Anhydrous Nucleic Acid Nanoparticles for Storage and Handling at Broad Range of Temperatures.

Recent advances in nanotechnology now allow for the methodical implementation of therapeutic nucleic acids (TNAs) into modular nucleic acid nanoparticles (NANPs) with tunable physicochemical properties which can match the desired biological effects, provide uniformity, and regulate the delivery of multiple TNAs for combinatorial therapy. Despite the potential of novel NANPs, the maintenance of their structural integrity during storage and shipping remains a vital issue that impedes their broader applications. Cold chain storage is required to maintain the potency of NANPs in the liquid phase, which greatly increases transportation costs. To promote long-term storage and retention of biological activities at higher temperatures (e.g., +50 °C), a panel of representative NANPs is first exposed to three different drying mechanisms-vacuum concentration (SpeedVac), lyophilization (Lyo), and light-assisted drying (LAD)-and then rehydrated and analyzed. While SpeedVac primarily operates using heat, Lyo avoids temperature increases by taking advantage of pressure reduction and LAD involves a near-infrared laser for uniform drying in the presence of trehalose. This work compares and defines refinements crucial in formulating an optimal strategy for producing stable, fully functional NANPs and presents a forward advancement in their development for clinical applications.

Hyperspectral imaging system based on a single-pixel camera design for detecting differences in tissue properties.

Optical spectroscopy can be used to distinguish between healthy and diseased tissue. In this study, the design and testing of a single-pixel hyperspectral imaging (HSI) system that uses autofluorescence emission from collagen (400 nm) and nicotinamide adenine dinucleotide phosphate (475 nm) along with differences in the optical reflectance spectra to differentiate between healthy and thermally damaged tissue is discussed. The changes in protein autofluorescence and reflectance due to thermal damage are studied in ex vivo porcine tissue models. Thermal lesions were created in porcine skin (n=12) and liver (n=15) samples using an IR laser. The damaged regions were clearly visible in the hyperspectral images. Sizes of the thermally damaged regions as measured via HSI are compared to sizes of these regions as measured in white-light images and via physical measurement. Good agreement between the sizes measured in the hyperspectral images, white-light imaging, and physical measurements were found. The HSI system can differentiate between healthy and damaged tissue. Possible applications of this imaging system include determination of tumor margins during surgery/biopsy and cancer diagnosis and staging.

Light-Assisted Drying for the Thermal Stabilization of Nucleic Acid Nanoparticles and Other Biologics.

Cold-chain storage can be challenging and expensive for the transportation and storage of biologics, especially in low-resource settings. Nucleic acid nanoparticles (NANPs) are an example of new biological products that require refrigerated storage. Light-assisted drying (LAD) is a new processing technique to prepare biologics for anhydrous storage in a trehalose amorphous solid matrix at ambient temperatures. Small volume samples (10 µL) containing NANPs are irradiated with a 1064 nm laser to speed the evaporation of water and create an amorphous trehalose preservation matrix. In previous studies, samples were stored for 1 month at 4 °C or 20 °C without degradation. A FLIR SC655 mid-IR camera is used to record the temperature of samples during processing. The trehalose matrix was characterized using polarized light imaging to determine if crystallization occurred during processing or storage. Damage to LAD-processed NANPs was assessed after processing and storage using gel electrophoresis.

The Application of Light-Assisted Drying to the Thermal Stabilization of Nucleic Acid Nanoparticles.

Background: Cold-chain storage can be challenging and expensive for the transportation and storage of biologics, especially in low-resource settings. Nucleic acid nanoparticles (NANPs) are an example of new biological products that require refrigerated storage. Light-assisted drying (LAD) is a new processing technique to prepare biologics for anhydrous storage in a trehalose amorphous solid matrix at ambient temperatures. In this study, LAD was used to thermally stabilize four types of NANPs with differing structures and melting temperatures. Methods: Small volume samples (10 µL) containing NANPs were irradiated with a 1064 nm laser to speed the evaporation of water and create an amorphous trehalose preservation matrix. Samples were then stored for 1 month at 4°C or 20°C. A FLIR C655 mid-IR camera was used to record the temperature of samples during processing. The trehalose matrix was characterized using polarized light imaging (PLI) to determine if crystallization occurred during processing or storage. Damage to LAD-processed NANPs was assessed after processing and storage using gel electrophoresis. Results: Based on the end moisture content (EMC) as a function time and the thermal histories of samples, a LAD processing time of 30 min is sufficient to achieve low EMCs for the 10 µL samples used in this study. PLI demonstrates that the trehalose matrix was resistant to crystallization during processing and after storage at 4°C and at room temperature. The native-polyacrylamide gel electrophoresis results for DNA cubes, RNA cubes, and RNA rings indicate that the main structures of these NANPs were not damaged significantly after LAD processing and being stored at 4°C or at room temperature for 1 month. Conclusions: These preliminary studies indicate that LAD processing can stabilize NANPs for dry-state storage at room temperature, providing an alternative to refrigerated storage for these nanomedicine products.

Noninvasive laser coagulation of the human vas deferens: optical and thermal simulations.

BACKGROUND AND OBJECTIVES: Successful noninvasive laser coagulation of the canine vas deferens, in vivo, has been previously reported. However, there is a significant difference between the optical properties of canine and human skin. In this study, Monte Carlo (MC) simulations of light transport through tissue and heat transfer simulations are performed to determine the feasibility of noninvasive laser vasectomy in humans. MATERIALS AND METHODS: A laser wavelength of 1,064 nm was chosen for deep optical penetration in tissue. MC simulations determined the spatial distribution of absorbed photons inside the tissue layers (epidermis, dermis, and vas). The results were convolved with a 3-mm-diameter laser beam, and then used as the spatial heat source for the heat transfer model. A laser pulse duration of 500 milliseconds, pulse rate of 1 Hz, and cryogen spray cooling were incident on the tissue for 60 seconds. Average laser power (5-9 W), cryogen pulse duration (60-100 milliseconds), cryogen cooling rate (0.5-1.0 Hz), and increase in optical transmission due to optical clearing (0-50%) were studied. RESULTS: After application of an optical clearing agent (OCA) to increase skin transmission by 50%, an average laser power of 6 W, cryogen pulse duration of 60 milliseconds, and cryogen cooling rate of 1 Hz resulted in vas temperatures of approximately 58°C, sufficient for thermal coagulation, while 1 mm of the skin surface (epidermis and dermis) remained at a safe temperature of approximately 45°C. CONCLUSIONS: MC and heat transfer simulations indicate that it is possible to noninvasively thermally coagulate the human vas deferens without adverse effects (e.g., scrotal skin burns), if an OCA is applied to the skin prior to the procedure.

Light-assisted drying for anhydrous preservation of biological samples: optical characterization of the trehalose preservation matrix.

Protein-based drugs have been developed to treat a variety of conditions and assays use immobilized capture proteins for disease detection. Freeze-drying is currently the standard for the preservation of proteins, but this method is expensive and requires lengthy processing times. Anhydrous preservation in a trehalose amorphous solid matrix offers a promising alternative to freeze-drying. Light assisted drying (LAD) is a processing method to create an amorphous trehalose matrix. Proteins suspended in a trehalose solution are dehydrated using near-infrared laser light. The laser radiation accelerates drying and as water is removed the trehalose forms a protective matrix. In this work, LAD samples are characterized to determine the crystallization kinetics of the trehalose after LAD processing and the distribution of amorphous trehalose in the samples. These characteristics influence the long-term stability of the samples. Polarized light imaging revealed that LAD processed samples are stable against crystallization during low-humidity storage at room temperature. Scanning white light interferometry and Raman spectroscopy indicated that trehalose was present across samples in an amorphous form. In addition, differential scanning microcalorimetry was used to measure the thermodynamic characteristics of the protein lysozyme after LAD processing. These results demonstrate that LAD does not change the properties of this protein.

Noninvasive enhanced mid-IR imaging of breast cancer development in vivo.

Lumpectomy coupled with radiation therapy and/or chemotherapy is commonly used to treat breast cancer patients. We are developing an enhanced thermal IR imaging technique that has the potential to provide real-time imaging to guide tissue excision during a lumpectomy by delineating tumor margins. This enhanced thermal imaging method is a combination of IR imaging (8 to 10 µm ) and selective heating of blood (∼0.5°C ) relative to surrounding water-rich tissue using LED sources at low powers. Postacquisition processing of these images highlights temporal changes in temperature and the presence of vascular structures. In this study, fluorescent, standard thermal, and enhanced thermal imaging modalities, as well as physical caliper measurements, were used to monitor breast cancer tumor volumes over a 30-day study period in 19 mice implanted with 4T1-RFP tumor cells. Tumor volumes calculated from fluorescent imaging follow an exponential growth curve for the first 22 days of the study. Cell necrosis affected the tumor volume estimates based on the fluorescent images after day 22. The tumor volumes estimated from enhanced thermal imaging, standard thermal imaging, and caliper measurements all show exponential growth over the entire study period. A strong correlation was found between tumor volumes estimated using fluorescent imaging, standard IR imaging, and caliper measurements with enhanced thermal imaging, indicating that enhanced thermal imaging monitors tumor growth. Further, the enhanced IR images reveal a corona of bright emission along the edges of the tumor masses associated with the tumor margin. In the future, this IR technique might be used to estimate tumor margins in real time during surgical procedures.

Fiber-optic manipulation of urinary stone phantoms using holmium:YAG and thulium fiber lasers.

Fiber-optic attraction of urinary stones during laser lithotripsy may be exploited to manipulate stone fragments inside the urinary tract without mechanical grasping tools, saving the urologist time and space in the ureteroscope working channel. We compare thulium fiber laser (TFL) high pulse rate/low pulse energy operation to conventional holmium:YAG low pulse rate/high pulse energy operation for fiber-optic suctioning of plaster-of-paris (PoP) stone phantoms. A TFL (wavelength of 1908 nm, pulse energy of 35 mJ, pulse duration of 500 µs, and pulse rate of 10 to 350 Hz) and a holmium laser (wavelength of 2120 nm, pulse energy of 35 to 360 mJ, pulse duration of 300 µs, and pulse rate of 20 Hz) were tested using 270-µm-core optical fibers. A peak drag speed of ~2.5 mm/s was measured for both TFL (35 mJ and 150 to 250 Hz) and holmium laser (210 mJ and 20 Hz). Particle image velocimetry and thermal imaging were used to track water flow for all parameters. Fiber-optic suctioning of urinary stone phantoms is feasible. TFL operation at high pulse rates/low pulse energies is preferable to holmium operation at low pulse rates/high pulse energies for rapid and smooth stone pulling. With further development, this novel technique may be useful for manipulating stone fragments in the urinary tract.

Noninvasive thermal coagulation of deep subsurface tissue structures using a laser probe with integrated contact cooling.

Cooling methods are used during cosmetic laser surgery to preserve a superficial layer of the skin surface. This study investigates contact cooling for sparing a deeper layer of the tissue surface during laser irradiation of subsurface tissues, with the goal of developing noninvasive laser therapy applications beyond cosmetic surgery. A laser probe was designed and tested for simultaneous laser irradiation and contact cooling of liver tissue, ex vivo. Gross and histologic examination was used to quantify thermal lesion dimensions. Liver lesions of 5.8-mm-diameter were created, while preserving the tissue surface to a depth of 1.5 mm. In vivo animal studies are planned to optimize the laser and cooling parameters for potential clinical applications.