An engineered disulfide bond reversibly traps the IgE-Fc3-4 in a closed, nonreceptor binding conformation

IgE antibodies interact with the high affinity IgE Fc receptor, FcεRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcεRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of an IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcεRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.

Scale Up of PLA Nanoparticle Synthesis by Solvent Displacement and Functionalization of the Particles with a Silica Shell for Targeted Drug Delivery Applications

Biodegradable polymer nanoparticles have the properties necessary to address many of the issues associated with current drug delivery techniques including targeted and controlled delivery. A novel drug delivery vehicle is proposed consisting of a poly(lactic acid) nanoparticle core, with a functionalized, mesoporous silica shell. In this study, the production of PLA nanoparticles is investigated using solvent displacement in both a batch and continuous manner, and the effects of various system parameters are examined. Using Pluronic F-127 as the stabilization agent throughout the study, PLA nanoparticles are produced through solvent displacement with diameters ranging from 200 to 250 nm using two different methods: dropwise addition and in an impinging jet mixer. The impinging jet mixer allows for easy scale-up of particle production. The concentration of surfactant and volume of quench solution is found to have minimal impact on particle diameter; however, the concentration of PLA is found to significantly impact the diameter mean and polydispersity. In addition, the stability of the PLA nanoparticles is observed to increase as residual THF is evaporated. Lastly, the isolated PLA nanoparticles are coated with a silica shell using the Stöber Process. It is found that functionalizing the silica with a phosphonic silane in the presence of excess Pluronic F-127 decreases coalescence of the particles during the coating process. Future work should be conducted to fine-tune the PLA nanoparticle synthesis process by understanding the effect of other system parameters and in synthesizing mesoporous silica shells.

Effect Of Confined Impinging Jet Mixing Processing Parameters On Poly (Lactic Acid) Nanoparticle Morphology

Biodegradable nanoparticles are at the forefront of drug delivery research as they provide numerous advantages over traditional drug delivery methods. An important factor affecting the ability of nanoparticles to circulate within the blood stream and interact with cells is their morphology. In this study a novel processing method, confined impinging jet mixing, was used to form poly (lactic acid) nanoparticles through a solvent-diffusion process with Pluronic F-127 being used as a stabilizing agent. This study focused on the effects of Reynolds number (flow rate), surfactant presence in mixing, and polymer concentration on the morphology of poly (lactic acid) nanoparticles. In addition to looking at the parameters affecting poly (lactic acid) morphology, this study attempted to improve nanoparticle isolation and purification methods to increase nanoparticle yield and ensure specific morphologies were not being excluded during isolation and purification. The isolation and purification methods used in this study were centrifugation and a stir cell. This study successfully produced particles having pyramidal and cubic morphologies. Despite successful production of these morphologies the yield of non-spherical particles was very low, additionally great variability existed between redundant trails. Surfactant was determined to be very important for the stabilization of nanoparticles in solution but appears to be unnecessary for the formation of nanoparticles. Isolation and purification methods that produce a high yield of surfactant free particles have still not been perfected and additional testing will be necessary for improvement.¿

Mechanisms of nanoparticle-mediated photomechanical cell damage

Laser-assisted killing of gold nanoparticle targeted macrophages was investigated. Using pressure transient detection, flash photography and transmission electron microscopy (TEM) imaging, we studied the mechanism of single cell damage by vapor bubble formation around gold nanospheres induced by nanosecond laser pulses. The influence of the number of irradiating laser pulses and of particle size and concentration on the threshold for acute cell damage was determined. While the single pulse damage threshold is independent of the particle size, the threshold decreases with increasing particle size when using trains of pulses. The dependence of the cell damage threshold on the nanoparticle concentration during incubation reveals that particle accumulation and distribution inside the cell plays a key role in tissue imaging or cell damaging.

Nanoparticle-based test measures overall propensity for calcification in serum

Vascular and soft tissue calcification contributes to cardiovascular morbidity and mortality in both the general population and CKD. Because calcium and phosphate serum concentrations are near supersaturation, the balance of inhibitors and promoters critically influences the development of calcification. An assay that measures the overall propensity for calcification to occur in serum may have clinical use. Here, we describe a nanoparticle-based assay that detects, in the presence of artificially elevated calcium and phosphate concentrations, the spontaneous transformation of spherical colloidal primary calciprotein particles (CPPs) to elongate crystalline secondary CPPs. We used characteristics of this transition to describe the intrinsic capacity of serum to inhibit the precipitation of calcium and phosphate. Using this assay, we found that both the sera of mice deficient in fetuin-A, a serum protein that inhibits calcification, and the sera of patients on hemodialysis have reduced intrinsic properties to inhibit calcification. In summary, we developed a nanoparticle-based test that measures the overall propensity for calcification in serum. The clinical use of the test requires evaluation in a prospective study.

Anisotropically oriented electrospun matrices with an imprinted periodic micropattern: a new scaffold for engineered muscle constructs

Engineered muscle constructs provide a promising perspective on the regeneration or substitution of irreversibly damaged skeletal muscle. However, the highly ordered structure of native muscle tissue necessitates special consideration during scaffold development. Multiple approaches to the design of anisotropically structured substrates with grooved micropatterns or parallel-aligned fibres have previously been undertaken. In this study we report the guidance effect of a scaffold that combines both approaches, oriented fibres and a grooved topography. By electrospinning onto a topographically structured collector, matrices of parallel-oriented poly(ε-caprolactone) fibres with an imprinted wavy topography of 90 µm periodicity were produced. Matrices of randomly oriented fibres or parallel-oriented fibres without micropatterns served as controls. As previously shown, un-patterned, parallel-oriented substrates induced myotube orientation that is parallel to fibre direction. Interestingly, pattern addition induced an orientation of myotubes at an angle of 24° (statistical median) relative to fibre orientation. Myotube length was significantly increased on aligned micropatterned substrates in comparison to that on aligned substrates without pattern (436 ± 245 µm versus 365 ± 212 µm; p < 0.05). We report an innovative, yet simple, design to produce micropatterned electrospun scaffolds that induce an unexpected myotube orientation and an increase in myotube length.

Enhancing sealing of fetal membrane defects using tissue engineered native amniotic scaffolds in the rabbit model

OBJECTIVE: The purpose of this study was to compare the efficacy of native engineered amniotic scaffolds (AS) and polyesterurethane scaffolds (DegraPol) and document wound healing response when sealing iatrogenic fetal membrane defects in the rabbit model. STUDY DESIGN: Native AS were engineered from freshly harvested membranes of 23 days' gestational age (GA; term = 31-2 d). Acellularity of AS was assessed by histology, light and scanning electron microscopy. Fetal membrane defects were created by 14 gauge-needle puncture at GA 23 days and primarily closed with AS (n = 10) or DegraPol (n = 10) or left unclosed (positive controls; n = 10). Sixty-one sacs served as negative controls. At GA 30 days a second look hysterotomy was performed to assess presence of amniotic fluid (AF) and harvest plugging sites for microscopic evaluation. RESULTS: Engineered AS had a cell-free collagenous fiber network. AF was significantly higher only in the DegraPol group (78%; P < .05) compared to the AF in positive controls (17%). Integration of plugs in the fetal membrane defect was better with AS than DegraPol, with higher reepithelialization rates (AS: 52.5% +/- 6.5%; DegraPol: 11.6% +/- 2.6%; P < .001) and proliferation indices (AS: 0.47 +/- 0.03; DegraPol: 0.28 +/- 0.04; P = .001). In both treatment groups, cell proliferation in the myometrium was increased (P < .05). CONCLUSION: Native AS seal iatrogenic fetal membrane defects better than DegraPol. Within a week, there is abundant reepithelilization and minimal local inflammation. This yields the proof of principle that engineered native, amniotic membrane scaffolds enhance fetal membrane wound healing response.

Re-evaluation of pulmonary titanium dioxide nanoparticle distribution using the "relative deposition index": Evidence for clearance through microvasculature

ABSTRACT: BACKGROUND: Translocation of nanoparticles (NP) from the pulmonary airways into other pulmonary compartments or the systemic circulation is controversially discussed in the literature. In a previous study it was shown that titanium dioxide (TiO2) NP were "distributed in four lung compartments (air-filled spaces, epithelium/endothelium, connective tissue, capillary lumen) in correlation with compartment size". It was concluded that particles can move freely between these tissue compartments. To analyze whether the distribution of TiO2 NP in the lungs is really random or shows a preferential targeting we applied a newly developed method for comparing NP distributions. METHODS: Rat lungs exposed to an aerosol containing TiO2 NP were prepared for light and electron microscopy at 1 h and at 24 h after exposure. Numbers of TiO2 NP associated with each compartment were counted using energy filtering transmission electron microscopy. Compartment size was estimated by unbiased stereology from systematically sampled light micrographs. Numbers of particles were related to compartment size using a relative deposition index and chi-squared analysis. RESULTS: Nanoparticle distribution within the four compartments was not random at 1 h or at 24 h after exposure. At 1 h the connective tissue was the preferential target of the particles. At 24 h the NP were preferentially located in the capillary lumen. CONCLUSION: We conclude that TiO2 NP do not move freely between pulmonary tissue compartments, although they can pass from one compartment to another with relative ease. The residence time of NP in each tissue compartment of the respiratory system depends on the compartment and the time after exposure. It is suggested that a small fraction of TiO2 NP are rapidly transported from the airway lumen to the connective tissue and subsequently released into the systemic circulation.

Hydrogel-based engineered skeletal muscle grafts normalize heart function early after myocardial infarction

Tissue engineering represents an attractive approach for the treatment of congestive heart failure. The influence of the differentiation of myogenic graft for functional recovery is not defined. We engineered a biodegradable skeletal muscle graft (ESMG) tissue and investigated its functional effect after implantation on the epicardium of an infarcted heart segment. ESMGs were synthesized by mixing collagen (2 mg/mL), Matrigel (2 mg/mL), and rat skeletal muscle cells (10(6)). Qualitative and quantitative aspects of ESMGs were optimized. Two weeks following coronary ligation, the animals were randomized in three groups: ESMG glued to the epicardial surface with fibrin (ESMG, n = 7), fibrin alone (fibrin, n = 5), or sham operation (sham, n = 4). Echocardiography, histology, and immunostaining were performed 4 weeks later. A cohesive three-dimensional tissular structure formed in vitro within 1 week. Myoblasts differentiated into randomly oriented myotubes. Four weeks postimplantation, ESMGs were vascularized and invaded by granulation tissue. Mean fractional shortening (FS) was, however, significantly increased in the ESMG group as compared with preimplantation values (42 +/- 6 vs. 33 +/- 5%, P < 0.05) and reached the values of controlled noninfarcted animals (control, n = 5; 45 +/- 3%; not significant). Pre- and postimplantation FS did not change over these 4 weeks in the sham group and the fibrin-treated animals. This study showed that it is possible to improve systolic heart function following myocardial infarction through implantation of differentiated muscle fibers seeded on a gel-type scaffold despite a low rate of survival.