Lipid-Based Nanoparticles for RNA Delivery.

Various nanoparticle-based delivery systems have been developed for the encapsulation and protection of active cargoes. Lipid nanoparticles represent one of the most widely used nanoparticle-based delivery systems for in vitro and in vivo applications, especially for the delivery of ribonucleic acid (RNA). In this chapter, a simple bulk mixing method for the encapsulation of RNA is described along with characterization techniques for measuring encapsulation efficiency and nanoparticle physicochemical properties.

Design and characterization of lipid nanocarriers for oral delivery of immunotherapeutic peptides.

The use of therapeutic proteins and peptides is of great interest for the treatment of many diseases, and advances in nanotechnology offer a path toward their stable delivery via preferred routes of administration. In this study, we sought to design and formulate a nanostructured lipid carrier (NLC) containing a nominal antigen (insulin peptide) for oral delivery. We utilized the design of experiments (DOE) statistical method to determine the dependencies of formulation variables on physicochemical particle characteristics including particle size, polydispersity (PDI), melting point, and latent heat of melting. The particles were determined to be non-toxic in vitro, readily taken up by primary immune cells, and found to accumulate in regional lymph nodes following oral administration. We believe that this platform technology could be broadly useful for the treatment of autoimmune diseases by supporting the development of oral delivery-based antigen specific immunotherapies.

Theranostic biocomposite scaffold membrane.

Acute and chronic wounds affect millions and are associated with billions of dollars in healthcare costs. The use of healing markers, biochemical cues from biocompatible matrices and materials, and their correlation with wound healing has the potential to generate valuable diagnostic, prognostic, and therapeutic information. In this study, we developed a collagen-dextran oxygen-sensing biocomposite scaffold membrane in which a phosphorescent oxygen sensor was incorporated to monitor physiological oxygen using in vivo phosphorescence imaging in a preclinical mouse model of wound healing. The oxygen-sensing biocomposite scaffold membrane enabled the noninvasive and longitudinal monitoring of oxygenation changes in vivo in an approach compatible with commercially available preclinical in vivo imaging system instruments. This study provides a new and novel capability where a biocomposite material can serve as a biocompatible, biodegradable theranostic platform to promote and assess tissue oxygenation during wound healing.

Evaluation of the biocompatibility of regenerated cellulose hydrogels with high strength and transparency for ocular applications.

Prompt emergency treatment for ocular injury, particularly in a battlefield setting, is essential to preserve vision, reduce pain, and prevent secondary infection. A bandage contact lens that could be applied in the field, at the time of injury, would protect the injured ocular surface until hospital treatment is available. Cellulose, a natural polymer, is widely used in biomedical applications including bandage materials. Hydrogels synthesized from different cellulose sources, such as plants, cotton, and bacteria, can have the optical transparency and mechanical strength of contact lenses, by tailoring synthesis parameters. Thus, we optimized the fabrication of cellulose-based hydrogels and evaluated their in vivo biocompatibility and related physical properties. Our data demonstrate that along with tailorable physical properties, our novel cellulose-based hydrogels could be made with contact lens geometry, exhibit no significant signs of material toxicity after 22 days of in vivo testing, and show significant promise for use as a corneal bandage immediately following ocular trauma.

Influence of collagen source on fibrillar architecture and properties of vitrified collagen membranes.

Collagen vitrigel membranes are transparent biomaterials characterized by a densely organized, fibrillar nanostructure that show promise in the treatment of corneal injury and disease. In this study, the influence of different type I collagen sources and processing techniques, including acid-solubilized collagen from bovine dermis (Bov), pepsin-solubilized collagen from human fibroblast cell culture (HuCC), and ficin-solubilized collagen from recombinant human collagen expressed in tobacco leaves (rH), on the properties of the vitrigel membranes was evaluated. Postvitrification carbodiimide crosslinking (CX) was also carried out on the vitrigels from each collagen source, forming crosslinked counterparts BovXL, HuCCXL, and rHXL, respectively. Collagen membrane ultrastructure and biomaterial properties were found to rely heavily on both collagen source and crosslinking. Bov and HuCC samples showed a random fibrillar organization of collagen, whereas rH vitrigels showed remarkable regional fibril alignment. After CX, light transmission was enhanced in all groups. Denaturation temperatures after CX increased in all membranes, of which the highest increase was seen in rH (14.71°C), suggesting improved thermal stability of the collagen fibrils in the membranes. Noncrosslinked rH vitrigels may be reinforced through CX to reach levels of mechanical strength and thermal stability comparable to Bov.

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Banded structures in collagen vitrigels for corneal injury repair.

There is a growing interest in using collagen vitrigels for corneal injury repair. We recently reported the synthesis and thermal denaturation behavior of these gels. In this paper, the banded structure in these vitrified gels is studied by small-angle X-ray scattering (SAXS) one-dimensional (1-D) correlation function analysis and transmission electron microscopy (TEM). Results demonstrate that the collagen vitrigel possess banded structures similar to those of the starting type I collagen, with an average D-spacing of 64nm (by SAXS) or 57nm (by TEM). A combination of SAXS 1-D correlation function analyses and TEM show that overlap and gap distances ranged from 30 to 33nm and from 23 to 25nm, respectively. Changing the vitrification condition does not impact on the banded structure significantly.

Modulation of keratocyte phenotype by collagen fibril nanoarchitecture in membranes for corneal repair.

Type I collagen membranes with tailored fibril nanoarchitectures were fabricated through a vitrification processing, which mimicked, to a degree, the collagen maturation process of corneal stromal extracellular matrix in vivo. Vitrification was performed at a controlled temperature of either 5 °C or 39 °C at a constant relative humidity of 40% for various time periods from 0.5 wk up to 8 wk. During vitrification, the vitrified collagen membranes (collagen vitrigels, CVs) exhibited a rapid growth in fibrillar density through the evaporation of water and an increase in fibrillar stiffness due to the formation of new and/or more-stable interactions. On the other hand, the collagen fibrils in CVs maintained their D-periodicity and showed no significant difference in fibrillar diameter, indicating preservation of the native states of the collagen fibrils during vitrification. Keratocyte phenotype was maintained on CVs to varying degrees that were strongly influenced by the collagen fibril nanoarchitectures. Specifically, the vitrification time of CVs mainly governed the keratocyte morphology, showing significant increases in the cell protrusion number, protrusion length, and cell size along with CV vitrification time. The CV vitrification temperature affected the regulation of keratocyte fibroblasts' gene expressions, including keratocan and aldehyde dehydrogenase (ALDH), demonstrating a unique way to control the expression of specific genes in vitro.

Structure and properties of collagen vitrigel membranes for ocular repair and regeneration applications.

The frequency of ocular injuries on the battlefield has been steadily increasing during recent conflicts. Combat-related eye injuries are difficult to treat and solutions requiring donor tissue are not ideal and are often not readily available. Collagen vitrigels have previously been developed for corneal reconstruction, but increased transparency and mechanical strength are desired for improved vision and ease of handling. In this study, by systematically varying vitrification temperature, relative humidity and time, the collagen vitrigel synthesis conditions were optimized to yield the best combination of high transparency and high mechanical strength. Optical, mechanical, and thermal properties were characterized for each set of conditions to evaluate the effects of the vitrification parameters on material properties. Changes in denaturing temperature and collagen fibril morphology were evaluated to correlate properties with structure. Collagen vitrigels with transmittance up to 90%, tensile strength up to 12 MPa, and denaturing temperatures that significantly exceed the eye/body temperature have been synthesized at 40 °C and 40% relative humidity for one week. This optimal set of conditions enabled improvements of 100% in tensile strength and 11% in transmittance, compared to the previously developed collagen vitrigels.

High resolution stationary digital breast tomosynthesis using distributed carbon nanotube x-ray source array.

PURPOSE: The purpose of this study is to investigate the feasibility of increasing the system spatial resolution and scanning speed of Hologic Selenia Dimensions digital breast tomosynthesis (DBT) scanner by replacing the rotating mammography x-ray tube with a specially designed carbon nanotube (CNT) x-ray source array, which generates all the projection images needed for tomosynthesis reconstruction by electronically activating individual x-ray sources without any mechanical motion. The stationary digital breast tomosynthesis (s-DBT) design aims to (i) increase the system spatial resolution by eliminating image blurring due to x-ray tube motion and (ii) reduce the scanning time. Low spatial resolution and long scanning time are the two main technical limitations of current DBT technology. METHODS: A CNT x-ray source array was designed and evaluated against a set of targeted system performance parameters. Simulations were performed to determine the maximum anode heat load at the desired focal spot size and to design the electron focusing optics. Field emission current from CNT cathode was measured for an extended period of time to determine the stable life time of CNT cathode for an expected clinical operation scenario. The source array was manufactured, tested, and integrated with a Selenia scanner. An electronic control unit was developed to interface the source array with the detection system and to scan and regulate x-ray beams. The performance of the s-DBT system was evaluated using physical phantoms. RESULTS: The spatially distributed CNT x-ray source array comprised 31 individually addressable x-ray sources covering a 30 angular span with 1 pitch and an isotropic focal spot size of 0.6 mm at full width at half-maximum. Stable operation at 28 kV(peak) anode voltage and 38 mA tube current was demonstrated with extended lifetime and good source-to-source consistency. For the standard imaging protocol of 15 views over 14, 100 mAs dose, and 2 × 2 detector binning, the projection resolution along the scanning direction increased from 4.0 cycles/mm [at 10% modulation-transfer-function (MTF)] in DBT to 5.1 cycles/mm in s-DBT at magnification factor of 1.08. The improvement is more pronounced for faster scanning speeds, wider angular coverage, and smaller detector pixel sizes. The scanning speed depends on the detector, the number of views, and the imaging dose. With 240 ms detector readout time, the s-DBT system scanning time is 6.3 s for a 15-view, 100 mAs scan regardless of the angular coverage. The scanning speed can be reduced to less than 4 s when detectors become faster. Initial phantom studies showed good quality reconstructed images. CONCLUSIONS: A prototype s-DBT scanner has been developed and evaluated by retrofitting the Selenia rotating gantry DBT scanner with a spatially distributed CNT x-ray source array. Preliminary results show that it improves system spatial resolution substantially by eliminating image blur due to x-ray focal spot motion. The scanner speed of s-DBT system is independent of angular coverage and can be increased with faster detector without image degration. The accelerated lifetime measurement demonstrated the long term stability of CNT x-ray source array with typical clinical operation lifetime over 3 years.

Prospective-gated cardiac micro-CT imaging of free-breathing mice using carbon nanotube field emission x-ray.

PURPOSE: Carbon nanotube (CNT) based field emission x-ray source technology has recently been investigated for diagnostic imaging applications because of its attractive characteristics including electronic programmability, fast switching, distributed source, and multiplexing. The purpose of this article is to demonstrate the potential of this technology for high-resolution prospective-gated cardiac micro-CT imaging. METHODS: A dynamic cone-beam micro-CT scanner was constructed using a rotating gantry, a stationary mouse bed, a flat-panel detector, and a sealed CNT based microfocus x-ray source. The compact single-beam CNT x-ray source was operated at 50 KVp and 2 mA anode current with 100 microm x 100 microm effective focal spot size. Using an intravenously administered iodinated blood-pool contrast agent, prospective cardiac and respiratory-gated micro-CT images of beating mouse hearts were obtained from ten anesthetized free-breathing mice in their natural position. Four-dimensional cardiac images were also obtained by gating the image acquisition to different phases in the cardiac cycle. RESULTS: High-resolution CT images of beating mouse hearts were obtained at 15 ms temporal resolution and 6.2 lp/mm spatial resolution at 10% of system MTF. The images were reconstructed at 76 microm isotropic voxel size. The data acquisition time for two cardiac phases was 44 +/- 9 min. The CT values observed within the ventricles and the ventricle wall were 455 +/- 49 and 120 +/- 48 HU, respectively. The entrance dose for the acquisition of a single phase of the cardiac cycle was 0.10 Gy. CONCLUSIONS: A high-resolution dynamic micro-CT scanner was developed from a compact CNT microfocus x-ray source and its feasibility for prospective-gated cardiac micro-CT imaging of free-breathing mice under their natural position was demonstrated.

Design and characterization of a spatially distributed multibeam field emission x-ray source for stationary digital breast tomosynthesis.

Digital breast tomosynthesis (DBT) is a limited angle computed tomography technique that can distinguish tumors from its overlying breast tissues and has potentials for detection of cancers at a smaller size and earlier stage. Current prototype DBT scanners are based on the regular full-field digital mammography systems and require partial isocentric motion of an x-ray tube over certain angular range to record the projection views. This prolongs the scanning time and, in turn, degrades the imaging quality due to motion blur. To mitigate the above limitations, the concept of a stationary DBT (s-DBT) scanner has been recently proposed based on the newly developed spatially distributed multibeam field emission x-ray (MBFEX) source technique using the carbon nanotube. The purpose of this article is to evaluate the performance of the 25-beam MBFEX source array that has been designed and fabricated for the s-DBT system. The s-DBT system records all the projection images by electronically activating the multiple x-ray beams from different viewing angles without any mechanical motion. The configuration of the MBFEX source is close to the published values from the Siemens Mammomat system. The key issues including the x-ray flux, focal spot size, spatial resolution, scanning time, beam-to-beam consistency, and reliability are evaluated using the standard procedures. In this article, the authors describe the design and performance of a distributed x-ray source array specifically designed for the s-DBT system. They evaluate the emission current, current variation, lifetime, and focal spot sizes of the source array. An emission current of up to 18 mA was obtained at 0.5 x 0.3 mm effective focal spot size. The experimentally measured focal spot sizes are comparable to that of a typical commercial mammography tube without motion blurring. Trade-off between the system spatial resolution, x-ray flux, and scanning time are also discussed. Projection images of a breast phantom were collected using the x-ray source array from 25 different viewing angles without motion. These preliminary results demonstrate the feasibility of the proposed s-DBT scanner. The technology has the potential to increase the resolution and reduce the imaging time for DBT. With the present design of 25 views, they demonstrated experimentally the feasibility of achieving 11 s scanning time at full detector resolution with 0.5 x 0.3 mm source resolution without motion blur. The flexibility in configuration of the x-ray source array will also allow system designers to consider imaging geometries that are difficult to achieve with the conventional single-source rotating approach.

A carbon nanotube field emission cathode with high current density and long-term stability.

Carbon nanotube (CNT) field emitters are now being evaluated for a wide range of vacuum electronic applications. However, problems including short lifetime at high current density, instability under high voltage, poor emission uniformity, and pixel-to-pixel inconsistency are still major obstacles for device applications. We developed an electrophoretic process to fabricate composite CNT films with controlled nanotube orientation and surface density, and enhanced adhesion. The cathodes have significantly enhanced macroscopic field emission current density and long-term stability under high operating voltages. The application of this CNT electron source for high-resolution x-ray imaging is demonstrated.