles moving 50-fold faster than PRV. The mobility of the particles measured was found to be related but not correlated to their surface charge. Furthermore, PEGylated PRV (-13.8±0.9 mV) was observed to diffuse 13-fold faster than native PRV. These findings clearly show that the mobility of PRV was significantly hindered in porcine tracheal respiratory mucus, and that the obstruction of PRV was due to complex mucoadhesive interactions including charge interactions rather than size exclusion.     

Altering Mucus Rheology to “Solidify” Human Mucus at the Nanoscale

The ability of mucus to function as a protective barrier at mucosal surfaces rests on its viscous and elastic properties, which are not well understood at length scales relevant to pathogens and ultrafine environmental particles. Here we report that fresh, undiluted human cervicovaginal mucus (CVM) transitions from an impermeable elastic barrier to non-adhesive objects sized 1 µm and larger to a highly permeable viscoelastic liquid to non-adhesive objects smaller than 500 nm in diameter. Addition of a nonionic detergent, present in vaginal gels, lubricants and condoms, caused CVM to behave as an impermeable elastic barrier to 200 and 500 nm particles, suggesting that the dissociation of hydrophobically-bundled mucin fibers created a finer elastic mucin mesh. Surprisingly, the macroscopic viscoelasticity, which is critical to proper mucus function, was unchanged. These findings provide important insight into the nanoscale structural and barrier properties of mucus, and how the penetration of foreign particles across mucus might be inhibited.     

Diffusion of macromolecules and virus-like particles in human cervical mucus

To determine whether or not large macromolecules and viruses can diffuse through mucus, we observed the motion of proteins, microspheres, and viruses in fresh samples of human cervical mucus using fluorescent recovery after photobleaching and multiple image photography. Two capsid virus-like particles, human papilloma virus (55 nm, approximately 20,000 kDa) and Norwalk virus (38 nm, approximately 10,000 kDa), as well as most of the globular proteins tested (15-650 kDa) diffused as rapidly in mucus as in saline. Electron microscopy of cervical mucus confirmed that the mesh spacing between mucin fibers is large enough (20-200 nm) for small viruses to diffuse essentially unhindered through mucus. In contrast, herpes simplex virus (180 nm) colocalized with strands of thick mucus, suggesting that herpes simplex virus, unlike the capsid virus particles, makes low-affinity bonds with mucins. Polystyrene microspheres (59-1000 nm) bound more tightly to mucins, bundling them into thick cables. Although immunoglobulins are too small to be slowed by the mesh spacing between mucins, diffusion by IgM was slowed by mucus. Diffusion by IgM-Fc(5 mu), the Fc pentamer core of an IgM with all 10 Fab moieties removed, was comparably slowed by mucus. This suggests that the Fc moieties of antibodies make low-affinity bonds with mucins.     

Transport of polymeric nanoparticle gene carriers in gastric mucus

Nanoparticle transport through mucosal barriers is often restricted owing to mucoadhesion and the highly viscoelastic nature of mucus gels, which may limit efficient drug and gene delivery. We formulated sub-200 nm particulates from poly(d,l-lactic-co-glycolic) acid (PLGA) and the cationic surfactant dimethyldioctadecylammonium bromide (DDAB). Subsequently, anionic DNA was condensed to the surface to obtain gene carriers with transfection rates 50-fold higher than those of naked DNA in vitro. Using the method of multiple particle tracking (MPT), we measured the transport rates of dozens of individual PLGA-DDAB/DNA nanoparticles in real time in reconstituted pig gastric mucus (PGM) that possessed physiologically relevant rheological properties. The average transport rate of PLGA-DDAB/DNA nanoparticles was 10-fold higher than those of similar size polystyrene nanoparticles. Improved transport rates, stability in mucus, and ability to transfect cells make PLGA-DDAB/DNA nanoparticles candidates for mucosal DNA vaccines and gene therapy.     

Mucin/poly(acrylic acid) interactions: a spectroscopic investigation of mucoadhesion

Studies using infrared, (1)H and (13)C nuclear magnetic resonance, and X-ray photoelectron spectroscopies and differential scanning calorimetry support the hypothesis that hydrogen bonds, formed between the carboxylic acid functionality of the mucoadhesive material poly(acrylic acid) and the glycoprotein component of mucus, play a significant role in the process of mucoadhesion. There are fewer H-bonded interactions between the components than within the bulk of the pure mucoadhesive agent. The pH of the medium influences the structures of both the poly(acrylic acid) and the mucus, which, in turn, determine the nature and the extent of mucoadhesive interactions.     

Enhanced viscoelasticity of human cystic fibrotic sputum correlates with increasing microheterogeneity in particle transport

Current biochemical characterizations of cystic fibrosis (CF) sputum do not address the high degree of microheterogeneity in the rheological properties of the mucosal matrix and only provide bulk-average particle diffusion coefficients. The viscoelasticity of CF sputum greatly reduces the diffusion rates of colloidal particles, limiting the effectiveness of gene delivery to underlying lung cells. We determine diffusion coefficients of hundreds of individual amine-modified and carboxylated polystyrene particles (diameter 100-500 nm) embedded in human CF sputum with 5 nm and 33 ms of spatiotemporal resolution. High resolution multiple particle tracking is used to calculate the effective viscoelastic properties of CF sputum at the micron scale, which we relate to its macroscopic viscoelasticity. CF sputum microviscosity, as probed by 100- and 200-nm particles, is an order of magnitude lower than its macroviscosity, suggesting that nanoparticles dispersed in CF sputum are transported primarily through lower viscosity pores within a highly elastic matrix. Multiple particle tracking provides a non-destructive, highly sensitive method to quantify the high heterogeneity of the mucus pore network. The mean diffusion coefficient becomes dominated by relatively few but fast-moving particles as particle size is reduced from 500 to 100 nm. Neutrally charged particles with a diameter <200 nm undergo more rapid transport in CF sputum than charged particles. Treatment with recombinant human DNase (Pulmozyme) reduces macroviscoelastic properties of CF sputum by up to 50% and dramatically narrows the distribution of individual particle diffusion rates but surprisingly does not significantly alter the ensemble-average particle diffusion rate.     

Recent Developments in Nanotechnology and Risk Assessment Strategies for Addressing Public and Environmental Health Concerns

Nanotechnology is a novel emerging technology that allows the manipulation of materials at the scale comparable to the size of a single molecule (i.e., < 100 nm). There have been many new developments in this technology, resulting in complex exposure and health risk implications. Nanotechnology offers major benefits to humankind; however, there is growing concern regarding the potential adverse interactions of engineered nanoparticles at cellular or sub-cellular levels. The nanotech community is therefore experiencing growing calls for legislation to minimize or prevent exposure to nanoparticles. This article focuses on recent developments in nanotechnology including current manufacturing techniques, uses of nanoscale particles, and implications for particle toxicity and human exposure pathways. Current risk assessment methods are reviewed in the context of nanoparticle exposure routes and regulation for human and environmental health protection. This study provides a better understanding of the factors governing risks from nanoparticles and current strategies for protecting environmental and public health.     

Increased nanoparticle penetration in collagenase-treated multicellular spheroids

The extracellular matrix of solid tumors presents a transport barrier that restricts nanoparticle penetration, thereby limiting the efficacy of nano-sized delivery vehicles for cancer imaging and therapy. In this study, the effect of nanoparticle size and collagenase treatment on penetration of carboxylated polystyrene nanoparticles was systematically assessed in a multicellular spheroid model. Penetration of the nanoparticles into the spheroid core was limited to particles smaller than 100 nm. Collagenase treatment of spheroids resulted in significantly increased penetration of nanoparticles up to 100 nm with only a minor increase in particle penetration observed for particles larger than 100 nm. Collagenase was immobilized onto the surface of nanoparticles for site-specific degradation of ECM proteins. Collagenase-coated, 100 nm nanoparticles demonstrated a 4-fold increase in the number of particles delivered to the spheroid core compared with control nanoparticles. Thus, nanoparticle delivery to solid tumors may be substantially improved by the incorporation of ECM-modulating enzymes in the delivery formulation.     

Mucoadhesive Chitosan-Pectinate Nanoparticles for the Delivery of Curcumin to the Colon

In the present study, we report the properties of a mucoadhesive chitosan-pectinate nanoparticulate formulation able to retain its integrity in the milieu of the upper gastrointestinal tract and subsequently, mucoadhere and release curcumin in colon conditions. Using this system, we aimed to deliver curcumin to the colon for the possible management of colorectal cancer. The delivery system comprised of a chitosan-pectinate composite nanopolymeric with a z-average of 206.0 nm (±6.6 nm) and zeta potential of +32.8 mV (±0.5 mV) and encapsulation efficiency of 64%. The nanoparticles mucoadhesiveness was higher at alkaline pH compared to acidic pH. Furthermore, more than 80% release of curcumin was achieved in pectinase-enriched medium (pH 6.4) as opposed to negligible release in acidic and enzyme-restricted media at pH 6.8. SEM images of the nanoparticles after exposure to the various media indicate a retained matrix in acid media as opposed to a distorted/fragmented matrix in pectinase-enriched medium. The data strongly indicates that the system has the potential to be applied as a colon-targeted mucoadhesive curcumin delivery system for the possible treatment of colon cancer.     

Assembly of collagen fibril meshes using gold nanoparticles functionalized with tris(hydroxymethyl)phosphine-alanine as multivalent cross-linking agents

We report on the use of tris(hydroxymethyl)phosphine-alanine (THPAL) functionalized gold nanoparticles as a multivalent cross-linking agent to assemble collagen fibrils into a mesh-like structure. Atomic force microscopy (AFM) was used for characterization of the structure after adsorption onto an atomically flat mica substrate, revealing a mesh-like construct in which the collagen fibrils and the gold nanoparticles interact to form interconnected nodes measuring from 100 to 500 nm. As expected, the density of the collagen mesh can be increased with a higher initial concentration of gold nanoparticles. The maximum thickness of the meshes (～ 20 nm) obtained through cross-sectional height measurements confirms that the adsorbed structure consists of a single layer of collagen fibrils/gold nanoparticles assembled in two-dimensions. We propose that the capability of gold nanoparticles functionalized with the THPAL to bind to several collagen fibrils combined with the large persistence length of the fibrils, which was reported to be in the hundreds of nanometer range, are determinant factors for the preferential 2D growth of the mesh in solution.     

Viscoelastic properties and dynamics of porcine gastric mucin

Gastric mucin is a glycoprotein known to undergo a pH-dependent sol-gel transition that is crucial to the protective function of the gastric mucus layer in mammalian stomachs. We present microscope-based dynamic light scattering data on porcine gastric mucin at pH 6 (solution) and pH 2 (gel) with and without the presence of tracer particles. The data provide a measurement of the microscale viscosity and the shear elastic modulus as well as an estimate of the mesh size of the gel formed at pH 2. We observe that the microscale viscosity in the gel is about 100-fold lower than its macroscopic viscosity, suggesting that large pores open up in the gel reducing frictional effects. The data presented here help to characterize physiologically relevant viscoelastic properties of an important biological macromolecule and may also serve to shed light on diffusive motion of small particles in the complex heterogeneous environment of a polymer gel network.     

The Impact of Vehicles on the Mucoadhesive Properties of Orally Administrated Nanoparticles: a Case Study with Chitosan-4-Thiobutylamidine Conjugate

The aim of this study was to evaluate the impact of various vehicles on mucoadhesive properties of thiolated chitosan nanoparticles both in vitro and in vivo. Nanoparticles (NPs) were prepared by in situ gelation technique followed by labeling with fluorescein diacetate. Comparative studies on mucoadhesion were done with these thiolated chitosan NPs and unmodified chitosan NPs (control). The obtained nanoparticles displayed a mean diameter of 164.2 ± 6.9 nm and a zeta potential of 21.5 ± 5 mV. In an in vitro adhesion study, unhydrated thiolated NPs adhered strongly to freshly excised porcine small intestine, which was more than threefold increase compared to the control. In contrast, in the presence of various vehicles (PEG 300, miglyol 840, PEG 6000, cremophor EL, and caprylic triglyceride), the mucoadhesive properties of thiolated NPs were comparatively weak. Thiolated NPs suspended in caprylic triglyceride, for example, had a percent mucoadhesion of 22.50 ± 5.35% on the mucosa. Furthermore, results from in vivo mucoadhesion studies revealed that the dry form of nanoparticles exhibits the strongest mucoadhesion, followed by nanoparticles suspended in PEG 300, miglyol, and 100 mM phosphate buffer, in that order. Three hours after administration, the gastrointestinal residence time of the dry form of thiolated NPs was up to 3.6-fold prolonged. These findings should contribute to the design of highly effective oral mucoadhesive nanoparticulate drug delivery systems.     

Alpha-Fe2O3 elicits diameter-dependent effects during exposure to an in vitro model of the human placenta

Iron oxide nanoparticles offer unique possibilities due to the change in their physico-chemical parameters when synthesized on the nanoscale (10^?9 m) compared to their bulk forms. While novel uses exist for these materials when synthesized as nanoparticles, their unintended effects on the human body and specifically during pregnancy remain ill defined. In this study, an iron oxide nanoparticle, α-Fe₂O₃, was employed and the potential toxicity due to exposure was assessed in the widely used model human placental cell line BeWo b30. These cells were grown as epithelia, and subsequently assessed for their epithelial integrity, reactive oxygen species production and cellular viability, ultrastructural and morphological disruption, and genotoxicity as a result of exposure to α-Fe₂O₃ nanoparticles. Transepithelial electrical resistance indicated that exposure to the large (50 and 78 nm), but not small (15 nm) diameter particles of α-Fe₂O₃ nanomaterial resulted in leakiness of the epithelium. Exposure to the large diameters of 50 and 78 nm resulted in increases in cell death and reactive oxygen species. Disruption of junctional integrity as monitored by immunolocalization of the tight junction protein ZO-1 was found to occur as a consequence of exposure to large diameter NPs. It was found that there was reduction in the number of microvilli responsible for increased surface area for nutrient absorption after exposing the epithelia to large diameter NPs. Finally, genotoxicity as assessed by DNA microarray and confirmed by QPCR indicated that the large diameter particles (78 nm) induce apoptosis in these cells. These data indicate that large (50 and 78 nm), but not small (15 nm) α-Fe₂O₃ nanoparticles disrupt the barrier function of this epithelium as assessed by in vitro analysis.     

Aqueous diffusion pathways as a part of the ventricular cell ultrastructure

The physical organization of the ventricular myocyte includes barriers for the movement of objects of varying dimensions ranging from ions to solid particles. There are two kinds of diffusion in the cell: lateral (in membranes) and aqueous. Here we examine the size constraints of aqueous diffusion pathways and discuss their impact on cellular physiology. Calibrated gold nanoparticles were used to probe the accessibility of the entire transverse-axial tubular system (TATS), the sarcoplasm, and intracellular structures. The TATS tubules, although up to 300 nm in diameter, permitted only particles 3 nm; 3), the mitochondrial voltage-dependent anion channel and the nuclear pore complex in ventricular cells could not be penetrated by particles >/=6 nm; and 4), there is a difference in size clearance between transversal and longitudinal sarcoplasmic diffusional pathways.     

Increased mobility of metal oxide nanoparticles due to photo and thermal induced disagglomeration

Significant advances have been made on our understanding of the fate and transport of engineered nanomaterials. One unexplored aspect of nanoparticle aggregation is how environmental stimuli such as light exposure and temperature variations affect the mobility of engineered nanoparticles. In this study, TiO(2), ZnO, and CeO(2) were chosen as model materials for investigating the mobility of nanoparticles under three external stimuli: heat, light and sonication. Sunlight and high power sonication were able to partially disagglomerate metal oxide clusters, but primary particles bonded by solid state necks were left intact. A cycle of temperature increase from 25°C to 65°C and then decrease back was found to disagglomerate the compact clusters in the heating phase and reagglomerate them as more open fractal structures during the cooling phase. A fractal model summing the pair-wise DLVO interactions between primary particles within two fractal agglomerates predicts weak attractions on the order of a few kT. Our study shows that common environmental stimuli such as light exposure or temperature variation can disagglomerate nanoparticle clusters and enhance their mobility in open waters. This phenomenon warrants attention since it is likely that metal oxide nanoparticles will experience these natural stimuli during their transport in the environment.     

Presence of nanosilica (E551) in commercial food products: TNF-mediated oxidative stress and altered cell cycle progression in human lung fibroblast cells

Silica (E551) is commonly used as an anti-caking agent in food products. The morphology and the dimension of the added silica particles are not, however, usually stated on the food product label. The food industry has adapted nanotechnology using engineered nanoparticles to improve the quality of their products. However, there has been increased debate regarding the health and safety concerns related to the use of engineered nanoparticles in consumer products. In this study, we investigated the morphology and dimensions of silica (E551) particles in food. The silica content of commercial food products was determined using inductively coupled plasma optical emission spectrometry. The result indicates that 2.74–14. 45 μg/g silica was found in commercial food products; however, the daily dietary intake in increase causes adverse effects on human health. E551 was isolated from food products and the morphology, particle size, crystalline nature, and purity of the silica particles were analyzed using XRD, FTIR, TEM, EDX and DLS. The results of these analyses confirmed the presence of spherical silica nanoparticles (of amorphous nature) in food, approximately 10–50 nm in size. The effects of E551 on human lung fibroblast cell viability, intracellular ROS levels, cell cycle phase, and the expression levels of metabolic stress-responsive genes (CAT, GSTA4, TNF, CYP1A, POR, SOD1, GSTM3, GPX1, and GSR1) were studied. The results suggest that E551 induces a dose-dependent cytotoxicity and changes in ROS levels and alters the gene expression and cell cycle. Treatment with a high concentration of E551 caused significant cytotoxic effects on WI-38 cells. These findings have implications for the use of these nanoparticles in the food industry.     

Anti-<Emphasis Type="Italic">Helicobacter pylori</Emphasis> effect of mucoadhesive nanoparticles bearing amoxicillin in experimental gerbils model

The purpose of the present study was to design mucoadhesive gliadin nanoparticles (GNP) containing amoxicillin and to evaluate their effectiveness in eradicating Helicobacter pylori. GNP-bearing amoxicillin (AGNP) was prepared by desolvation method. The effect of process variables such as gliadin concentration and initial drug loading on particle size, shape, percent payload, percent entrapment efficiency, in vitro release profile, and mucoadhesive property of GNP was assessed. Rhodamine isothiocyanate-entrapped GNP formulations were prepared to evaluate their in vivo gastric mucoadhesive property in albino rats. With increasing gliadin concentration, the mucoadhesive property of GNP increased. Typically, the maximum amount of nanoparticles remaining was 82±4%, which represented a stronger mucoadhesive propensity and specificity of GNP toward the stomach. In vitro antimicrobial activity of AGNP was evaluated by growth inhibition studies on an isolated H pylori strain. The time required for complete eradication was higher in AGNP than in amoxicillin because of the controlled drug delivery of amoxicillin from AGNP. In vivo clearance of H pylori following oral administration of AGNP to infected Mongolian gerbils was examined. Amoxicillin and AGNP both showed anti-H pylori effects in this experimental model of infection, but the required dose for complete eradication was less in AGNP than in amoxicillin. In conclusion, AGNP eradicated H pylori from the gastrointestinal tract more effectively than amoxicillin because of the prolonged gastrointestinal residence time attributed to mucoadhesion. A dosage form containing mucoadhesive nanoparticles bearing a potential antibiotic should be useful for the complete eradication of H pylori.     

Local magnetism in palladium bionanomaterials probed by muon spectroscopy

Palladium bionanomaterial was manufactured using the sulfate-reducing bacterium, Desulfovibrio desulfuricansm, to reduce soluble Pd(II) ions to cell-bound Pd(0) in the presence of hydrogen. The biomaterial was examined using a Superconducting Quantum Interference Device (SQUID) to measure bulk magnetisation and by Muon Spin Rotation Spectroscopy (μSR) which is uniquely able to probe the local magnetic environment inside the sample. Results showed behaviour attributable to interaction of muons both with palladium electrons and the nuclei of hydrogen trapped in the particles during manufacture. Electronic magnetism, also suggested by SQUID, is not characteristic of bulk palladium and is consistent with the presence of nanoparticles previously seen in electron micrographs. We show the first use of μSR as a tool to probe the internal magnetic environment of a biologically-derived nanocatalyst material.     

Optimization of PEGylation Conditions for BSA Nanoparticles Using Response Surface Methodology

Chemical coupling of polyethylene glycol (PEG) to proteins or particles (PEGylation), prolongs their circulation half-life by greater than 50-fold, reduces their immunogenicity, and also promotes their accumulation in tumors due to enhanced permeability and retention effect. Herein, phase separation method was used to prepare bovine serum albumin (BSA) nanoparticles. PEGylation of BSA nanoparticles was performed by SPA activated mPEG through their free amino groups. Effect of process variables on PEGylation efficiency of BSA nanoparticles was investigated and optimized through response surface methodology with the amount of free amino groups as response. Optimum conditions was found to be 32.5 g/l of PEG concentration, PEG-nanoparticle incubation time of 10 min, incubation temperature of 27°C, and pH of 7 for 5 mg of BSA nanoparticles in 1 mL phosphate buffer. Analysis of data showed that PEG concentration had the most noticeable effect on the amount of PEGylated amino groups, but pH had the least. Mean diameter and zeta potential of PEGylated nanoparticles under these conditions were 217 nm and −14 mV, respectively. In conclusion, PEGylated nanoparticles demonstrated reduction of the negative surface charge compared to the non modified particles with the zeta potential of −31.7 mV. Drug release from PEGylated nanoparticles was almost slower than non-PEGylated ones, probably due to existence of a PEG layer around PEGylated particles which makes an extra resistance in opposition to drug diffusion.