Successful administration of therapeutic proteins via the oral route has long eluded the drug delivery community; a variety of factors, both physical and physiological, have hindered the myriad ...approaches to increasing the bioavailability of orally administered therapeutic proteins, including: 1) pre-systemic degradation by enzymes and 2) poor penetration of the intestinal mucosa and epithelium. Even when bypassing the harsh, acidic environment of the stomach, the intestines pose significant obstacles to systemic uptake. For example, the lining of the gastrointestinal tract comprises a thick wall of epithelial cells covered by a layer of polysaccharides and mucus. In this review, we will discuss the biology underlying intestinal uptake of protein-containing, biodegradable nanoparticles, review insulin delivery as the most accepted model for oral delivery of proteins, and present a variety of new material systems enabling novel approaches to oral protein delivery which we believe will bring to bear the next therapeutic advances in our field.
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Of the new direct oral anticoagulants, direct factor Xa inhibitors are limited by the absence of a proven reversal agent. We assessed the safety, tolerability and impact on anticoagulation reversal ...of ciraparantag (PER977) alone and following a 60 mg dose of the FXa inhibitor edoxaban. Escalating, single IV doses of ciraparantag were administered alone and following a 60 mg oral dose of edoxaban in a double-blind, placebo-controlled fashion to healthy subjects. Serial assessments of the pharmacokinetics and pharmacodynamic effects of ciraparantag were performed. Eighty male subjects completed the study. Following edoxaban (60 mg), a single IV dose of ciraparantag (100 to 300 mg) demonstrated full reversal of anticoagulation within 10 minutes and sustained for 24 hours. Fibrin diameter within clots was restored to normal 30 minutes after a single dose of 100 to 300 mg ciraparantag as determined by scanning electron microscopy and change in fibrin diameter quantified by automated image analysis. Potentially related adverse events were periorbital and facial flushing and cool sensation following IV injection of ciraparantag. Renal excretion of ciraparantag metabolite was the main elimination route. There was no evidence of procoagulant activity following ciraparantag as assessed by D-dimer, prothrombin fragments 1.2, and tissue factor pathway inhibitor levels. In conclusion, ciraparantag in healthy subjects is safe and well tolerated with minor, non-dose limiting adverse events. Baseline haemostasis was restored from the anticoagulated state with doses of 100 to 300 mg ciraparantag within 10-30 minutes of administration and sustained for at least 24 hours.
Ciraparantag, an anticoagulant reversal agent, is a small molecule specifically designed to bind noncovalently by charge-charge interaction to unfractionated heparin and low-molecular-weight heparin. ...It shows binding characteristics that are similar to those of direct oral anticoagulants (DOACs). A dynamic light-scattering methodology was used to demonstrate ciraparantag's binding to the heparins and DOACs and its lack of binding to a variety of proteins including coagulation factors and commonly used drugs. Ciraparantag reaches maximum concentration within minutes after IV administration with a half-life of 12 to 19 minutes. It is primarily hydrolyzed by serum peptidases into 2 metabolites, neither of which has substantial activity. Ciraparantag and its metabolites are recovered almost entirely in the urine. In animal models of bleeding (rat tail transection and liver laceration), a single IV dose of ciraparantag given at peak concentrations of the anticoagulant, but before the bleeding injury, significantly reduced the blood loss. Ciraparantag, given after the bleeding injury, also significantly reduced blood loss. It appears to have substantial ability to reduce blood loss in animal models in which a variety of anticoagulants are used and has potential as a useful DOAC reversal agent.
Abstract Major bleeding with low molecular weight heparin (LMWH) therapy occurs in up to 5% of patients and its anticoagulation is only partially reversed by protamine sulfate. We studied the ability ...of ciraparantag (PER977), a novel agent that reverses LMWH in preclinical studies, to reverse LMWH in healthy volunteers. Methods In this phase 1/2 trial, 4 cohorts of 10 healthy volunteers received escalating doses of ciraparantag (100 to 300 mg) or placebo (8:2 ratio) approximately 4 h after a single subcutaneous dose of enoxaparin, 1.5 mg/kg. Safety, pharmacokinetic and pharmacodynamic effects were assessed. Results Complete reversal of enoxaparin anticoagulation, measured by a reduction of whole blood clotting time, was observed in all subjects who received a single ciraparantag dose ranging from 100 mg to 300 mg. The anticoagulation reversal occurred rapidly after bolus injection and persisted for the duration of the study. At 12 h and 24 h, the differences in whole blood clotting time in the treated group compared to placebo were no longer significant, consistent with the decline in enoxaparin concentrations and anticoagulation effects. No procoagulant signals were detected as measured by D-dimer, F1.2, and tissue factor pathway inhibitor levels. Ciraparantag was well tolerated with only transient, minor side effects. Conclusion Ciraparantag reverses the whole blood clotting time induced by enoxaparin in a dose related manner and produces no procoagulant signal or deleterious adverse events in doses up to 300 mg.
Tracking cells after therapeutic transplantation is imperative for evaluation of implanted cell fate and function. In this study, ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) ...were surface functionalized with water-soluble chitosan, a cationic polysaccharide that mediates enhanced endocytic uptake, endosomal escape into the cytosol, and subsequent long-term retention of nanoparticles. NP surface and chitosan were independently fluorescently labeled. Our NPs enable NP trafficking studies and determination of fate beyond uptake by fluorescence microscopy as well as tracking of labeled cells as localized regions of hypointensity in T(2)*-weighted magnetic resonance imaging (MRI) images. Adult rat neural stem cells (NSCs) were labeled with NPs, and assessment of NSC proliferation rates and differentiation potential revealed no significant differences between labeled and unlabeled NSCs. Significantly enhanced uptake of chitosan NPs in comparison to native NPs was confirmed by transmission electron microscopy, nuclear magnetic resonance (NMR) spectroscopy and in vitro cellular MRI at 11.7 Tesla. While only negligible fractions of native NPs enter cells, chitosan NPs appear within membranous vesicles within 2 hours of exposure. Additionally, chitosan-functionalized NPs escaped from membrane-bound vesicles within days, circumventing NP endo-lysosomal trafficking and exocytosis and hence enabling long-term tracking of labeled cells. Finally, our labeling strategy does not contain any NSC-specific reagents. To demonstrate general applicability across a variety of primary and immortalized cell types, embryonic mouse NSCs, mouse embryonic stem cells, HEK 293 kidney cells, and HeLa cervical cancer cells were additionally exposed to chitosan-USPIO NPs and exhibited similarly efficient loading as verified by NMR relaxometry. Our efficient and versatile labeling technology can support cell tracking with close to single cell resolution by MRI in vitro, for example, in complex tissue models not optically accessible by confocal or multi-photon fluorescence microscopy, and potentially in vivo, for example, in animal models of human disease or injury.
The intrinsic abilities of mammalian neural stem cells (NSCs) to self-renew, migrate over large distances, and give rise to all primary neural cell types of the brain offer unprecedented opportunity ...for cell-based treatment of neurodegenerative diseases and injuries. This thesis discusses development of technologies in support of autologous NSC-based therapies, encompassing harvest of brain tissue biopsies from living human patients; isolation of NSCs from harvested tissue; efficient culture and expansion of NSCs in 3D polymeric microcapsule culture systems; optimization of microcapsules as carriers for efficient in vivo delivery of NSCs; genetic engineering of NSCs for drug-induced, enzymatic release of transplanted NSCs from microcapsules; genetic engineering for drug-induced differentiation of NSCs into specific therapeutic cell types; and synthesis of chitosan/iron-oxide nanoparticles for labeling of NSCs and in vivo tracking by cellular MRI. Sub-millimeter scale tissue samples were harvested endoscopically from subventricular zone regions of living patient brains, secondary to neurosurgical procedures including endoscopic third ventriculostomy and ventriculoperitoneal shunt placement. On average, 12,000 +/- 3,000 NSCs were isolated per mm 3 of subventricular zone tissue, successfully demonstrated in 26 of 28 patients, ranging in age from one month to 68 years. In order to achieve efficient expansion of isolated NSCs to clinically relevant numbers (e.g. hundreds of thousands of cells in Parkinson's disease and tens of millions of cells in multiple sclerosis), an extracellular matrix-inspired, microcapsule-based culture platform was developed. Initial culture experiments with murine NSCs yielded unprecedented expansion folds of 30x in 5 days, from initially minute NSC populations (154 +/- 15 NSCs per 450 μm diameter capsule). Within 7 days, NSCs expanded as almost perfectly homogenous populations, with 94.9% +/- 4.1% of cultured cells staining positive for Nestin, a marker for NSCs, 81.4 +/- 3.7% of cells staining positive for KI67, a proliferation marker, and 0% of cultured cells staining positive for GFAP, a marker indicative of undesired astrocytes. The same microcapsules used for expansion were designed to contain NSCs beyond delivery to the brain, maintaining NSC phenotype and suppressing undesired astroglial differentiation during the acute phase of inflammation beyond surgical implantation. In vitro, >80% of encapsulated cells challenged with 0.1 % fetal calf serum over five days in culture showed persistent Nestin expression, compared to <20% under the same conditions outside of microcapsules, indicating that the microcapsule interior can preserve phenotype in the presence of serum concentrations at least an order of magnitude greater than those estimated to be present in cerebrospinal fluid (CSF) after surgical implantation. In order to release transplanted NSCs on cue from microcapsules after the acute inflammatory response, NSCs were genetically engineered using the Tet-on® drug-inducible gene expression system to produce and secrete the enzyme alginase in response to the inducer drug doxycycline. Engineered NSCs, exposed to 1 μg/ml doxycycline, produced sufficient alginase to digest alginate, a structural component of the microcapsule wall, within 8 hours, effectively dissolving microcapsules and releasing encapsulated NSCs. In order to direct differentiation of transplanted NSCs towards therapeutically valuable cell types (e.g. dopaminergic neurons in case of Parkinson's disease and oligodendrocytes in case of multiple sclerosis), NSCs were genetically engineered to inducibly express the proneural transcription factors NGN1 and Olig1 on demand. Induced expression of NGN1 yielded >90% neurons, induced expression of Olig1 yielded >80% oligodendrocytes, compared to neuron/oligodendrocyte yields <10% for GFP-expressing controls. NSCs with the capacity to inducibly express these transcription factors showed preservation of therapeutically valuable migratory capacity (average RMS migration rate of approximately 40 μm/hr before induction). Differentiating NSCs, however, showed largely arrested migration within 12 hours of induction for Olig1 cells and 36 hours of induction for NGN1 cells. Finally, tracking of NSCs at the single cell level via high-resolution (11.7 T) cellular MRI, was made possible through development of contrast-enhancing, chitosan-functionalized ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles that are rapidly uptaken by NSCs. Chitosan, a positively charged derivative of chitin, promotes electrostatically-driven attachment of chitosan-USPIO nanoparticles to negatively charged domains on the outer leaflet of the cellular membrane, enhancing uptake by clathrin-mediated endocytosis (>10x increase in uptake efficiency relative to unmodified USPIO). Uptaken USPIOs remained in cells for at least 8 days due to charge-induced endosomal escape of nanoparticles into the cytosol. In combination, all developed technologies offer a basis for clinical evaluation of autologous neural stem cell replacement therapies, the future of which promises to shift the present paradigm for treatment of neurodegenerative diseases and injuries.
The nature of a striking pattern of extended defects formed by very high dose implantation of helium in a complex oxide, for example, ferroelectricz-cut LiNbO{sub 3}, is studied. After irradiation, a ...high concentration of defects is found to collect and create a network of thick prismatic planar defects, which have typical dimensions of -1.5 {micro}m and 200 nm parallel and perpendicular to the z axis, respectively. This defect network and its effect on the lattice were studied using a set of x-ray and electron microscopy probes of the lattice structure and spatial variation. Optical microscopy shows that there is strong temperature dependence for forming the network; the density of these extended defects reaches a maximum value for an annealing temperature of 250 C but is fully eliminated by a temperature of 380 C. High-resolution transmission electron microscopy studies indicate that these extended defects are probably localized twinning and dislocation pileups due to plastic deformation of the lattice to relieve He-implantation-induced stress. During this deformation, He accumulates at the twin boundaries. The study also shows that the He interstitials evolve into bubbles causing high stress and resulting in a formation of thick prismatic planar defects. Finally, a mechanism is proposed for defect creation and elimination.
We report on the use of thin, i.e. 10 microm-thick, single-crystal LiNbO3, in low-voltage electrooptic prism scanners. These devices are fabricated by electric-field poling of a series of ...electrooptic prisms in a bulk crystal followed by high-energy ion implantation and subsequent etching of the poled samples. Such a single-crystal thin-film scanner, while having the same scanning functionality as with a bulk device, has an order-of-magnitude reduction in its required voltage; for example, a series of two prisms, of 2mm in total length, yields a deflection angle of 0.7 at 100V compared to more than 1.7kV for the same device in standard 200 microm-thick LiNbO3 wafers.
Fabrication of a bilayer HfO
2/single-crystal LiNbO
3 film is demonstrated using deep high-energy He
+ implantation in a LiNbO
3 wafer, followed by HfO
2 atomic layer deposition, and, then, selective ...etching exfoliation from the bulk LiNbO
3 crystal. The properties and morphology of these exfoliated bilayer films are characterized using a set of thin-film probes. Pre-exfoliation film patterning and one model application, in surface-refractive-index tuning of guided waves in a free-standing LiNbO
3 film, are also demonstrated.
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