Single particle tracking reveals spatial and dynamic organization of the Escherichia coli biofilm matrix

Biofilms are communities of surface-adherent bacteria surrounded by secreted polymers known as the extracellular polymeric substance. Biofilms are harmful in many industries, and thus it is of great interest to understand their mechanical properties and structure to determine ways to destabilize them. By performing single particle tracking with beads of varying surface functionalization it was found that charge interactions play a key role in mediating mobility within biofilms. With a combination of single particle tracking and microrheological concepts, it was found that Escherichia coli biofilms display height dependent charge density that evolves over time. Statistical analyses of bead trajectories and confocal microscopy showed inter-connecting micron scale channels that penetrate throughout the biofilm, which may be important for nutrient transfer through the system. This methodology provides significant insight into a particular biofilm system and can be applied to many others to provide comparisons of biofilm structure. The elucidation of structure provides evidence for the permeability of biofilms to microscale objects, and the ability of a biofilm to mature and change properties over time.National Science Foundation (U.S.) (CBET-1335938)Cystic Fibrosis Foundation (HANES07XX0)Massachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund)Burroughs Wellcome Fund (Preterm Birth Research Grant)National Institute of Allergy and Infectious Diseases (U.S.) (F30 Fellowship 1F30AI110053-01)National Institute of Allergy and Infectious Diseases (U.S.) (Training Grant in Toxicology 5 T32 ES7020-37

Biotargeted nanomedicines for cancer: six tenets before you begin

Biotargeted nanomedicines have captured the attention of academic and industrial scientists who have been motivated by the theoretical possibilities of the ‘magic bullet’ that was first conceptualized by Paul Ehrlich at the beginning of the 20th century. The Biotargeting Working Group, consisting of more than 50 pharmaceutical scientists, engineers, biologists and clinicians, has been formed as part of the National Cancer Institute’s Alliance for Nanotechnology in Cancer to harness collective wisdom in order to tackle conceptual and practical challenges in developing biotargeted nanomedicines for cancer. In modern science and medicine, it is impossible for any individual to be an expert in every aspect of biology, chemistry, materials science, pharmaceutics, toxicology, chemical engineering, imaging, physiology, oncology and regulatory affairs. Drawing on the expertise of leaders from each of these disciplines, this commentary highlights six tenets of biotargeted cancer nanomedicines in order to enable the translation of basic science into clinical practice

DNA nanoparticle-mediated thymulin gene therapy prevents airway remodeling in experimental allergic asthma

Thymulin has been shown to present anti-inflammatory and anti-fibrotic properties in experimental lung diseases. We hypothesized that a biologically active thymulin analog gene, methionine serum thymus factor, delivered by highly compacted DNA nanoparticles may prevent lung inflammation and remodeling in a mouse model of allergic asthma. The DNA nanoparticles are composed of a single molecule of plasmid DNA compacted with block copolymers of poly-L-lysine and polyethylene glycol (CK30PEG), which have been found safe in a human phase I/II clinical trial. Thymulin plasmids were detected in the lungs of ovalbumin-challenged asthmatic mice up to 27days after administration of DNA nanoparticles carrying thymulin plasmids. A single dose of DNA nanoparticles carrying thymulin plasmids prevented lung inflammation, collagen deposition and smooth muscle hypertrophy in the lungs of a murine model of ovalbumin-challenged allergic asthma, leading to improved lung mechanics. In the present model of chronic allergic asthma, highly compacted DNA nanoparticles using thymulin analog gene modulated the inflammatory and remodeling processes improving lung mechanics.Instituto de Investigaciones Bioquímicas de La Plat

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations

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

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

Systematic assessment of microneedle injection into the mouse cornea

<p>Abstract</p> <p>Background</p> <p>Corneal intrastromal injection is an important mode of gene-vector application to subepithelial layers. In a mouse model, this procedure is substantially complicated by the reduced corneal dimensions. Furthermore, it may be difficult to estimate the corneal area reached by the volume of a single injection. This study aimed to investigate intrastromal injections into the mouse cornea using different microneedles and to quantify the effect of injecting varying volumes. A reproducible injection technique is described.</p> <p>Methods</p> <p>Forty eyes of 20 129 Sv/J mice were tested. India ink was intrastromally injected using 30° beveled 33 G needles, tri-surface 25° beveled 35 G needles, or hand-pulled and 25° beveled glass needles. Each eye received a single injection of a volume of 1 or 2 μL. Corneoscleral buttons were fixed and flat mounted for computer-assisted quantification of the affected corneal area. Histological assessment was performed to investigate the intrastromal location of the injected dye.</p> <p>Results</p> <p>A mean corneal area of 5.0 ±1.4 mm² (mean ± SD) and 7.7 ±1.4 mm² was covered by intrastromal injections of 1 and 2 μL, respectively. The mean percentage of total corneal area reached ranged from 39% to 53% for 1 μL injections, and from 65% to 81% for 2 μL injections. Injections using the 33 G needles tended to provide the highest distribution area. Perforation rates were 8% for 30° beveled 33 G needles and 44% for tri-surface beveled 35 G needles. No perforation was observed with glass needle; however, intrastromal breakage of needle tips was noted in 25% of these cases.</p> <p>Conclusions</p> <p>Intracorneal injection using a 30° beveled 33 G needle was safe and effective. The use of tri-surface beveled 35 G needles substantially increased the number of corneal perforations. Glass needles may break inside the corneal stroma. Injections of 1 μL and 2 μL resulted in an overall mean of 49% and 73% respectively of total corneal area involved.</p

Preparation of particles for inhalation

Particles incorporating a surfactant and/or a hydrophilic or hydrophobic complex of a positively or negatively charged therapeutic agent and a charged molecule of opposite charge for drug delivery to the pulmonary system, and methods for their synthesis and administration are provided. In a preferred embodiment, the particles are made of a biodegradable material and have a tap density less than 0.4 g/cm and a mass mean diameter between 5 mu m and 30 mu m, which together yield an aerodynamic diameter of the particles of between approximately one and three microns. The particles may be formed of biodegradable materials such as biodegradable polymers. For example, the particles may be formed of poly(lactic acid) or poly(glycolic acid) or copolymers thereof. Alternatively, the particles may be formed solely of a therapeutic or diagnostic agent and a surfactant. Surfactants can be incorporated on the particule surface for example by coating the particle after particle formation, or by incorporating the surfactant in the material forming the particle prior to formation of the particle. Exemplary surfactants include phosphoglycerides such as dipalmitoyl phosphatidylcholine (DPPC). The particles can be effectively aerosolized for administration to the respiratory tract to permit systemic or local delivery of wide a variety of therapeutic agents. Formation of complexes of positively or negatively charged therapeutic agents with molecules of opposite charge can allow control of the release rate of the agents into the blood stream following administration

Preparation de particules pour inhalation

Cette invention se rapporte à des particules, dans lesquelles sont incorporés un tensioactif et/ou un complexe hydrophyle ou hydrophobe constitué par un agent thérapeutique chargé positivement ou négativement et par une molécule chargée de charge opposée pour l'administration de médicaments dans le système pulmonaire, ainsi qu'à des procédés pour la synthèse et l'administration de ces particules. Dans un mode de réalisation préféré, les particules sont constituées d'un matériau biodégradable et possèdent une densité après tassement inférieure à 0,4 g/cm et un diamètre médian massique compris entre 5 mu m et 30 mu m, qui produisent ensemble un diamètre aérodynamique des particules compris approximateivement entre 1 et 3 microns. Ces particules peuvent être formées de matériaux biodégradables, tels que des polymères biodégradables. Ces particules peuvent par exemple être formées d'acide polylacétique ou d'acide polyglycolique ou de copolymères de ceux-ci. Dans une variante, ces particules peuvent être formées uniquement d'un agent thérapeutique ou diagnostique et d'un tensioactif. Les tensioactifs peuvent être incorporés sur la surface des particules, par exemple par enrobage de la particule après sa formation, ou par incorporation du tensioactif dans le matériau formant la particule avant sa formation. Ces tensioactifs sont par exemple des phosphoglycérides, tels que la dipalmitoyl-phosphatidylcholine (DPPC). Ces particules peuvent être efficacement atomisées pour être administrées à l'appareil respiratoire, afin de permettre l'apport systémique ou local d'une grande variété d'agents thérapeutiques. La formation de complexes d'agents thérapeutiques chargés positivement ou négativement avec des molécules de charge opposée peut permettre de régler le taux de libération de ces agents dans le sang après leur administration